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2013-02-06
Cloning, Expression, and Functional
Characterization of TL1A-Ig
Samia Q. Khan
University of Miami, [email protected]
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UNIVERSITY OF MIAMI CLONING, EXPRESSION, AND FUNCTIONAL
CHARACTERIZATION OF TL1A-IG
By
Samia Q. Khan
A DISSERTATION
Submitted to the Faculty
of the University of Miami
in partial fulfillment of the requirements
for the degree of Doctor of Philosophy
Coral Gables, Florida
May 2013
©2013 Samia Q. Khan All Rights Reserved UNIVERSITY OF MIAMI A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy CLONING, EXPRESSION, AND FUNCTIONAL
CHARACTERIZATION OF TL1A-IG
Samia Q. Khan Approved:
________________
Eckhard R. Podack, M.D., Ph.D.
Professor of Immunology and Microbiology
_________________
M. Brian Blake, Ph.D.
Dean of the Graduate School
________________
Kerry L. Burnstein, Ph.D.
Professor of Molecular & Cell Pharmacology
_________________
Wasif N. Khan, Ph.D.
Professor of Immunology and Microbiology
_________________
Robert Levy, Ph.D.
Professor of Immunology and Microbiology
_________________
Dao Nguyen, M.D.
Associate Professor of Surgery
KHAN, SAMIA Q. (Ph.D., Cancer Biology) Cloning, Expression, and Functional Characterization
(May 2013)
of TL1A-Ig
Abstract of a dissertation at the University of Miami Dissertation supervised by Professor Eckhard R. Podack No. of pages in text. (124) TNFRSF25, a member of the TNF receptor superfamily, is a type I
transmembrane protein that has a cytoplasmic death domain. TNFRSF25 is expressed
primarily by T lymphocytes, including CD4+, CD8+, and NKT cells and is efficiently
upregulated after T cell stimulation. The natural ligand for TNFRSF25, TL1A, is a type II
transmembrane protein can be subsequently cleaved as a soluble trimeric protein by a
member of metalloproteases.
Our lab previously reported that TNFRSF25 stimulation using an agonist
antibody, 4C12, expands pre-existing CD4+FoxP3+ Tregs in vivo. To determine how the
physiological ligand differs from the antibody, we generated the soluble mouse TL1A-Ig
fusion protein (TL1A-Ig) that forms a dimer of TL1A-trimers in solution with an
apparent m.w. of 528 kDa. In vitro, TL1A-Ig mediated rapid proliferation of Foxp3+Treg
and a population of CD4+FoxP3- conventional T cells (Tconv). TL1A-Ig also blocked de
novo biogenesis of inducible Tregs (iTregs) and it attenuated the suppressive function of
Treg. Treatment with TL1A-Ig in vivo induced the proliferation and activation of
memory Tconvs, as well as, the expansion of Tregs such that they became 30-35% of all
CD4+ T cells in the peripheral blood within 5 days of treatment. Treg proliferation was
dependent upon TCR engagement with MHC class II. Elevated Tregs were maintained
for at least 20 days with daily injections of TL1A-Ig. TL1A-Ig-expanded Treg cells
expressed high levels of activation/memory markers KLRG1 and CD103 and were highly
suppressive ex vivo. TL1A-Ig mediated Treg expansion in vivo was protective against
allergic lung inflammation, a mouse model for asthma, by reversing the ratio of Tconv
cells to Tregs in the lung and blocking eosinophil exudation into the bronchoaveolar
fluid. The potential of TNFRSF25 agonists was also tested in transplant tolerance.
TNFRSF25 mediated Treg cell expansion in vivo significantly prolonged allogeneic skin
graft survival. Our findings shed light on the role of TNFRSF25 signaling in CD4+ T cell
subsets and the role of TL1A/TNFRSF25 in mediating inflammatory responses and
resolving inflammation in tissue. Thus, TL1A-Ig fusion proteins are highly active and
tightly controllable agents to stimulate Treg proliferation in vivo, and are uniquely able to
maintain high-levels of expanded Treg for long periods of time by repeated
administration.
This dissertation is dedicated to my parents and my husband.
My accomplishments are a result of their unconditional love,
support and unwavering faith in me.
iii Acknowledgements
I am thankful to my mom and dad, Imtiaz and Qamar, who taught me the
importance of education, hard work and persistence. As I come to the end of my five and
a half year journey of graduate school, I realize how important the emotional support,
patience and encouragement of my parents, and very recently my husband, was to make
this accomplishment possible. I am proud to be a woman who left her home at a young
age to pursue her dream of higher education. Imran, my husband and my best friend,
thank you for being patient with me and for being by my side through the toughest time
of my graduate career. This accomplishment would have been impossible without my
family.
To the Podack Lab: Thank you everyone for your help and critical analysis of my
experimental data, for sharing your wisdom and technical skills and making the lab a
wonderful place to work.
And finally, to my mentor, Dr. Eckhard Podack, MD, PhD, and the person who
ignited my passion for science: Thank you for teaching me classical scientific and correct
techniques, for your guidance and support, for pushing me harder, for sharing your love
for science and giving me the freedom to ask questions about the unknown. No words can
describe my gratitude for your priceless gift of knowledge.
Thank you all!
iv Table of Contents
LIST OF FIGURES
viii
LIST OF TABLES
xi
CHAPTER 1: INTRODUCTION
1
1.1 The immune system: innate and adaptive responses
1
1.2 Subsets of T lymphocytes
3
1.3 TNF superfamily members
12
1.3.1 Expression, structure, and signaling of TNFSF members
13
1.3.2 Multiple functions of TNF superfamily members
20
1.3.2.1 Development & maintenance of lymphoid structure
20
1.3.2.2 Defense against microbial infections
21
1.3.2.3 Mediators of cell death
22
1.3.2.4 TNF superfamily members in cellular responses
22
1.3.3
1.3.2.4.1
CD4+ and CD8+ T effector cells
23
1.3.2.4.2
Other immune cells
24
1.3.2.4.3
Regulatory T cells
26
TNF superfamily members in disease and immunotherapy
27
1.3.3.1 TNF superfamily members in autoimmune and
inflammatory diseases and cancer
28
1.3.3.2 Protein therapeutics targeting TNF super family members
1.4 TNF-like ligand 1A (TL1A)/TNF receptor superfamily 25 (TNFRSF25)
30
33
1.4.1
TL1A
33
1.4.2
TNFRSF25
35
1.4.3
TL1A/TNFRSF25 in immunological responses
36
1.5 Aims of the dissertation
38
CHAPTER 2. RESULTS
40
2.1 Construction and characterization of TL1A-Ig
40
2.1.1
Genetic engineering and preliminary studies of four TL1A-Ig constructs
v 40
2.1.2
GS selection system for the generation of high TL1A-Ig expressing clone
43
2.1.3
Biochemical and gel filtration analysis of TL1A-Ig
45
2.2 In vitro functional activity of TL1A-Ig
50
2.2.1
TL1A-Ig binds and stimulates TNFRSF25 overexpressed on tumor cells
50
2.2.2
TL1A-Ig binds and co-stimulates TNFRSF25 expressed on T cells
52
2.3 In vivo functional activity of TL1A-Ig
55
2.3.1
Half-lives of TL1A-Ig and 4C12
55
2.3.2
TL1A-Ig induces rapid proliferation of CD4+FoxP3+ Treg cells in vivo
56
2.3.3
Long-term administration of TL1A-Ig maintains elevated Treg levels
58
2.3.4
Characterization of T cell subsets in TL1A-Ig treated mice.
60
2.3.5
FoxP3+ Tregs from TL1A-Ig treated mice are highly suppressive ex vivo
64
2.3.6
Depletion of gut commensal bacteria by antibiotics treatment
66
2.3.7
TNFRSF25 costimulation and induction of ovalbumin specific Tregs in vivo
68
2.3.8
Suppression of allergic lung inflammation by TL1A-Ig expanded Tregs
70
2.3.9
Prolongation of allogeneic skin transplant by TL1A-Ig expanded Tregs
73
CHAPTER 3: DISCUSSION
76
CHAPTER 4: MATERIAL AND METHOD
88
4.1
Mice
88
4.2
Antibodies and reagents
88
4.3
Plasmid Construction
89
4.4
Cells and Culture Conditions
90
4.5
Transfection and selection
90
4.6
Production and Purification Conditions
91
4.7
ELISA quantification assays
92
4.8
Coomassie Blue stain and Western blot analysis
93
4.9
Gel filtration and dot blot analysis
93
4.10
In vitro binding assays to mouse TNFRSF25 expressing cells
94
4.11
In vitro caspase assays
95
vi 4.12
In vitro T cell culture
95
4.13
In vitro suppression assays
96
4.14
Antibiotics
97
4.15
Induction of allergic lung inflammation
97
4.16
Skin transplantation
98
4.17
Tumor inoculation and immunization
98
4.18
Flow cytometry analysis and FACs sorting
98
4.19
Cell adoptive transfer
99
4.20
Histology
99
4.21
Statistics
100
Appendix 1: Combination therapy of gp96-Ig vaccine and TNFRSF25
agonists to enhance anti-tumor immunity
101
REFERENCES
105
vii List of Figures
Figure 1.
T cell lineage and subsets
3
Figure 2.
APCs deliver 3 kinds of signals
3
Figure 3.
Summary of the 4 CD4 T helper cell fates
5
Figure 4.
Suppressive mechanisms of regulatory T cells
8
Figure 5:
The TNF/TNFR superfamily
12
Figure 6:
Structural organization of the TNF/TNFR family members
15
Figure 7:
Primary FADD-dependent DD signaling complexes
18
Figure 8:
TRADD-dependent DD signaling complex
18
Figure 9:
Present and future therapeutics based on members of the
TNF superfamily and their receptors
28
Figure 10:
Diagram of TL1A
34
Figure 11:
Schematic diagram of TL1A-Ig-pBMGSneo cloning strategy
40
Figure 12:
Construction and expression of TL1A-Ig fusion proteins
41
Figure 13:
Selection of the highest TL1A-Ig producing cell clone
42
Figure 14:
TL1A-Ig exists as a higher molecular weight protein in supernatant
43
Figure 15:
Construction of the TL1A-Ig-pVitro plasmid
44
Figure 16:
Characterization of purified TL1A-Ig by SDS-PAGE and western blot analysis
46
Figure 17:
Characterization of purified TL1A-Ig Prep 1 by gel filtration
and SDS-PAGE analysis
Figure 18:
47
Characterization of purified TL1A-Ig Prep 2 by gel filtration
and SDS-PAGE analysis
Figure 19:
47
Characterization of purified TL1A-Ig Prep 3 by gel filtration
and SDS-PAGE analysis
Figure 20:
48
Characterization of purified TL1A-Ig Prep 4 by gel filtration
and SDS-PAGE analysis
Figure 21:
49
Characterization of purified TL1A-Ig Prep 5 by gel filtration
and SDS-PAGE analysis
49
viii Figure 22:
TL1A-Ig and 4C12 bind to their receptor, TNFRSF25,
expressed on P815 cells
50
Figure 23:
TL1A-Ig induces apoptosis in TNFRSF25 expressing P815 cells
51
Figure 24:
TL1A-Ig binds to and stimulates endogenous TNFRSF25 on activated T cells
52
Figure 25:
TL1A-Ig induces proliferation and induction of iTregs
53
Figure 26:
MHCII antibody does not abrogate TL1A-Ig mediated Treg proliferation
54
Figure 27:
Treg cells maintain residual TCR signaling from self-peptide recognition in vivo
55
Figure 28:
Half-lives of 4C12 and TL1A-Ig are 13.5 hrs and 4 days
55
Figure 29:
TL1A-Ig mediates rapid proliferation of CD4+FoxP3+ Treg cells in vivo
56
Figure 30:
TNFRSF25 mediated Treg expansion is dependent on MHC Class II
57
Figure 31:
Daily injections of TL1A-Ig lead to sustained Treg
expansion in vivo for over 20 days
58
Figure 32:
H&E staining of tissue sections from mice injected daily with TL1A-Ig
59
Figure 33.
Characterization of T cell subsets in TL1A-Ig treated mice
60
Figure 34.
TL1A-Ig treatment increases expression of memory markers on Tconvs
61
Figure 35.
TL1A-Ig treatment leads to Treg expansion in vivo by inducing
proliferation of CD25hi and CD25int Tregs
63
Figure 36.
TL1A-Ig treatment enhanced activation and memory formation of Treg cells
64
Figure 37.
Suppressive activity of in vivo expanded Treg
65
Figure 38.
Antibiotics treatment results in ceca dilation
66
Figure 39.
Antibiotics treatment does not abrogate TL1A-Ig mediated
proliferation and activation of memory T cells
Figure 40.
67
Antibiotics treatment does not abrogate TL1A-Ig mediated Treg
activation and formation of memory Tregs
68
Figure 41.
TL1A-Ig does not abrogate the induction of Tregs in vivo
69
Figure 42.
Therapeutic protocol for the acute model of allergic lung inflammation
70
Figure 43.
TL1A-Ig mediated Treg expansion in the peripheral blood, lungs and spleens
71
Figure 44.
TL1A-Ig treatment reduces eosinophils in the BALF
72
ix Figure 45.
TL1A-Ig treatment reduces lymphocyte infiltration and airway
mucus secretion in the lungs
73
Figure 46.
TNFRSF25 agonists prolong allogeneic skin grafts
74
Figure 47.
Protocol for TL1A-Ig and gp96-Ig vaccine treatment for OTI cell expansion
Figure 48.
TL1A-Ig boosts clonal expansion of CD8+ T cells and Tregs
when combined with gp96-Ig vaccine
Figure 49.
102
TNFRSF25 agonist TL1A-Ig does not enhance gp96-Ig mediated
rejection of established tumors
101
103
x List of Tables
Table 1.
Cellular expression of some TNF ligands and receptors
Table 2.
Leukocyte subsets with similar frequencies in IgG-treated
13
and TL1A-Ig-treated mice
61
Table 3.
TL1A-Ig decreases Tconv:Treg ratio and (TCM +TEM):Treg ratio
62
Table 4.
Summary of comparative studies of 4C12 and TL1A-Ig
78
xi CHAPTER 1: Introduction
1.1 The immune system: innate and adaptive arms
The immune system consists of molecules and cells that work together to protect
the individual from microbial infection and maintain homeostasis. The immune system is
comprised of the innate and adaptive immune responses. Together these responses fulfill
four crucial tasks: immunological recognition (the detection of an infection), containment
and elimination of infection, immune regulation (keeping the immune response under
control so that there is no damage to the body leading to conditions of allergy and
autoimmune disease), and generation of immunological memory that protects the
individual from recurring disease due to the same pathogen. The earlier evolved innate
immune system, found in all plants and animals, provides a non-specific immediate
defense response to infection that develops in hours---faster than adaptive responses.
Microbes that overcome physical and chemical barriers first encounter toxic chemicals
and powerful degradative enzymes produced by phagocytic innate cells such as
macrophages, neutrophils, eosinophils, basophils, and natural killer (NK) cells. Innate
dendritic cells engulf pathogens, process and present pathogen antigen to T lymphocytes
leading to the initial activation of the adaptive immune response.
The adaptive immune system, although overlapping with the innate immune
response, requires days to develop since this depends on the formation and proliferation
of antigen specific clones of B and T lymphocytes that recognize and respond to
individual antigens by means of highly specialized antigen receptors. The activated
lymphocytes produced in the initial adaptive response provide the vertebrate immune
system with the ability to “remember” specific pathogens (immunological memory) and
1 2 subsequently mount an immune response that is faster and greater in magnitude.
Adaptive immunity includes antibodies that provide humoral immunity and T cells that
provide cellular immunity, both of which complement each other. All cellular
components of the innate and adaptive immune system derive from the hematopoietic
stem cells of the bone marrow. B cells mature in the bone marrow, while T cell
progenitors migrate to the thymus and mature there. Lymphocytes circulate in the blood
and lymphatic system and are found in primary lymphoid organs, the bone marrow and
thymus, and peripheral secondary lymphoid organs including spleen, lymph nodes, and
the mucosal lymphoid tissues found in the gut, nasal, respiratory and urogenital tracts and
other mucosa.
Ligation to antigen causes B cells to proliferate and differentiate into effector
plasma cells that produce antibodies, while T cells recognize antigen presented by APCs
activate and differentiate into one of the effector cell subsets involved in killing
(cytotoxic T cells), activation (helper T cells that activate B cells to produce antibody and
to undergo class switching and affinity maturation, and regulate other lymphocytes), or
regulation (regulatory T cells that suppress other lymphocyte activity and help control
immune responses). Some of the antigen-activated B and T cells formed during an
immune response also differentiate into memory cells that are responsible for the
“adaptive” long-lasting immunity (1).
3 1.2 Subsets of T lymphocytes
Figure 1: T cell lineages and subsets
(adapted from (2))
Figure 2: APCs deliver 3 kinds of signals
(adapted from (1))
After maturation, naïve T lymphocytes emerge out of the thymus and are
composed of two main classes that are distinguished by the mutually exclusive
expression of cell-surface co-receptor proteins CD4 or CD8 (Figure 1) (2). Thymic T
cells are also defined by the expression of a T cell receptor (TCR), αβ or γδ (depending
on the TCR chains present), which is responsible for recognizing antigens presented by
the products of major histocompatibility gene complex (MHC) family. αβ T cells are
classically divided either into CD4+ T helper (Th) cells or CD8+ cytotoxic lymphocytes
(CTL) that recognize peptides presented by MHC class II or MHC class I on antigen
presenting cells (APCs), respectively. APCs deliver 3 kinds of signals for clonal
expansion and differentiation of naïve T cells (Figure 2) (1). Signal 1 enables T cell
4 activation and includes antigen specific signals derived from the ligation of a specific
antigen-MHC molecule. The clonal expansion of antigen-specific T cells is provided by
costimulatory signals (signal 2) that are primarily involved in promoting or inhibiting the
survival and expansion of the T cells and belong to the B7 family (i.e CD28). CD28dependent costimulation of activated T cells induces expression and production of IL-2
and the higher affinity IL-2 surface receptor CD25. Other signal 2 molecules belong to
the tumor necrosis factor (TNF) receptor superfamily. Finally, cytokines, including
members of the TNF superfamily, provide signal 3 that is one determining factor for the
subtype of effector T cell generated. The milieu of cytokines in the microenvironment
and the subsequent activation of specific transcription factors are the two main factors
determining the final effector T cell fate (3).
Some of the T-cell populations emerging out of the thymus are distinct lineages of
cells such as “natural” regulatory T cells (nTreg), natural killer T cells (NKT cells), and
γδ T cells (Figure 1). More “innate-like” T cells subsets, NKT and γδ T cells can be
activated by cytokines or TCR stimulation. NKT cells have an αβ TCR and recognize
antigen in the context of a CD1d, a non-classical MHC I molecule, while γδ T cells are
not MHC restricted although they are capable of generating more unique antigen
receptors than αβ T cells and B cells combined (4). The latter’s ability to recognize
soluble protein and non-protein antigens of endogenous and exogenous origins also
distinguishes them from αβ T cells (4). Naive CD4+ αβ T cells can differentiate into four
different lineage of effector T cells after their initial activation by antigen: Th1, Th2,
Th17 or induced regulatory T cell (iTreg) (Figure 3) (5). The heterogeneity of CD4+ T
cells enables an optimized immune response according to the nature of the infection.
5 Figure 3: Summary of the 4 CD4 T helper cell fates: their functions, their
unique products, their characteristic transcription factors, and cytokines
critical for their fate determination. (adapted from (5)).
Th1
cells
provide
immunity
against
intracellular
pathogens
including
mycobacteria and can induce some autoimmune and inflammatory diseases. The
principle cytokines produced by Th1 cells are IFN-γ, lymphotoxin α (LTα), and IL-2.
High levels of IFN-γ production by Th1 cells is important in enhancing the microbicidal
activity of phagocytes that mediate pathogenic clearance. In addition, Th1 cells also
facilitate the production of opsonizing and complement fixing antibodies including IgG2a
antibodies in mice, and IgM, IgA, IgG1 and IgG3 antibodies in humans (3). Although
generally overlooked, the humoral response promoted by Th1 cells plays an important
role in protection against intracellular pathogens. Collaboration between interferons and
IL-12, produced by Toll-like receptor (TLR) activated dendritic cells that recently
encountered microbes, are potent inducers of Th1 polarization. IFN-γ and IL-12 activate
the signal transducer and activator of transcription (STAT-1), resulting in the
upregulation of T-box expressed in T cells (T-bet), which is the master regulator of Th1
cells. T-bet in turn induces early T-cell IFN-γ production and upregulates IL-12Rβ2
6 expression, which renders Th1 cells more hyperresponsive to IL-12, resulting in a
positive feedback production of IFN-γ through STAT-4 activation. STAT-4 causes
upregulation of IL-18Rα. IL-18 and IL-12 synergize in inducing more IFN-γ, showing
that IL-18 is crucial in Th1 responses but not required for Th1 differentiation. Together
IFN-γ and IL-12 fully differentiate Th1 cells (3, 5). Interestingly, to block differentiation
toward alternative effector pathways, T-bet also interacts with several other T helper cell
lineage-defining transcription factors (6-9).
Th2 cells help B cells mediate host defense against extracellular parasitic
infections such as helminthes and extracellular bacteria and this population is implicated
in the induction and persistence of asthma and allergic diseases. Cytokines produced by
Th2 cells are involved in IgE class switching in B cells (IL-4 and IL-13), recruitment of
eosinophils (IL-5), activation of mast cells and lymphocytes and production of mucus by
epithelial cells (IL-9), suppression of Th1 cell proliferation and dendritic cell function
(IL-10), and expulsion of helminthes and induction of airway hypersensitivity (IL-13) (3,
5). Th2 cells induce IgG1 and IgE antibodies in mice and IgM, IgG4 and IgE in humans.
IgE antibody binds and crosslinks FcεRI receptors of basophils and mast cells resulting in
the secretion of active molecules including serotonin and histamine and the production of
several cytokines and chemokines including IL-4, IL-13, TNF-α, RANTES and eotaxins
(3, 5). Th2 cells also produce IL-25 that serves both as an activation and amplification
factor for Th2 responses. IL-4, the positive feedback cytokine of Th2 cell differentiation,
activates STAT-6, which is the major signal transducer in IL-4 mediated Th2
differentiation. STAT-6 activation in turn is necessary and sufficient for inducing high
expression levels of the Th2 master regulator gene, GATA-3, which serves as a positive
7 feedback loop to produce more IL-4 in collaboration with IL-2 induced activation of
STAT-5 (3, 5). Together, GATA-3 and STAT-5 have been shown to fully differentiate
Th2 cells in vitro (10).
A more recently characterized subset includes Th17 cells that mediate host
immune responses against extracellular bacteria and fungi and are pathogenic in
induction of organ-specific autoimmune diseases. Th17 cells produce several cytokines
including IL-17a, IL-17f, IL-21 and IL-22. Studies show that many organ-specific
diseases that were previously attributed to Th1 cells were indeed caused by Th17 cells
that are responsive to IL-23, which shares a subunit (p40) and a chain of its receptor (IL12Rβ1) with IL-12 (3). IL-17a can induce several pro-inflammatory cytokines, including
IL-6, and chemokines such as IL-8 to induce inflammatory responses. Transforming
growth factor-β (TGF-β) is required for initiation, while IL-6 is a critical cofactor for
Th17 differentiation. IL17a and IL17f recruit and activate neutrophils. IL-21 is a
stimulatory factor for Th17 differentiation, a positive feedback amplifier, and can replace
IL-6 in inducing transcription factor retinoic acid-related orphan receptor (RORγt), which
in turns mediates IL-17 production. RORα, STAT-3 (a major inducer of IL-6, IL-21 and
IL-23) and IRF4 have been found to contribute to Th17 cell differentiation (3, 5, 11).
Regulatory T cells play a vital role in preventing the development of autoimmune
diseases, maintaining self-tolerance, and regulating the development and intensity of
immune responses to foreign antigens, including allergens (12). Tregs rely on several
mechanisms including cytokine production, cytolysis, IL-2 consumption, and reduction
of costimulation and antigen presentation, to suppress the functions of a full array of cell
types including CD4+ Th cells, CD8+ cytotoxic T lymphocyte (CTL) granule release, B-
8 cell antibody production and affinity maturation, and APC function and maturation(13,
14) (Figure 4).
Figure 4: Suppressive mechanisms of regulatory T cells (adapted from (13))
Tregs secrete suppressive cytokines including transforming growth factor-β (TGF-β), IL10 and IL-35. TGF-β is necessary for the survival of nTregs in the periphery and it also
maintains peripheral tolerance by inhibiting proliferation and differentiation of selfreactive CD4 and CD8 T cells. Mechanistically, it appears that membrane bound TGF-β
on Tregs, in combination with CTLA-4 and CD80, interacts with TGF-β receptor on
effector T cells that causes a block in the upregulation of IL-2 receptor and therefore their
inability to respond to IL-2 required for survival (15). In combination with IL-2 and
retinoic acid (RA), TGF-β promotes the differentiation of naïve CD4+ T cells into iTregs
in the periphery. IL-10 downregulates IL-12 production and co-stimulatory molecules in
macrophages, thereby reducing the generation of Th1 type CD4+ T cells (16). IL-35 has
been reported to suppress immune responses by expanding Treg cells, inhibiting
proliferation of CD4+CD25- effector cells and differentiation of Th17 cells (17). The
9 expression of Galectin-1 by Treg cells also causes cell cycle arrest, apoptosis, and
inhibition of production of proinflammatory cytokines by target cells. Treg cells can be
lytic for activated CD4+ T cells, CD8+ T cells and NK cells by mechanisms that are
dependent on perforin, granzyme A, granzyme B, or a combination of these factors (18).
Since a large population of thymic derived Treg expresses CD25, the receptor for IL-2,
studies have suggested that Tregs may compete with FoxP3- T cells for IL-2 and inhibit
the differentiation and proliferation of FoxP3- T cells, resulting in apoptosis that is
dependent on proapoptotic factor Bim. Tregs can condition dendritic cells to secrete
immunosuppressive molecule indoleamin 2,3 dioxygenese (IDO) and downregulate
costimulatory molecules, including CD80 and CD86, and MHC-II on the cell surface—
factors that that suppress activation of effector T cells. Tregs also use the catalytic
inactivation of ATP (an indicator of tissue destruction that upregulates CD86 on DCs) by
expressing CD39 and/or CD73 to produce adenosine and to hinder upregulation of
costimulatory molecules and maturation of DCs. A combination of these suppressive
mechanisms, in combination with homeostatic proliferation of Tregs, leads to the
regulation of effector T (Teff) cells during an immune response. Indeed, studies indicate
that during a primary immune response to foreign antigen, the expansion and contraction
of Treg cells was equal to that of Teff, while the relative accumulation of Treg at the peak
of the response was greater than Teff, reflecting extensive proliferation of Treg in the cell
pool (19). Although proliferating polyclonal nTregs may provide bystander suppression
(19), studies also indicate accumulation of more potent antigen-specific iTregs that have
the ability to attenuate effector responses in an antigen-dependent manner (20).
10 Both thymic nTreg and peripheral iTreg differentiation require TCR signaling that
results in the upregulation of CD25 on the cell surface, making the precursor cells more
receptive to IL-2 signaling in the microenvironment. IL-2 signaling leads to the activation
of transcription factor STAT-5. Additionally, CD28 signaling, via ligation to CD80 and
CD86, leads to the induction of the key Treg transcription factor, FoxP3 in the thymus.
FoxP3, an invaluable marker for Treg, is required for their differentiation, maintenance
and suppressive function (21, 22). These signaling pathways lead to the recruitment of
several transcription factors including NFAT, cRel, Creb and STAT-5 to the FoxP3
promoter that induces its expression. Although TGF-β is important for the differentiation
of iTreg, it is also important for nTreg development (23). In the absence of
proinflammatory cytokines and presence of IL-6, TGF-β induces iTreg differentiation
from CD4+ T conventional cells (Tconv) by activating Smad-3. Concurrent TCR
signaling induces NFAT activation, and together Smad-3 and NFAT collaborate to
promote FoxP3 expression. As is the case for nTregs, TGF-β and IL-2-mediated STAT-5
activation is also required for iTregs survival and function after differentiation (5).
CD8+ T cells are a crucial part of the adaptive immune system since they have the
intrinsic capability to respond to low levels of peptide-MHC I molecules and mediate
direct killing of antigen-presenting and MHC I positive cells. They mediate host immune
response against intracellular pathogens (viruses) and tumor cells. Like CD4+ T cells,
naïve CD8+ T cells require detection of peptide-MHC I on APCs and costimulation
signals provided by CD28-CD86 interaction in the presence of cytokines that leads to the
activation, proliferation and differentiation of naïve CD8 cells into cytotoxic T
lymphocytes (CTL) (24). The differentiation of CD8 cells parallels that of their CD4 T
11 cell counterparts. For example, IL-12 induces T-bet expression leading to Tc1
differentiation, while GATA-3 is induced by IL-4 for Tc2 differentiation (25). More
recently it has been shown that the IL-17 producing subset of CD8 T cells displays a
suppressed cytotoxic function along with low levels of the CTL markers and undergoes a
similar differentiation program as Th17 cells (26).
Differentiated effector CD8 T cells utilize different mechanisms, including
cytotoxic and non-cytotoxic, to attack their target cells. These methods include the use of
cytotoxic molecules such as perforin and granzyme mediated direct contact-dependent
cytolysis (27), expression of TNF member Fas ligand (CD95L) that induces apoptosis in
a Fas-FasL dependent manner (27) and immediate release of pro-inflammatory cytokines
such as IFN-γ and TNF-α to sustain local inflammation that attracts other immune cells
(28).
Recent studies have assigned an additional duty to effector CD8 T cells which
includes a regulatory role in preventing excessive tissue injury by secretion of IL-10,
which is an immunosuppressive cytokine produced by multiple cell types including CD4+
Treg cells (29-31). Fully differentiated CD8 T cells produce high levels of IL-10 at
infection sites in different viral infection models (29-31). At the peak of the viral immune
response, IL-10 produced by CD8 cells is found mostly in peripheral sites, while CD4+ T
cells derived IL-10 dominates in secondary lymphoid organs (29-31). IL-10+CD8+ T cells
quickly diminish after the viral infection is cleared, while IL-10+CD4+ T cells remain,
suggesting differences between the mechanisms that regulate IL-10 produced in CD8+
and CD4+ T cells (32). CD8+ T cells derived IL-10 prevents bystander tissue damage
during viral immune response but does not interfere with viral clearance.
erfamily. The
in blue and
h their recepTNFR superhe appropriate
ns within the
d as red cylinTRAF adaptors
12 1.3 TNF superfamily members
More than a century ago, German physician P Burns reported tumor regression in
patients after a bacterial infection. Later studies identified molecular factors released by
lymphocytes and macrophages during infections that caused the cytolysis of several cell
types, but more importantly tumor cells. Lymphotoxin (LT) and tumor necrosis factor
(TNF) were identified as these products that were able to lyse several cell types, most
importantly tumorThe
cellsintriguing
(33, 34). Identification
of the
the protein
sequence
revealed that LT
biology of
TNF/TNFR
superfamily
and
TNF
exhibited
50% OX40L
aminoGITRL
acid sequence
and bound
to the?same?receptor.
CD30L
CD40L 4-1BBL
BAFF
CD27L
RANKL homology
APRIL
TWEAK
Moreover, the cDNA cloning and sequence homology analysis revealed that these two
cytokines are the prototypic members of a gene superfamily that regulates essential
biological functions in mammals and most importantly immune responses at different
CD27 CD30 CD40 4-1BB
levels
(35, 36).
CD95L
TL1A
OX40
GITR
OPG
TRAIL
RANK BAFFR TACI BCMA Fn14
EDA
TNF
LTα
LTαβ
TROY RELT
LIGHT BTLA
The intriguing biology of the TNF/TNFR superfamily
CD95 DcR3 DR3 DR4 DR5 DcR1 DcR2
OPG
CD27L CD30L CD40L 4-1BBL OX40L GITRL
EDAR XEDAR TNFR1 TNFR2 LTβR HVEM DcR3
RANKL
BAFF
APRIL
TWEAK
?
?
Further mechanisms to control the induction of apopurn can associate with TNFRtosis mediated by TNFR family members is achieved by
TRAF1 and receptor interacttargeting downstream caspases.32 Expression of inhibitors
tivate the nuclear factor-jB
of apoptosis (IAPs) like IAP-1, IAP-2 and X-linked X-IAP
inal kinase (JNK) pathways,
specifically inactivate effector caspases33,34 and are uppoptosis.22,23 Mice with a genunable to activate NF-jB in
regulated in an NF-jB-dependant manner.35
TheOX40
second
group
of BAFFR
receptors,
including
TNFRII,
ation leading to TNF CD27
induced
RELT
CD30 CD40 4-1BB
GITR
OPG
RANK
TACI BCMA
Fn14 TROY
ncy in turn led to the inability
CD27, CD 30, CD40, LTbR, OX40, 4-1BB, BAFFR, B-cell
Figure
5: The TNF/TNFR
superfamily.
The (BCMA),
TNF-related ligands
are shown
in blue
arrows
y in response to TNF.25
Therematuration
antigen
receptor
activator
ofand
NF-jB
CD95Linteractions
TL1A
TRAIL
EDA
TNF
LIGHT
BTLA
LTα
LTαβ are
indicate
with
their
receptors.
The
ectodomains
of
the
TNFR
superfamily
shown
in
erfamily.
The
s to signalling
complexes that
(RANK), transmembrane activator and calcium-signal
grey
with
the
appropriate
number
of
cysteine
rich
domains
(CRDs).
Death
domains
within
the
in
blue and
caspase
cascade and cytoplasmic
the NF- domain modulating
cyclophilin
ligandfrom
(CAML)
interactor (TACI),
are indicated as red
cylinders (adapted
(37)).
h pathways
their recep-(Fig. 2a). This balFn14, herpes virus entry mediator (HVEM), activation
TNFR superous
levels including regulation
induced TNF-receptor (AITR), X-linked EDA-A2 recephe appropriate
ion, soluble decoy receptor
tor (XEDAR) and the member of the TNFR family
ns within the
ic ligand induction.26
(TROY) contain TNF-receptor associated factor (TRAF)d as red cylinpendent
activation of some
interacting motifs (TIMs)
in their cytoplasmic domain
RAF adaptors
TNFR family members
the
(Fig.
1).
Activation
of
TIM
TNFR
CD95 DcR3 DR3 DR4 DR5 DcR1 DcR2
OPG EDAR XEDAR containing
TNFR1 TNFR2 LTβR
HVEMfamily
DcR3
SODD) proteins associate conmembers leads to the recruitment of TRAF family
DR3 but not to other death
members and the subsequent activation of signal trans-
13 Today, a total of 19 ligands in TNF superfamily family have been identified along with
29 interacting receptors (37-39)(Figure 5).
1.3.1 Expression, structure, and signaling of TNFSF members
The cell expression patterns of TNF ligands/receptors are well characterized
(Table 1)(40). Cells of the immune system including B cells, T cells and dendritic cells
primarily express both ligands and receptors. TNFRs are usually expressed by activated T
cells while their ligands are induced or upregulated on APCs after immune activation.
Receptor
Cells
Ligand
Cells
CD27 (TNFRSF7)
CD4+ and CD8+ T
cells
B cells (subset)
NK cells (subset)
FOXP3+ Treg cells
NKT cells
Hematopoietic
progenitors
CD70 (TNFSF7)
APCs (DCs and B cells) CD4+
and CD8+ T cells
Mast cells
NK cells
Smooth muscle
Thymic epithelium
DR3 (TNFRSF25)
CD4+ and CD8+ T
cells
NK cells
NKT cells
FOXP3+ Treg cells
LTi cells
TL1A (TNFSF15)
APCs (DCs, B cells,
macrophages)
CD4+ and CD8+ T cells
Endothelial cells
OX40 (CD134 and TNFRSF4)
CD4+ and CD8+ T
cells NK cells
NKT cells
FOXP3+ Treg cells
Neutrophils
OX40L (CD252 and TNFSF4)
APCs (DCs, B cells,
macrophages)
CD4+ and CD8+ T cells
LTi cells
NK cells
Mast cells
Endothelial cells
Smooth muscle
4-1BB (CD137 and TNFRSF9)
CD4+ and CD8+ T
cells NK cells
NKT cells
Mast cells
Neutrophils
FOXP3+ Treg cells
DCs
Endothelial cells
Eosinophils
Osteoclast
precursors
4-1BBL (TNFSF9)
APCs (DCs, B cells,
macrophages)
CD4+ and CD8+ T cells
Mast cells
NK cells
Smooth muscle
Hematopoietic progenitors
Osteoclast precursors
CD30 (TNFRSF8)
CD4+ and CD8+ T
cells B cells
NK cells
Macrophages
Eosinophils
CD30L (CD153 and TNFSF8)
T cells
B cells Mast cells Monocytes
Neutrophils Eosinophils
CD40 (TNFRSF5)
Basophils,
APCs (DCs, B cells,
Macrophages)
Epithelial cells
Endothelial cells
Smooth muscle cells
Fibroblasts
CD40L (CD154 and TNFSF5)
CD4+ and CD8+ T cells B cells
Eosinophils
Mast cells
Monocytes Macrophages
NK cells
Endothelial cells Smooth muscle
cells Epithelial cells
Table 1: Cellular expression of some TNF ligands and receptors (adapted from ((40))
14 This indicates that antigen recognition by the T cells results in the interaction and the
bidirectional activity of the ligand/receptor pair, which in turn promotes the effector
responses of T cells and other immune cells. These responses can be enhanced by TNF
ligand molecules upregulated on non-immune cells, adding to the effector function and
inflammatory response that may lead to tissue inflammation in the disease context (38).
For example, CD27, OX40, 4-1BB, and TNFRSF25 expression is either induced or
upregulated within 24 hours following the antigen mediated activation of naïve T cells,
and more rapidly by memory T cells. Their respective ligands CD70, OX40L, 4-1BBL
and TL1A are induced on APCs. Macrophages and dendritic cells display increased
TL1A after stimulation with TLR ligands and Fc receptor cross-linking (41, 42). TL1A
up-regulation is also reported on endothelial cells after stimulation with inflammatory
cytokines such as IL-1 and TNF. Furthermore, both CD4+ and CD8+ T cells can also
express both TNF receptors and ligands, which suggests that T-cell interaction amongst
themselves can also influence T cell populations.
The majority of the TNF ligands are type II transmembrane proteins (intracellular
N-terminus and extracellular C-terminus) that are biologically active as non-covalently
bound self-assembling homotrimers, whose individual chains fold as compact “jellyroll”
β sandwiches and interact at hydrophobic interfaces (43-45) (Figure 6). Some of these
ligands, like TL1A, are functional as a transmembrane protein and as a soluble form
cleaved off from the cell membrane by proteolytic cleavage by metalloproteinases. Other
ligands, such as LTα and APRIL, are secreted only as soluble molecules but can be
recruited to the cell membrane to form surface anchored trimeric molecules that
contribute to regulatory specificity and complexity (46).
15 Figure 6: Structural organization of the co-stimulatory TNF/TNFR family members.
TNF ligands are shown as homotrimeric type II transmembrane proteins, while TNF
receptors are depicted as type I transmembrane monomers that are thought to associate in
trimers when interacting with their ligands. The total amino-acid length and number of
intracellular amino acids (parentheses) are also indicated (adapted from (45)).
TNF ligands are characterized by the presence of a 150 amino acid long
conserved C-terminus TNF homology domain (THD) containing a framework of
aromatic and hydrophobic residues (Figure 6). The THD is responsible for the
trimerization and binding of the ligand to the receptor. Although 20-30% amino acid with
sequence homology in their interacting protein interfaces account for the trimeric
assembly of the ligands, very little similarity in the sequence of the external surfaces of
the ligand trimers leads to individual receptor specificity (43, 47). Some of the ligands
can bind more than one receptor with high affinity. Why nature chose these molecules to
be trimeric is unclear. One explanation would be that trimers require more contact sites
than dimers, allowing them to bind with higher avidity (combined strength of multiple
16 bonds) to receptors (48). Elongated receptor chains interact with all the three sides of the
inverted bell shaped ligand, hence creating a 3:3 symmetric complex. When the ligand
ligates to the receptor, the intracellular cytoplasmic tails forms a 3:3 internal complex
with signaling proteins such as TRAF2 and FADD (explained in detail in this section),
resulting in an overall protein complex with a 3-fold symmetry (48).
The TNF receptors are primarily type I transmembrane proteins (intracellular Cterminus and extracellular N-terminus), while there are exceptions including BCMA,
TACI, BAFFR and XEDAR that are type III transmembrane proteins and lack a signal
peptide (49). Some TNFRs can be secreted as soluble proteins due to proteolytic
processing (e.g. CD27, CD30, TNFR1, TNFRSF25), alternative splicing of the exon
encoding the transmembrane domain (e.g. Fas and 4-1BB) or merely because they lack a
membrane-interacting domain sequence in the encoding gene (e.g. Decoy-R3; Decoy
TNFRSF25). The extracellular domains of TNF receptors are characterized by the
presence of cysteine-rich domain (CRD) (Figure 6), which are 40 pseudorepeats
typically containing 1-6 cysteine residues interacting to form disulfide bonds (50).
TNFRs can have significantly varying numbers of CRD, from B-cell activating factor
receptor (BAFFR) with only 1 partial CRD, to TNFR1 and TNFRII which contain the
typical 3 CRD and up to 6 CRDs found in CD30 (37). The elongated chains of the
receptor fit into the grooves between the monomers of the trimeric ligand, suggesting that
the trimeric ligand cross-links the three receptor monomers into the 3:3 stoichiometry.
This school of thought contradicts more recent studies that report TNFR pre-assembly
without the requirement of binding to trimeric ligand and signaling requires the
rearrangement of preassembled receptors (51, 52). Additionally, trimerization of some
17 members of the TNF receptor superfamily, such as Fas, TNFR II and TRAIL receptors is
not sufficient to trigger a response hence higher oligomerization is required (53-55),
while trimerization of TNFR1 and Tweak receptors is enough to mediate signaling (53,
55). Higher oligomeric complexes of CD40L form molecular clusters when interacting
with the receptor, resulting in more effective signaling as compared to single trimeric unit
(56).
TNF ligand mediated TNF receptor activation can lead to the recruitment of
several different intracellular adaptor proteins, which in turn activate several signal
transduction pathways. TNFR family members are categorized into three groups based on
the their intracellular sequences. The first group (e.g Fas TNFRI, TNFRSF25) contains
an intracellular death domain (DD), a 45 amino acid long sequence. Activation of DD
leads to the recruitment of intracellular adaptors including Fas-associated death domain
(FADD) (57)(Figure 7) and TNFR-associated death domain (TRADD) (Figure 8) that
can lead to caspase dependent apoptosis or nuclear factor (NF)κB dependent survival.
Ligation of TNF receptors CD95, DR4, and DR5 on type 1 cells (Figure 7),
including thymocytes, drives intracellular DD clustering and binding of the DD to
FADD, which recruits caspase-8 and caspase-10, to form the death-inducing signaling
complex (DISC). The DISC mediates the autocatalytic processing of the caspases and
releases them in the cytoplasm to recruit effector caspases 3,6, and 7, which in turn
cleave cellular substrates leading to apoptotic cell death (58, 59). Apoptosis in type II
cells (B cells) requires caspase 8 to process Bid, which engages a cell- intrinsic
mitochondrial pathway (60) (Figure 7).
18 FADD: CD95, DR4, DR5
TRADD: TNFR1 and TNFRSF25
Figure 8: TRADD-dependent DD signaling complex
(adapted from (61)).
Figure 7: Primary FADDdependent
DD
signaling
complexes (adapted from (57)).
Interestingly, some TNF receptor signaling, including TNFRSF25 and TNFR1,
are capable of activating two pathways simultaneously which leads to induction of
apoptosis and activation of NF-κB (Figure 8)(61). The DD binds to TRADD, which
provides a scaffold for the binding of RIP1, TRAF2 (or TRAF5) and c-IAP 1 and 2
creating “complex 1” (62-64). This structure has the ability to activate the NFκB and jun
N-terminal kinase (JNK) and p38 pathways. Activation and translocation of NFκB to the
nucleus induces transcription of genes encoding proinflamatory cytokines and
chemokines as well as anti-apoptotic proteins including c-FLIP and c-IAP (65).
Activation of JNK and p38 pathways stimulates genes that regulate proliferation,
differentiation, inflammation or apoptosis. TNFR1 ligation leads to receptor
internalization and conformation changes of RIP1 and TRAF2 that allow TRADD to
recruit FADD to make complex II (66, 67). RIP1 associates with FADD to activate
19 caspase-8 and trigger apoptosis downstream of TNFR1. Anti-apoptotic molecules like cFLIP and c-IAP that emanate from complex-1 signaling negatively regulate this pathway.
The second group of TNF receptors (e.g TNFR2, CD40, CD30, CD27) lacks the
DD and have one or more TRAF-interacting motifs (TIMS) in their cytoplasmic domain.
CD40 and TNFR2 are the most well-characterized members of this group in terms of the
their association and functional relationships with TRAFs. Ligation of CD40 by
multimeric CD40L results in the redistribution of CD40 to membrane lipid rafts and
conformational changes recruiting TRAFs to at least two binding sites on the cytoplasmic
domain of CD40. TRAFs then recruit several other TRAF-interacting kinases and
together activate several signal transduction pathways such as NF-κB, JNK, p38,
extracellular signal-related kinase (ERK) and phoshoinositide 3-kinase (PI3K) (68, 69).
For example, TRAF-2 interacts with germinal center kinase in B cells and contributes to
CD40-induced c-Jun-NH2-kinase activation and cell proliferation. Moreover, some
CD40-TRAF interactions also have been reported to be inhibitory (70). CD40 signaling
targets several genes that regulate apoptosis, cell cycle progression, cytokine production,
expression of cell surface immune modulators, and other TNF family members and other
pathways. Extracellular secondary signals also cooperate with CD40 signaling
introducing overlapping responses or triggering others. CD40 can also activate kinases,
signal transducers, and activators of transcription pathway via TRAF-independent
mechanisms.
The third group of TNF receptors includes TRAIL-R3, TRAIL-R4, decoy-R3
(decoy TNFRSF25) and osteoprotegerin (OPG) none of which contain functional
intracellular signaling domains. Instead, these molecules compete with the other 2
20 signaling groups of receptors for their ligand and function by hindering the activation of
signal transduction pathways by other TNF receptors (68).
The functional modularity of TNF receptors, which depends on several
intracellular molecules activating various interconnected signaling pathways downstream,
allows these proteins to modulate various aspects of the immune system, including
morphogenesis and maintenance of lymphoid structure, lymphocyte homeostasis, innate
and adaptive immunity and immune surveillance.
1.3.2 Multiple functions of TNF superfamily members
Signaling through TNF receptors via interaction with their respective ligands play
various
roles
that
include
promoting
cell
activation,
proliferation,
survival,
differentiation, and apoptosis, mediating protection against bacterial infection, and
orchestrating the development of multicellular structures such as lymphoid organs, hair
follicles, bones, mammary glands, as well as nonpermanent inflammatory foci during
acute inflammation. Here we will discuss how the expression of TNF family members
controls the immune system and inflammatory responses at different levels.
1.3.2.1 Development and maintenance of lymphoid structure
The expression of TNF family members mediates crosstalk between cells and
orchestrates the development of secondary lymphoid organs including lymph nodes and
Peyer’s patches. These are areas of aggregated lymphocytes, antigen-presenting cells, and
other immune-influencing cells positioned throughout the body with strategic vascular
connections to gateways of foreign antigen entry. Interestingly, mice that are deficient in
LTβR or LTβ(α1β2) do not develop lymph nodes, Peyer’s patches, and have defective
spleens and humoral immunity (71). RANK and RANKL deficient mice also lack
21 peripheral and mesenteric lymph nodes but they develop Payer’s patches and their splenic
structure is intact (72-74). Additionally, ectopic expression of LTα1β2 or LTα3, or B cell
chemokines influenced by expression of LTα1β2 or LTα3 on B cells, stimulates
vasculature adhesion molecules and generates ectopic lymphoid tissue creating plasticity
in lymphoid organ boundaries (48, 75-77). Deficiency of LTβR or any of its receptors
leads to the perivascular infiltration of B and T cells into multiple tissues (48, 78, 79). In
summary, the expression of TNF members establishes spatial constraints required for
lymphoid organ definition.
1.3.2.2 Protection against microbial infections
The expression of TNF superfamily signaling and the ability to produce an
appropriate Th1 type cytokine are both required for protection against microbial
infections. Neutralizing TNF revealed that the host immune response against pathogens is
severely impaired in the absence of TNF. Genetic deletion of TNFR1 in mice resulted in
increased susceptibility to infections with intracellular bacteria such as Listeria
monocytogenes, Mycobacterium tuberculosis, M. avium, and Salmonella. TNFR1deficient mice are incapable of hindering the replication of L. monocytogenes in
phagocytes although other antimicrobial defense systems, including generation of oxygen
radicals and reactive nitrogen intermediates are not defective in these mice (37, 80, 81). It
is not surprising that anti-TNF therapies, including antibodies specific for TNF and
soluble TNFRs, approved for treatment of Crohn’s disease and rheumatoid arthritis,
increase the risk of development of diseases such as tuberculosis due to TNF’s important
role in killing M. tuberculosis in macrophages.
22 1.3.2.3 Mediators of cell death
The ability to induce cell death in different immune or non-immune cells is one of
the unique properties that TNF family members have evolved with great adaptive value.
Six of the DD containing TNF members, including TNFRSF25, have the ability to
directly induce apoptosis by activating caspases, while non-DD containing TNFSF
members have the capacity to modulate the response to DD or directly influence cell
survival. For example, signaling through TNFR2 directly enhances TNFR1-induced T
cell death while CD40 also augments CD95 mediated B cell death (82, 83). As mentioned
earlier, signaling through CD95 on CD8+ T cells is a major calcium-independent
mechanism of CTLs that results in cell-mediated cytotoxicity in response to infectious
agents (84). Importantly, this mechanism is also vital in maintaining immune homeostasis
by regulating the balance between different lymphocyte subsets in the limited space of
lymphoid organs. Genetic deficiencies in CD95 mediated apoptosis in humans and mice
leads to drastic loss of lymphocyte homeostasis and autoimmunity (85, 86), while
dysregulation of TNF has also been associated with human systemic lupus erythematosus
and other autoimmune diseases (48, 87).
1.3.2.4 TNF superfamily members in cellular responses
TNF superfamily members can stimulate conventional T cells and antigen
presenting cells, influence antibody production by B cells, mediate communication
between CD4+ and CD8+ T cells, which can potentially amplify signals that are initiated
by APCs and promote immune responses. The ligand/receptor duo can also facilitate
interactions between T cells and NK cells, between NKT cells and APCS presenting lipid
antigens, and between T cells and other immune cells or tissue cell. TNF superfamily
23 member interactions are also crucial during the clonal expansion/effector phases of
primary and at least some secondary immune responses.
1.3.2.4.1 CD4+ and CD8+ T effector cells
TNFSF receptor costimulation can simply decrease the activation threshold of
TCR stimulated T cells, or can provide additional signals to promote cell division,
augment cell survival, or induce effector function such as cytokine secretion or
cytotoxicity. These effects can occur by TNF/TNFR directly acting on T cells or on
APCs with which they interact. The studies of LIGHT-HVEM show that blocking
LIGHT can inhibit T-cell proliferation and cytokine secretion in allogeneic mixed
lymphocyte reactions (MLR). In vitro studies show that LIGHT-deficient splenocytes
that respond to alloantigen have reduced production of Th1 and Th2 cytokines and weak
generation of CD8+ CTL activity (88), while in vivo studies indicate that blocking LIGHT
reduces the generation of alloreactive CTLs (89). Similarly, interruption of CD27-CD70
interaction decreases the cytokine production and proliferation response in the initial
stages of T cell response (90-92). T cells that lack CD27 initially divide normally, but
they proliferate slowly after initial activation. In vivo, CD27 deficient mice have reduced
numbers of antigen specific CD4+ and CD8+ T cells in the peak of primary response
(days 4-8), and fewer memory T cells develop over 3 or more weeks (93). Unlike CD27CD70, early proliferation is unimpaired in OX40 deficient naïve CD4+ T cell populations,
but reduced proliferation and increased apoptotic death occurs 4-5 days after activation
resulting in reduced long-term T cells (94).
OX40L-OX40 has been extensively studied using transgenic mice overexpressing
OX40L or with agonistic antibodies. The overexpression of OX40L on dendritic cells
24 (95) and T cells (96) results in the accumulation of antigen responding CD4+ T cells and
autoimmune-like symptoms that are associated with pathogenic T cells. Mice treated with
agonist antibody for OX40 after immunization results in the accumulation of a larger
number of antigen specific CD4+ T cells and effector CD8+ CTLs in the primary
response, followed by an increase in the number of memory T cells (97, 98). Similar
observations have been made in transgenic systems overexpressing TL1A and with the
use of agonistic antibody to TNFRSF25, which will be discussed in further detail.
1.3.2.4.2 Other immune cells
Since TNF family members are upregulated by antigen driven lymphocytes
during the priming phase, it is not surprising that these molecules play an important role
in the T-cell and DC interaction. The general idea is that TNF ligands are expressed on
activated T cells, while the paired receptor is highly expressed on DC that requires prestimulation through CD40 or through pattern recognition receptors (PPR). CD40L and
LTαβ are highly induced on antigen specific CD4+ T cells that interact with antigen
bearing DC in the context of infection or immunization. These TNF superfamily
members enable DC to cross-present cognate antigen to CD8+ T cells, by signaling
through CD40, which is highly upregulated on DCs, and LTβR that is constitutively
expressed on DCs. Blocking of CD40-mediated signaling causes defects in CD8+ T cell
clonal expansion, function, and memory(99), while defective LTβR on DCs results in
reduced production of type 1 IFN production required for optimal CD8+ T cell
priming(100).
The interactions of TNF superfamily members also regulate the functions of other
immune cells that enhance immune responses mediated by T cell counterparts. It has
25 been reported that OX40/OX40L, CD70/CD27, TNFRSF25/TL1A have the ability to
either directly enhance NK effector functions of cytotoxic ability or cytokine production
or indirectly mediate NK driven conventional T cell activation and differentiation (101105). For example, IFN-γ production by NKT cells induces upregulation of CD70 on
dendritic cells that subsequently primes a Tconv response. Lei Fang et al. in Dr. Podack’s
laboratory reported that signaling through TNFRSF25 is required to co-stimulate IL-13
production by glycosphingolipid-activated NKT cells. In vivo, antibody blockade of
TL1A inhibits lung inflammation and production of Th2 cytokines such as IL-13, even
when administered days after airway antigen exposure. To further support these results,
in vivo blockade of TNFRSF25 by a dominant-negative (DN) transgene, DN TNFRSF25,
results in resistance to lung inflammation in mice. Allergic lung inflammation-resistant,
NKT-deficient mice become susceptible to disease when wild-type NKT cells are
adoptively transferred, but not after transfer of DN TNFRSF25 transgenic NKT cells
(106).
Members of TNF superfamily also perform critical roles in peripheral B cell
maturation, homeostasis, and antigen dependent proliferation and antibody production.
The expression of CD27, CD70, and OX40L is induced on most B cells and can promote
B cell proliferation and antibody production. CD40 promotes B cell survival,
costimulation proliferation, enhances T-cell collaboration, and enables immunoglobulin
class switch recombination (CSR) (107). Resting B cell constitutively express CD40 that
is upregulated after interaction with CD40L on activated T cells in an antigen dependent
manner. In the absence of CD40, germinal center formation is disrupted and antibody
responses are largely limited to low affinity IgM produced in a T-cell independent
26 manner (108, 109). BAFF is the most critical soluble factor for peripheral B-cell
maturation and survival. Genetic disruption of BAFF or BAFF-R results in a similar
phenotype of a peripheral B cell block (110-112).
The expression of OX40/OX40L, 41BB/41BBL and CD70 can be induced on
mast cells. OX40L expression on mast cells has been shown to directly costimulate T-cell
activation (113, 114) and 41BB signaling in mast cells increases the productions of proinflammatory meditators by Tconv (115). Stimulation through OX40 and 41BB on
neutrophils has been reported to contribute to tissue inflammation by enhancing cell
survival or the production of pro-inflammatory molecules (116). Interestingly,
constitutive expression of TNFRSF25 has also been detected on a CD4+CD3- lymphoid
tissue inducer cells (LTi) and signaling through TNFRSF25 upregulates OX40L on these
cells. This mechanism has been described to further promote T cell responses and
prolong survival of memory T cells (117, 118).
1.3.2.4.3 Regulatory T cells
The triggering of different TNF receptors can influence the differentiation,
function and costimulation proliferation capacity of the Treg population. Signaling
triggered by OX40 and TNFRSF25 has been found to inhibit the development of FoxP3+
Treg cells that differentiate from naïve CD4+ T cells in the presence of TGF-β and RA
(119-121). Furthermore, signaling through OX40, TNFRSF25, 4-1BB, and GITR also
block the suppressive ability of Treg to inhibit the proliferation of Tconv cells (120-127).
Several studies report that this phenomenon seems to be dependent either only on the
direct effect of blocking Treg function or together with the indirect effect on Tconv
making them resistant to Treg mediated suppression. Transgenic mice over-expressing
27 TL1A on dendritic cells or T cells have higher number of FoxP3+ Tregs despite the fact
that TL1A suppresses generation of iTregs (128), while soluble TL1A induces the
proliferation of Tregs in vitro (129).
Given the overlap between either functional suppression or induction of Tregs
between TNFRSF25 and these other TNFSF members, Dr. Schreiber et al. in our
laboratory tested Treg expansion in vivo after stimulation of TNFRSF25, OX40, 4-1BB,
GITR, or CD27 with agonists. In all cases, we used well-characterized agonistic
monoclonal antibodies to the respective receptor to trigger specific signaling. These
studies demonstrated that TNFRSF25 was unique among the TNFRSF members
examined in its ability to selectively induce expansion of Tregs in naïve mice. Signaling
through TNFR25 induced rapid and selective expansion of preexisting Tregs in vivo such
that they became 30%-35% of all CD4+ T cells in the peripheral blood within 4 days of
treatment. TNFRSF25-induced Treg proliferation was dependent upon TCR engagement
with MHC class II, IL-2 receptor, and Akt signaling, but not upon costimulation by CD80
or CD86; it was unaffected by rapamycin. TNFRSF25-expanded Tregs remained highly
suppressive ex vivo, and Tregs expanded by TNFRSF25 in vivo were protective against
allergic lung inflammation, a mouse model for asthma, by reversing the ratio of effector
T cells to Tregs in the lung, suppressing IL-13 and Th2 cytokine production, and
blocking eosinophil exudation into bronchoalveolar fluid (130).
1.3.3 TNF superfamily members in disease and immunotherapy
The TNF members regulate immune responses at different levels that modulate
both the inflammatory and regulatory aspects of immunity. Hence, it is not surprising that
homeostatic disruption of these molecules have been implicated in inflammatory and
28 autoimmune diseases and other diseases including heart failure, bone reabsorption, AIDS,
Alzheimer’s disease, and other disorders, hence targeting these molecules may be
extremely useful for clinical exploitation (Figure 9).
Figure 9. Present and future therapeutics based on members of
the TNF superfamily and their receptors (adapted from (39))
1.3.3.1 TNF superfamily members in cancer, autoimmune and inflammatory
diseases
Although TNF was discovered as a cytokine that kills tumor cells, its now know
that TNF mediates the proliferation, invasion and metastasis of tumor cells. Studies report
that TNF is highly carcinogenic and mice deficient in TNF are resistant to skin
carcinogenesis (131). CD30 expression is upregulated in various hematological
malignancies, including Reed-Sternberg cells in Hodgkin's disease (HD), anaplastic large
cell lymphoma (ALCL) and subsets of Non-Hodgkin's lymphomas (NHLs). Increased
CD30L expression was also found on mast cells within HD tumors.
In the autoimmune disease of rheumatoid arthritis (RA), elevated levels of the
soluble form of CD30L were observed in patient synovial fluid together with increased
levels of CD30 and CD30L in biopsies with evidence that these molecules contribute to
29 mast cell activation (132). Additionally, synovial CD30+ T cells produce large amounts
of IFN-γ, Th2 cytokine IL-4, and inflammatory IL-10 suggesting that T cells may play a
role in RA-induced inflammatory responses (133). Similarly, synovial fluids from RA
patients also has increased numbers of OX40+ T cells and reduced incidence of collagen
type II induced (CII) RA was correlated with decreased expression of OX40 on T cells in
mice (134). APRIL and BLyS protein levels are also increased the serum and the
synovial fluid of RA patients indicating that they might be produced locally at the sites of
inflammation(135, 136). A recent study reported that APRIL is present in the highest
levels in germinal-center-positive synovitis and is produced by CD83+ DCs. Blocking
APRIL and BLyS was most effective in inhibiting germinal-center-positive synovitis and
other forms of synovitis correlated with the presence of TACI+ T cells. Seyler et al.
propose that APRIL and BLyS regulate both B and T cell function and may have both
pro-and anti-inflammatory regulation in different forms of RA (137)
In asthma, its has been reported that OX40 and OX40L are upregulated in the
bronchial submucosa in mild asthmatics compared to healthy individuals (138), while
CD30 expression on Th2 cells is also elevated in patients with allergic asthma (139). In
patients with severe asthma, higher sputum LIGHT concentrations correlate with the
most impaired lung function (140).
Mutations in both ligands and receptors of TNF superfamily have been found in
humans. For example, people diagnosed with autoimmune lymphoproliferative syndrome
(LPS) have mutations in CD95 and CD95L resulting in increased risk of developing B
and T cell lymphomas, similar to the phenotype in lprl or gld mice that also have
mutations in CD95 or CD95L. Their risk of non-Hodgkin’s lymphoma and HD, is greater
30 than expected due to the defective lymphocyte apoptosis, implicating that CD95mediated apoptosis is important for preventing B and T cell lymphomas (141).
From the studies of patient cohorts, genetic mutations in humans and mice and
murine disease models, it is very clear that TNF superfamily members and their receptors
have a wide-ranging role in many cellular and physiological functions that can be
therapeutically targeted and translated into new generation therapies.
1.3.3.2 Protein therapeutics targeting TNF super family members
Several TNFSF members drive cell division, differentiation, and survival, while
others suppress responses by promoting cell death (which is not the focus of this section).
Agonistic and blocking reagents such as antibodies and Fc fusion proteins can be used to
target these molecules to modulate immune responses for beneficial disease outcomes.
Strategies include blocking one or more of these interactions to reduce pathogenic
immune responses in autoimmune and inflammatory disease, or enhancing signaling that
is triggered by the ligand receptor interactions to stimulate a more robust immune
response and promote anti-tumor immunity. In addition to blocking TNF receptor
signaling, newly discovered methods include TNFR mediated direct costimulation and
proliferation of endogenous nTreg and iTreg to dampen immune responses in
inflammation, allergy and autoimmunity, or to expand this immunosuppressive cell
population prior to transplantation for induction of immunological tolerance.
One method of decreasing immunopathology and inhibiting disease progression
in inflammatory or autoimmune diseases should aim to suppress the immune responses of
T cells, APCs, NK cells and NKT cells. In order to do so, the logical method would be to
prevent TNFR interaction with their ligands, which would decrease the expansion and
31 survival of pathogenic cell populations and/or decrease their production of proinflammatory cytokines. For example, antibodies and Fc fusion proteins that target and
block TNF, a proinflammatory TNFSF molecule, have been highly successful in patients
for treatment of several autoimmune diseases including RA and Crohn’s disease. These
studies show that neutralizing TNF-TNFR interactions can result in strong suppression of
disease symptoms, which are usually the result of decreased activity of CD4+ or CD8+ T
cells, or in other cases impaired NK and NKT cell function.
The second therapeutic approach to dampen inflammation is through the
administration of depleting antibodies. These therapies target the ligand or receptor and
directly eliminate the pathogenic cells that express a specific molecule. OX40 is highly
upregulated on enchephalitogenic myelin basic protein (MBP)-specific T cells at the site
of inflammation during the onset of experimental autoimmune encephalomyelitis (EAE).
In vivo, injection of an OX40 immunotoxin (anti-OX40 antibody coupled to toxin) at the
onset of disease eliminates MBP-specific T cells in the CNS, resulting in the amelioration
of EAE (142). Similarly, injection of anti-OX40 coated liposomes in mice that had
Mycobaterium tuberculosis (Mt) induced arthritis, resulted in the elimination of MTspecific T cells and resolution of disease (143). However, the depletion strategy can also
affect TNFR expressing Treg cells that are essential for the induction of immunological
tolerance associated with transplantation and autoimmunity and for the protection against
other inflammatory diseases such as asthma. Elimination of both Treg and pathogenic
cells could result in short-term benefit and inhibition of disease, yet future lack of Treg
function can lead to re-occurrence of disease when new pathogenic cells are uncontrolled
(38).
32 The third therapeutic approach relies on the stimulatory rather than the
neutralizing or depleting reagents to inhibit the activity of pathogenic effector cells. In
this case, agonistic antibodies or Fc fusion proteins would be targeted at a TNF receptor
expressed on Treg cells.
Natural and inducible CD4+ Treg cells can constitutively
express OX40, CD27, 4-1BB, or TNFRSF25 in mice, or these molecules can be induced
on these cell populations in humans. Since signaling through TNF molecules can increase
the frequency and function of these T cell subsets, therapeutic strategies can be utilized to
skew the balance in favor or against Treg. In allergic lung inflammation, an inflammatory
disease that is mainly driven by CD4+ T cells (144, 145), treatment with either
TNFRSF25 agonist, 4C12, prior to airway antigen challenge (ova aerosolization) induced
the preferential accumulation of Tregs recognizing self-antigen, but not Tconvs, within
the lungs and reduced eosinophilia and mucus production in the bronchoalveolar space.
To take advantage of Treg expansion by TNFRSF25 signaling, it is important to inject
the agonist at a time when no exogenous antigen is present that may activate other T cell
subsets. The sequence of exposure of CD4+ T cells to cognate antigen and TNFRSF25
costimulation determines whether an inflammatory response is induced by Tconvs or
suppressed by Tregs. Dr. Wolf et al. in our laboratory reported that TNFRSF25 mediated
expansion of endogenous Treg population prior to transplantation results in prolongation
of cardiac allograft survival in a mouse model of fully major histocompatibility complexmismatched ectopic heart transplants (146). Similarly, enhanced inflammation in a model
of corneal infection with HSV was observed when 4C12 was administered 6 days post
infection, while TNFRSF25 stimulation prior to infection reduced the severity of
subsequent stromal keratitis lesions, which was attributed to the expansion of the Treg
33 population that additionally expressed activation markers such as CD103 needed to
access inflammatory sites (147).
1.4
TNF-like ligand 1A (TL1A)/TNF receptor superfamily member 25
(TNFRSF25)
TNFRSF25 (also known as DR3) is a member of the TNF superfamily that is
expressed primarily on lymphocytes and is the receptor of TNF-like ligand, TL1A (also
known as TNFSF15). TNFRSF25 costimulates T-cell activation through the
intracytoplasmic DD and the adapter proteins TRADD, which is described above. TL1A
costimulates T cells to produce various cytokines and can promote expansion of Treg in
vitro and vivo. Studies done in our laboratory using transgenic mice that lack TNFRSF25
or TL1A or mice that are treated with blocking antibodies to TL1A report that
TNFRSF25 play a specific role in enhancing effector T cell proliferation at the site of
tissue inflammation in autoimmune disease models (106). Transgenic overexpression of
TL1A in mice results in an IL-13 dependent pathology in the small intestine marked by
goblet cell hyperplasia (129). Together, these studies suggest that TNFRSF25 and TL1A
are therapeutic targets for therapies designed to modulate the immune system to improve
clinical outcome in inflammation, autoimmunity, allergy, transplantation, and cancer.
1.4.1 TL1A
TL1A was identified and characterized as the ligand for both TNFRSF25 and
decoy receptor TNFRSF25 by Mignone et. al in 2002. TL1A is a longer splice variant of
TL1 (also called VEGI), which was previously identified as an endothelium-derived
factor that inhibited endothelial cell growth in vitro and tumor progression in vivo (148151). TL1A is a trimeric type II transmembrane protein that can be cleaved by
34 metalloproteases and released as a functional soluble trimer. TL1A lacks an N-terminal
signal peptide and the 5’ end carboxyl domain reveals highest sequence identity to TNF
(24.6%), followed by FasL (22.9%) and LTα (22.2%). The C-terminal 151 aa of TL1A
(aa 101-251) and that of TL1 (aa 24-174) are identical and there is no sequence
homology at the N-terminal. The cloned mouse TL1A cDNA encode proteins of 252 aa
(Figure 10) and shares 66.1% sequence homology to human TL1A. The 2.02 kb TL1A
cDNA has been mapped to chromosome 9q32. Detailed sequence alignment revealed that
TL1A is encoded by 4 coding exons utilizing consensus splicing sites(152).
ICD:%(aa%1)40)%
TM%
ECD:%(aa%65)252)%
Figure 10. Diagram of TL1A. ICD: intracellular domain; TM: Transmembrane; ECD:
extracellular domain.
Migone et al. found that TL1A was expressed pre-dominantly by human
umbilical, dermal microvascular and uterus myometrial endothelial cells (HUVEC) cells.
Very low TL1A expression has been detected on human aortic endothelial cells, adult
fibroblasts, aortic smooth muscle cells, skeletal muscle cells, adult keratinocytes, tonsillar
B cells, T cells, NK cells, monocytes, or dendritic cells. TL1A mRNA levels were
detected in human tissues including prostate, kidney, placenta, and stomach while low
levels were observed in intestine, lung, spleen, and thymus. TL1A is undetectable in most
cancer cell lines including 293T and HeLa cells (152). TL1A expression can be induced
through stimulation with Toll-like receptor (TLR) ligands, such as lipopolysaccharide
(LPS), and crosslinking of Fc receptors on dendritic cells and macrophages, as well as
exposure to other cytokines including IL-1 and TNF in endothelial cells (41, 105, 152).
Such stimulation for TL1A expression is highly transient, declining back to baseline
35 levels within 1 day. TL1A can be induced in a slower and more sustained manner on T
cells after TCR stimulation that could be stabilized by the autocrine positive feedback
stimulation with TNFRSF25 (152).
1.4.2 TNFRSF25
TNFRSF25 was initially cloned by a number of research groups through
homology with TNFR1 and it was initially given several other names including LARD,
TRAMP, APO-3, and WSL-1. The coding part of the gene spans 5 kb with 10 exons.
Mouse TNFRSF25 has 63% homology at the protein level to human TNFRSF25, with
94% homology in the death domains and 52% in the extracellular domains. Additionally,
hTNFRSF25 expression on human tissues does not correspond to mTNFRSF25
expression in mouse tissues. For example, hTNFRSF25 is found in intestine, while
mTNFRSF25 is found in the kidney. There are at least 13 splice variants for TNFRSF25
in humans making it complicated to define the precise function of the receptor. There are
2 splice variants of mTNFRSF25, including the full length receptor, soluble receptor that
lacks the transmembrane domain and the transmembrane receptor lacking one of the
CRD regions in the extracellular domain (153). Signaling through TNFRSF25 may
induce caspase dependent apoptosis or activation of NFκB as described in section 1.3.1
(pg. 13).
TNFRSF25 is expressed constitutively in the form of randomly spliced transcripts
mainly in lymphoid cells (63, 153, 154) and also in other cell types including tumor cells
(155, 156). TNFRSF25 is detected at low levels on naive CD4 T cells and CD8 T cells
and TCR-activated T cells upregulate expression. NKT cells express high levels of the
receptor. A subpopulation of CD11c+ cells and NK cells are also TNFRSF25 positive,
36 whereas B cells are negative (106). Expression is also detected on activated human
monocytes (157). Combination of IL-12 and IL-18 upregulated TNFRSF25 expression on
NK and CCR9+CD4+ peripheral blood (PB) cells, whereas there is a minimal effect in
other T cell subsets. PB CCR9+CD4+ T cells represent a small subset of circulating T
cells with mucosal T cell characteristics and a Th1 cytokine profile. Papadakis et al.
reported that the TL1A/TNFRSF25 pathway plays a dominant role in the ultimate level
of IL-12/IL18 induced IFN-γ production by CCR9+ mucosal and gut homing PB T cells
that could play an crucial role in Th1 mediated intestinal diseases such as Crohn’s.
1.4.3
TL1A/TNFRSF25 in immunological responses
Although TL1A induces apoptosis in TNFRSF25 expressing cell lines (41),
TL1A/TNFRSF25 interactions have been mainly associated with costimulation of Th1
cells with TNFRSF25 being expressed on activated T cell and TL1A being induced on
APCs such as DCs. This pair of molecules has been implicated in the pathogenesis of
several diseases marked by gut inflammation. Polymorphisms in TL1A, GWAS and
selective association studies have linked the duo with inflammatory bowel disease (IBD),
ulcerative colitis, and Crohn’s disease (158-160).
In 2011 three individual laboratories reported that overexpression of TL1A on T
cells or APCs results in T cell dependent inflammatory small bowel pathology (128, 129,
161). Meylan et al. reported mild splenomegaly and mild-to-moderate mesenteric
lymphadenopathy in adult mice resulting in higher cellularity. Flow cytometry analysis
revealed an accumulation of FoxP3+ Treg, an increased number of CD4+ T cells in the
mLN and to a lesser extent in the spleen, and a mild increase in CD8 T cell frequency in
the mLN. They also reported an increase in the frequency of CD69+ cells (recent
37 activation marker) and memory cells (CD62LlowCD44hi) gated on the CD4+ T cells. B cell
and myeloid cell frequency were normal in the transgenic mice. TL1A mediated
costimulation driven proliferation and activation of CD4+ T cells in the absence of
exogenous antigen was due to recognition of microflora or other environmental mucosal
antigens. These mice develop a Th2-cytokine, IL-13, dependent IBD pathology, and
blocking of TL1A-TNFRSF25 abrogated induced colitis(128). Studies looking at Th2
driven models of asthma report that TNFRSF25 deficient mice or anti-TL1A treated
wild-type mice have reduced airway inflammation and mucus production, which is
associated with inhibition of Th2 cytokine expression and reduced frequency of CD4+ T
cells and invariant natural killer cells (iNKT cells) in the lung.
The activity of TL1A/TNFRSF25 has also been studied extensively in Th17
mediated inflammatory diseases. TNFRSF25 is expressed on Th17 cells, and in studies of
murine EAE, genetic deletion of TNFRSF25 or TL1A significantly reduces the numbers
of pathogenic CD4+ T cells and the abrogation of clinical disease symptoms (152, 162).
Similar inhibition of TNFRSF25/TL1A signaling protects mice against arthritis, while
injections with recombinant TL1A exacerbates the disease (163), which correlates with
the elevated expression of TL1A observed in human RA synovial fluid (164). In all these
studies, the activity of TL1A is due to the immunomodulation of antigen activated
pathogenic CD4+ T cells that contribute to disease.
Although TL1A/TNFRSF25 has been implicated in exacerbating inflammatory
diseases, our laboratory has recently identified a role for TL1A/TNFRSF25 in
maintaining health and resolving inflammation. TNFRSF25 is constitutively expressed on
the surface of Treg cells and costimulation of TNFRSF25 by an agonistic antibody,
38 4C12, results in the expansion of pre-existing Treg that are protective against allergic
lung inflammation in a mouse model for asthma, by reversing the ratio of effector T cells
to Tregs in the lung, suppressing IL-13 and Th2 cytokine production, and blocking
eosinophil exudation into bronchoalveolar fluid. The protective role of TNFRSF25
expanded Tregs in allergic lung inflammation does not contradict our previous studies
implicating TL1A in the exacerbation of allergic lung inflammation. In our most recent
studies, TNFRSF25 signaling preceded antigen exposure, whereas in previous studies,
TNFRSF25 signals followed antigen challenge. Since TNFRSF25 costimulates antigen
TCR-activated CD4+FoxP3- Tconvs and Tregs, the sequence of antigen exposure and
TNFRSF25 signaling determines whether an inflammatory response is suppressed by
self-antigen activated Tregs or induced by Tconvs (130).
1.5 Aims of the dissertation
Over the past several years, TNF superfamily members have been identified as
important costimulators of activated T cells. TCR activated T cells progress to anergy and
even apoptosis without co-stimulatory signaling. Our previous study, using clone 4C12,
reported that TNFRSF25 has a non-redundant function as regulator of Treg cells and its
activity is unique among TNFR family members. These studies demonstrated that
TNFRSF25 signaling provides costimulation for the proliferation of pre-existing Tregs in
vivo, which receive continuous TCR signals by self-antigen presented by MHC II.
Given the evidence that agonistic antibodies are antigenic and do not always
precisely mimic the function of TNF ligands on different cells (165, 166), a comparison
to the functional effects of the natural ligand is necessary. Transgenic overexpression of
TL1A resulted in modulation of both CD4+ Tconv and Treg-cell response in intestinal
39 goblet cell hyperplasia and intestinal inflammation (128, 161, 167, 168) indicating
important regulatory functions especially in the mucosa. The murine TL1A Ig fusion
protein (TL1A-Ig) enabled comparison to the agonistic antibody and offers flexibility of
experimental design for further dissection of TL1A-effects on Treg and Tconv.
Recombinant TL1A-Ig fusion protein may serve as an attractive alternate to the
heterologous hamster antibody 4C12 that is cleared out due to its antigenic nature.
Additionally, trimeric ligand Ig fusion proteins have a shorter biological half –life and the
ability to oligomerize receptors on the cell surface, mediating stronger signaling. Our
initial aim, therefore, was to genetically engineer the physiological ligand fusion TL1AIg protein for TNFRSF25 and compare the function of the agonists in vitro and in vivo.
CHAPTER 2: Results
2.1 Construction and characterization of TL1A-Ig
2.1.1 Genetic engineering and preliminary studies of four TL1A-Ig constructs
The cloning strategy for the construction of four TL1A-Ig fusion proteins is
described in Figure 11. The fusion proteins differed by the length of the extracellular
domain (ECD) and by the presence or absence of the putative cleavage site. The cDNA of
each construct comprised of the murine IgG λ-chain peptide leader sequence followed by
the hinge, CH2 and CH3 domains of mIgG1 and then the ECD of TL1A (Figure 12a).
PCR mIgG1
PCR TL1A
(4 constructs)
Ligate into
pCRII-TOPO
Ligate into
pCRII-TOPO
1. Cut Fc-mIgG1pCRII and receiving
vector pBluescript II
SK with EcorI and
XhoI; ligate
2. Cut mTL1A-pCRII
and receiving vector
Fc-mIgG1-pBluescript
II SK with EcorI and
NotI; ligate
Sequence
Fc-mIgG1-TL1ApBluescript II
Cut Fc-mIgG1-TL1ApBluescript II and
receiving vector
pBCMGSneo with
XhoI and NotI, ligate
SalI
XhoI
Cut Fc-mIgG1-TL1ApBMGSneo with XhoI and ligate
synthesized dimer oligomers for
Ig κ leader squence flanked with
(5’)SalI and (3’) XhoI
Neo
mIgG1-TL1A
pBCMGSneo
~15.5 kbp
NotI
Neo
SalI
XhoI site removed
when SalI ligates
Ig κ leader
sequence-mIgG1TL1A
pBCMGSneo
~15.5 kbp
NotI
Restriction digests
to confirm correct
orientation
Figure 11. Schematic diagram of TL1A-Ig-pGMGSneo cloning strategy.
40 41 Each of the fusion protein constructs was transfected and expressed in 3T3-NIH cells.
Non-reducing and reducing western blot analysis was done for all fusion proteins in
supernatant using an HRP conjugated anti-mIgG1 antibody for immunoblotting (Figure
12b). The non-reduced gel showed that constructs TL1A-Ig (FL ECD), (-3aa ECD), and
(-5aa ECD) form dimers (100 kDa), while construct TL1A-Ig (ECD -11a) that lacks a
protease cleavage site separates as a monomer (50 kDa). Since the IgG1 had been
mutated to replace the three cysteines of the hinge portion to serines (169), we were
expecting all constructs to separate as monomers in non-reducing conditions due to the
lack of disulfide-bond formation. All TL1A-Ig fusion proteins have an apparent,
monomeric molecular weight of approximately 50 kDa upon separation under reducing
conditions.
All
constructs
were
a
intact
b
A
hinge*CH2*CH3#
B
hinge*CH2*CH3#
C
hinge*CH2*CH3#
D
hinge*CH2*CH3#
TL1A#
TL1A#
TL1A#
TL1A#
and
Non-reduced
stable.
Reduced
FL#ECD#TL1A*Ig#(aa#65*252)#
*3aa#ECD#TL1A*Ig#(aa#68*252)#
*5aa#ECD#TL1A*Ig#(aa#70*252)#
*11aa#ECD#TL1A*Ig#(aa#76*252)#
a
b
c
d
a
b
c
d
Figure 12. Construction and expression of TL1A-Ig fusion proteins. (a) Schematic diagram of the
four different TL1A-Ig fusion protein constructs. (b) TL1A-Ig variants were stably transfected into and
secreted by NIH-3T3 cells. Twenty ng of TL1A-Ig in supernatant (quantified by Elisa) was analyzed by
SDS-PAGE under reducing and non-reducing conditions, transferred to a nitrocellulose membrane and
detected by Western blot analysis with HRP anti-mIgG mAb.
With the aim of identifying the highest TL1A-Ig producing clone, all transfectants
were selected for high IgG1 expression using flow cytometry sorting followed by single
cell cloning under high antibiotics selection. Briefly, all TL1A-Ig producing cells were
expanded, harvested and stained with FITC anti-mIgG1. The highest IgG1 positive cells
(top 10%) were FACs sorted. Recovered TL1A-Ig secreting cells were single-cell cloned
42 (0.33 cells/well) under 3 mg/mL G418 selection. TL1A-Ig clones were screened by an
indirect IgG sandwich ELISA at each step to select for the highest TL1A-Ig producing
clone
(Figure
Post-transfection
IgG1
gp96-Ig
FL TL1A-Ig
-3aa TL1A-Ig
-5aa TL1A-Ig
-11aa TL1A-Ig
1.0
OD 405 nm
0.8
0.6
1.0
IgG1
gp96-Ig
0.8
FL TL1A-Ig
-3aa TL1A-Ig
-5aa TL1A-Ig
-11aa TL1A-Ig
0.6
0.4
0.4
0.2
0.2
0.0
20 21 22 23 24 25 26 27 28 29 210
Dilution
0.0
20 21 22 23 24 25 26 27 28 29 210
Dilution
c
Post-single cell cloning
1.0
0.8
OD 405 nm
Post-FLOW sort
b
OD 405 nm
a
13).
0.6
IgG1
gp96-Ig
FL TL1A-Ig-A
FL TL1A-Ig-B
FL TL1A-Ig-C
-3aa TL1A-Ig-A
-3aa TL1A-Ig-B
-5aa TL1A-Ig-A
-5aa TL1A-Ig-B
-11 aa TL1A-Ig-A
0.4
0.2
0.0
20 21 22 23 24 25 26 27 28 29 210
Dilution
Figure 13. Selection of the highest producing
TL1A-Ig cell clone. (a) Each of the four TL1A-Ig
expression plasmids was transfected and expressed in
3T3-NIH cells. Transfected cells were expanded and
seeded at 1 million cells per well in a 6 well plate for
24 hours. Supernatants were collected and quantified
for TL1A-Ig using an indirect IgG1 ELISA. (b)
TL1A-Ig expressing cells from (a) were expanded,
harvested and stained with FITC-anti-IgG1 antibody.
The top 10% cells expressing IgG1 were FLOW
sorted and supernatants were collected for TL1A-Ig
quantification using ELISA as described above. (c)
Highest TL1A-Ig expressing cells for each construct
were single cell-cloned at 0.3 cells/well in 96-well
plates in 10% FBS supplemented media with G418
selection. Supernatants were collected from wells that
turned yellow (indicating cell growth) to quantify for
TL1A-Ig using IgG1 directed ELISA. The cells
recovered from each well were expanded, harvested,
frozen, and stored at -135° C for future use.
Size exclusion chromatography was used to determine the apparent molecular
weights of unpurified TL1A-Ig fusion proteins (Figure 14). Briefly, 0.5 mL TL1A-Ig
supernatant was applied on a Superdex 200 gel filtration column equilibrated with Tris
buffer. Elution fractions were collected at a flow rate of 0.5 mL/min (200 µl/fraction) and
10 µl of each fraction were applied to a nitrocellulose membrane (dot blot) and blocked
43 with 5% filtered non-fat dry milk. TL1A-Ig was detected with an HRP conjugated antimouse IgG antibody (1:1000 dilution) and the SuperSignal West Pico Chemiluminescent
Substrate (Thermo Fisher Scientific, Rockford, IL). The signals from each blotted dot
were quantified using a densitometry program on the Versa Doc imaging system and the
readout was overlapped with the UV absorption profile (280 nm) chromatogram of
standards IgG and albumin separated on the same column (Figure 14a).
b
TL1A-Ig,
120 void
Albumin,
44
Exclusion#peak#
IgG,
150
100
60
6
8
10
8
10
440#
10
Exclusion#peak#
Buffer:#
150#mM#NaCl#
10#mM#Tris#
1#mM#EDTA# 669#
10%#glycerol#
#
12
12
8
mL#
80
6
5#frac(ons/mL#
Buffer:#
150#mM#NaCl#
10#mM#Tris#
1#mM#EDTA# 669#
#
Density ODu/mm2
Absorbance (280 nm)
a
14
Posi(ve# Nega(ve##
control# control#
Elution volume (mL)
8
440#
10
Thyroglobulin,#669#kDa#
Ferri7n,#440#kDa#
Figure 14. TL1A-Ig exists as a high molecular weight protein in supernatant (a) Representative
dot-blot densitometry analysis of elution fractions collected from gel filtration of TL1A-Ig secreted
from 3T3 cells in supernatant superimposed on the elution chromatogram of standards. (b) Supernatants
containing TL1A-Ig were applied on a Superdex 200 column and buffer (with or without 10% glycerol)
was used to transport the sample through the column.
All TL1A-Ig fusion proteins had a similar densitometry profile that suggested higher
molecular weight oligomerization of the protein causing it to elute in the void volume.
The addition of 10% glycerol to the Tris buffer did not change the elution profile (Figure
14b).
2.1.2 GS selection system for generation of high TL1A-Ig expressing clone
All four TL1A-Ig constructs had functional activity assessed by in vitro caspase
activation assays that will be discussed in section 2.2.1 (pg. 51). The highest producing
clone of TL1A-Ig construct (-3aa ECD aa 68-252 with cleave site) was selected for
44 purification and further analysis. The murine glutamine synthetase (mGS)/ methionine
sulfoxamine (MSX) gene amplification system was utilized to attain high TL1A-Ig
producing clones (170). Glutamine synthetase is an enzyme that is required for the
biosynthesis of glutamine from glutamate and ammonia. Mammalian cell lines utilize this
enzymatic pathway to make glutamine, which is required for survival. MSX binds to
endogenous GS enzyme and prevents the production of glutamine. Cell lines including
Chinese Hamster Ovarian cells (CHO) can be transformed with a plasmid with a gene of
interest and the GS gene. Gene amplification occurs when cells are subjected to
increasing concentrations of MSX. The transfected GS gene can function as a selectable
marker to select for gene amplified clones that produce high levels of recombinant
proteins. Using this system, CHO-K1 cells were transfected with the pVitro-2-hygro
vector (Invivogen, San Diego) double cassette plasmid expressing TL1A-Ig and mGS
(Figure 15a). The cDNA for the fusion protein comprised of the murine IgG k-chain
peptide leader sequence, followed by the hinge, CH2 and CH3 domain of mouse IgG1
and then the extracellular domain (ECD) of murine TL1A (amino acid 68-252) (Figure
15b). The pVITRO plasmid carries two human ferritin composite promoters, FerH
(heavy chain) and FerL (light chain).
a
b
hFerL mIgG1-mTL1A hFerH mGS
leader
mIgG1
peptide hinge-CH2-CH3
5’
62 bp
681 bp
mIgG1
N hinge-CH2-CH3
227 a.a
Figure 15. Construction of the TL1A-IgpVitro plasmid. (a) Schematic diagram of the
expression cassette of the TL1A-Ig fusion
protein. An expression cassette of mIgG1mTL1A is inserted 3′ to hFerL promoter in the
3’ pVitro2-hygro plasmid. The mouse glutamine
synthetase (mGS) cDNA is inserted 3′ to the
hFerH promoter, which provides a selection
marker for amplification in transfected
mammalian cells. (b) Schematic of the TL1AIg cDNA and the corresponding translated
monomeric protein structure.
mTL1A
ECD
558 bp
mTL1A
ECD
186 a.a
C
45 The transformants were single-cell cloned with 1 mg/mL hygromycin selection
and supernatants were analyzed by ELISA for mouse IgG as a read-out for TL1A-Ig. The
highest producing clone (40 µg/mL/ 106cells/ 24h) was subsequently passaged in
standard cell culture media and 10% FBS. FBS was slowly reduced and substituted with
increasing proportions of serum-free media. Serum-free subclones were subjected to
further selective pressure in glutamine-free media and with increasing concentrations of
MSX, an inhibitor of GS, resulting in gene amplification. After the MSX selection, the
subclone producing the highest level of TL1A-Ig (200 µg/mL/ 106cells/ 24h) was
identified and maintained for production of TL1A-Ig using a hollow fiber cell cartridge
and culture in serum free and glutamine free medium. By utilizing cartridges, we
increased the cells per volume per day that resulted in higher TL1A-Ig yields (up to 4.4
mg/mL/day). TL1A-Ig was purified to homogeneity by affinity chromatography by
binding to protein A and elution with diethylamine (0.1 M, 11.5 pH).
2.1.3 Biochemical and gel filtration analysis of TL1A-Ig
TL1A is a trimeric protein (171-174) while the IgG heavy chain forms dimers.
The theoretical m.w of monomeric TL1A-Ig is 47 kDa with the IgG contributing 26 kDa
and TL1A part of 21 kDa. Upon SDS-PAGE analysis, unreduced TL1A-Ig migrated as a
dimer of 100 kDa indicating that the IgG1 heavy chains did not disassociate. Reducing
conditions resulted in an apparent m.w band of approximately 50 kDa (Figure 16a). The
low m.w band in Figure 16a does not stain with either anti-IgG or anti-TL1A antibody
and represents a minor contaminant. By Western blot analysis, both anti-TL1A and antiIgG antibody detected the 50 kDa band indicating that all of TL1A is in association with
46 IgG
and
a
kDa
no
cleavage
2-ME
- +
2-ME
- +
occurred
2-ME
- +
150
100
75
50
37
25
150
100
75
50
37
25
(Figure
16a).
b
kDa
150
100
75
50
37
25
1
α-TL1A α -IgG
Western Blot
Coomassie Blue
2
3
4
Coomassie Blue
Figure 16. Characterization of purified TL1A-Ig by SDS-PAGE and western blot analysis (a)
Non-reduced (-2-ME) and reduced (+2-ME) TL1A-Ig samples were separated on SDS gel and stained
with Coommassie Blue or analyzed by immunoblotting using antibodies to mouse-TL1A or mouse-IgG
(b) SDS-PAGE separation of reduced TL1A-Ig after enzymatic deglycosylation. Protein samples were
treated with glycanases and separated on SDS-PAGE followed by staining with Coommassie Blue
Stain. Denatured TL1A-Ig was loaded in each lane. Lane 1: Untreated control, lane 2: treated with Nglycanase only, lane 3: treated with N-glycanase and sialidase A, and lane 4: treated with N-glycanase,
sialidase A and O-glycanase.
Multiple bands in non-reduced conditions may be explained by thiol-disulfide exchange
or post-translational glycosylation.
N-glycanase treatment indicates N-linked post-
translational glycosylation in CHO cells while O-glycanase and salidase had no effect
(Figure 16b).
To determine whether preparative conditions and purification methods affect the
final oligomerization and function of TL1A-Ig, we collected separate batches of TL1A-Ig
supernatants from 3T3 cell or CHO cell cultures and purified them using different affinity
chromatography columns and elution buffers. Since 3T3 cells produced low amounts of
TL1A-Ig (~5 µg/mL), we applied a large 5 L volume of TL1A-Ig containing supernatant
on
a
traditional
protein
A
column
for
purification
of
TL1A-Ig
Prep
1.
Void
Aldolase, Ovalbumin,
158
44
Hexameric TL1A-Ig,
528 kDa
TL1A7Ig#Prep1#
Thyroglobulin,
670
Protein#
Marker#(kDa)#
47 Absorbance (280 nm)
Dimeric TL1A-Ig,
175 kDa
75#
50#
37#
TL1A-Ig Prep 1
Hexameric
Dimeric
d
Elution volume (mL)
Figure 17. Characterization of purified TL1A-Ig Prep 1 by gel filtration and SDS-PAGE analysis.
After the volume passed through the column, bound TL1A-Ig was eluted using a basic
elution buffer diethylamine, (0.1 M, 11.5 pH) and neutralized with potassium
dihydrophosphate (1M, monobasic). The TL1A-Ig containing solution was dialyzed
against PBS overnight and the recovered protein was stored at -80° C. An aliquot of
TL1A-Ig Prep 1 was applied on a Superdex 200 gel filtration chromatography column to
determine the apparent molecular weights of 528 kDa (hexamer) and 175 kDa (dimer),
which suggests high frictional coefficient as depicted in schematic drawings of the
quarternary structure (Figure 17). Upon SDS-PAGE analysis and Coomassie blue
staining the reduced fusion protein migrated as bands (due to N-glycosylation as
described above) at 50 kDa with a faint band of contaminating albumin at approximately
Ovalbumin,
44
Aldolase,
158
Void
TL1A7Ig#Prep4#
TL1A3Ig(Prep(2(
Thyroglobulin,
670
Protein(
Protein#
Marker((kDa)(
(Marker#(kDa)#
Absorbance (280 nm)
65 kDa (Figure 17).
75#
50#
TL1A-Ig Prep 2
37#
Elution volume (mL)
Figure 18. Characterization of purified TL1A-Ig Prep 2 by gel filtration and SDS-PAGE analysis.
48 Purification on A protein A column followed by elution with an acidic glycine buffer (0.1
M, 2.5 pH) was done for TL1A-Ig Prep 2 (Figure 18). The gel filtration analysis revealed
higher molecular weight oligomerization of TL1A-Ig Prep 2, while SDS-PAGE analysis
of reduced TL1A-Ig resulted in expected bands at 50 kDa (Figure 18).
We encountered difficulty in handling large supernatant volumes due to the low
amount of TL1A-Ig secreted by 3T3 cells. Undesired protein aggregation clogged the
protein A column and frequent centrifugation and filtration of the supernatant was
laborious and resulted in loss of protein at each step. Hence, in the next protein
purification (TL1A-Ig Prep 3), ammonium sulfate precipitation was used to concentrate
the proteins and reduce the volume of TL1A-Ig supernatant (Figure 19). Briefly, solid
ammonium sulfate was added to the supernatant to achieve a 55% ammonium sulfate and
the solution was mixed overnight at 4° C. The supernatant was centrifuged and the
precipitate containing TL1A-Ig was resuspended in binding buffer (1.6 M Glycine, 3.2 M
NaCl, pH 9.0) and dialyzed against fresh binding buffer overnight. The solution from the
dialysis tube was centrifuged, filtered and applied on a protein A column for purification.
Size exclusion chromatography analysis resulted in majority of TL1A-Ig Prep 2 eluting
as a dimer of 175 kDa molecular weight. Upon SDS-PAGE analysis, reduced TL1A-Ig
bands
with
50
Thyroglobulin,
670
Void
kDa
molecular
Aldolase, Ovalbumin,
158
44
Hexameric TL1A-Ig,
528 kDa
Dimeric TL1A-Ig,
175 kDa
weight
(Figure
19).
Protein(
Protein(
Marker((kDa)(
Marker((kDa)(
(
TL1A7Ig(Prep2(
TL1A3Ig(Prep(3(
as
Absorbance (280 nm)
migrated
75(
50(
TL1A-Ig-Prep3
37(
Elution volume (mL)
Figure 19. Characterization of purified TL1A-Ig Prep 3 by gel filtration and SDS-PAGE analysis.
49 We next evaluated the affect of acidic elution using a protein G column for
homogenous purification of TL1A-Ig. The TL1A-Ig containing supernatant was directly
applied on the protein G column without prior preparative methods. Bound fusion protein
was eluted with an acidic glycine buffer (0.1 M, 2.5 pH) and neutralized with Tris (1 M,
9.0 pH). Size exclusion chromatography showed that TL1A-Ig purified under these
conditions resulted in dimeric oligomerization of TL1A-Ig. Separation of reduced TL1AIg on an SDS-PAGE gel resulted in clean bands at 50 kDa confirming that the recovered
Hexameric TL1A-Ig,
528 kDa
Dimeric TL1A-Ig,
175 kDa
75#
50#
TL1A-Ig-Prep 4
37#
Thyroglobulin,
670
Void
Protein#
Protein#
#Marker#(kDa)#
#Marker#(kDa)#
TL1A7Ig#Prep#4#
TL1A7Ig#Prep#5#
Void
Aldolase, Ovalbumin,
158
44
Absorbance (280 nm)
Thyroglobulin,
670
Protein#
#Marker#(kDa)#
TL1A7Ig#Prep#4#
Absorbance (280 nm)
TL1A-Ig was intact and the preparation had no contaminants (Figure 20).
Aldolase, Ovalbumin,
158
44
Hexameric TL1A-Ig,
528 kDa
75#
75#
50#
50#
Purified TL1A-Ig Prep 5
37#
37#
Elution volume (mL)
Elution volume (mL)
Figure 20. Characterization of purified TL1A-Ig
Prep 4 by gel filtration and SDS-PAGE analysis.
Figure 21. Characterization of purified
TL1A-Ig Prep 5 by gel filtration and SDSPAGE analysis.
After the establishment of the GS/MSX protein production system, we were able
to harvest volumetrically smaller and highly concentrated TL1A-Ig supernatants (up to
4.4 mg/mL). A small 75 mL batch of TL1A-Ig containing supernatant was passed over a
protein A column followed by elution with a basic diethylamine buffer (0.1 M, 11.5 pH).
The recovered TL1A-Ig p Prep 5 was analyzed using gel filtration that revealed a
molecular weight of 528 kDa (hexamer) (Figure 21). Separation under reduced
conditions on an SDS-PAGE gel resulted in bands at approximately 50 kDa molecular
weight. The lower contaminating band did not stain with anti-TL1A or anti-IgG1 as seen
in Figure 16a, confirming that the protein was intact and pure (Figure 21).
50 2.2 In vitro functional activity of TL1A-Ig
2.2.1 TL1A-Ig binds and stimulates TNFRSF25 overexpressed on tumor cells
TL1A-Ig bound to P815-TNFRSF25 cells with a similar profile to the
TNFRSF25-specific monoclonal antibody 4C12 but not to untransfected P815 cells as
determined by flow cytometry (Figure 22a-c). In addition, binding of TL1A-Ig to P815TNFRSF25 cells was competitively inhibited by pre-incubation with the monoclonal
TL1A blocking antibody, L4G6. TL1A-Ig purified using either acidic or basic elution
buffers maintained structural integrity and bound to TNFRSF25 (Figure 22b).
Hexameric and dimeric TL1A-Ig separated by gel filtration also bound to TNFRSF25
(data
not
a
shown).
b
TL1A-Ig acidic elution
FL TL1A-Ig
-3 aa TL1A-Ig
-5 aa TL1A-Ig
-11 aa TL1A-Ig
Untransfected P815
2° Ab control
mIgG1
TL1A-Ig basic elution
L4G6 treated TL1A-Ig
Untrasfected P815
2° Ab control
mIgG1
c
Unstained
Hamster IgG
Untransfected P815
2° Ab control
4C12
Figure 22: TL1A-Ig and 4C12 bind to their receptor, TNFRSF25, expressed on P815. (a) P815
cells were stably transfected with expression vectors encoding mouse TNFRSF25. TNFRSF25-P815
cells were incubated with isotype mIgG1, (a) TL1A-Ig in culture supernatant or (b) purified TL1A-Ig
or (c) hamster IgG or 4C12 and stained with fluorochrome conjugated anti-IgG secondary antibody.
Negative controls included: Untransfected P815 cells incubated with TL1A-Ig followed by secondary
antibody, TNFRSF25-P815 cells stained with secondary antibody only, and TNFRSF25-P815 cells
incubated with TL1A-Ig pretreated with L4G6 (TL1A blocking Ab) followed by incubation with
+
secondary antibody. TNFRSF25 cells were then visualized by flow cytometry.
Migone et al. reported that TL1A induces NF-kappaB activation and apoptosis in
TNFRSF25-expressing cell lines(41). During apoptosis, the activation of specific
51 proteases, called caspases (cysteinyl-aspartic-acid-proteases), is one of the initial
intracellular biochemical events. To determine whether the four TL1A-Ig fusion proteins
can activate caspases, tumor P815 cells over-expressing TNFRSF25 were exposed to
titrating concentrations of TL1A-Ig fusion proteins in supernatants harvested from
transfected
60
40
20
0
0.01
300
3
Rhodamine counts (10 )
c
200
b
No fusion protein
FL TL1A-Ig
-3 aa TL1A-Ig
-5aa TL1A-Ig
-11 aa TL1A-Ig
3
1
Concentration (ng/mL)
No fusion protein
mIgG1
TL1A-Ig CHO cell sup.
Purified TL1A-Ig
4C12
100
0
0.0001
0.01
1
100
Rhodamine counts (10 )
80
cultures
3
3
Rhodamine counts (10 )
100
cell
Rhodamine counts (10 )
a
3T3
100
Concentration (ng/mL)
80
60
23a). No fusion protein
Untransfected P815
L4G6 treated TL1A-Ig
Basic elution TL1A-Ig
Acidic elution TL1A-Ig
Hexameric TL1A-Ig
Dimeric TL1A-Ig
4C12
40
20
0
0.0001
100
(Figure
0.01
1
100
Concentration (ng/mL)
10000
Figure 23. TL1A-Ig induced apoptosis in
TNFRSF25 expressing P815 cells. TNFRSF25P815 cells were exposed to titrating concentrations
of (a) unpurified TL1A-Ig constructs secreted from
3T3 cells, (b) acidic or basic purified TL1A-Ig,
hexameric or dimeric TL1A-Ig separated using gel
filtration (3T3 cells), or (c) unpurified or purified
TL1A-Ig secreted from CHO cells. Negative
controls: TNFRSF25-P815 cells cultured without
fusion protein, TNFRSF25-P815 cells exposed to
titrating concentration of IgG1, untransfected P815
10000
cells incubated with purified TL1A-Ig, and
TNFRSF25-P815 cells incubated with TL1A-Ig
pretreated with L4G6. Cells were incubated with
caspase substrate solution and free Rhodamine 110
was determined fluorimetrically. These data are
representative of more than 5 experiments.
Caspase activity was also observed when TNFRSF25-P815 cells were exposed to TL1AIg purified using either acidic or basic elution buffers, or hexameric and dimeric TL1A-Ig
that was separated by gel filtration (Figure 23b). Pre-incubation of TL1A-Ig with L4G6
completely inhibited apoptosis of P815-TNFRSF25 cells, and apoptosis was not observed
when untransfected P815 cells were exposed to TL1A-Ig. TL1A-Ig was able to induce
52 caspase activation at a 100-fold lower concentration by weight than 4C12 (Figure 23c),
suggesting high avidity binding and induction of ligand-mediated oligomerization on the
receptor resulting in enhanced TNFRSF25 signaling relative to 4C12. As expected,
adding titrating concentrations of control mIgG1 did not induce apoptosis.
2.2.2 TL1A-Ig binds to and stimulates endogenous TNFRSF25 expressed by T
cells
Resting Tconv, Treg and CD8+ T cells express low levels of TNFRSF25 that was
detected using a triple sandwich staining method with 4C12 (Figure 24a).
a
b
Iso. Conrol
Unstained
Tconv
Iso. Control
Treg
Treg
CD8+ Teff
CD8+ Teff
% Max
% Max
Tconv
4C12
TNFRSF25
c
TNFRSF25
d
Tconvs
CPM (103)
300
***
***
40
200
ns
***
30
20
100
0
Tregs
50
nv
nv nv
nv
nv
10
0
nv nv
nv
nv
nv
αCD3
--- + --- --- + + --- + + --- +
IL-2
--- --- + --- + --- + + --- + +
--- --- + --- + --- + + --- + +
--- --- --- + --- + + + --- --- ---
--- --- --- + --- + + + --- --- ---
--- --- --- --- --- --- --- --- +
--- --- --- --- --- --- --- --- +
TL1A-Ig
4C12
+ +
--- + --- --- + + --- + + --- +
+ +
Figure 24. TL1A-Ig binds to and stimulates endogenous TNFRSF25 expressed on activated T cells
(a) FIR splenocytes were harvested and sequentially incubated with isotype hamster IgG or 4C12,
+
biotin labeled anti-hamster IgG, and finally streptavidin Ab (b) CD4 T cells and CD8+ T cells were
magnetically purified from FIR mice and activated using plate bound anti-CD3 for 4 days. Cells were
harvested and incubated with isotype IgG or TL1A-Ig followed by staining with FITC anti-mouse IgG.
(a) 4C12 bound or (b) TL1A-Ig bound TNFRSF25 was analyzed using flow cytometry by gating on
+
+
+
+
+
+
CD4 FIR Tconv and CD4 FIR Treg or CD8+ Teff cells. (c) CD4 CD25 Tconv or (d) CD4 FIR Treg
cells were cultured in proliferation assays with indicated stimuli: anti-CD3 (2 µg/mL; 2C11), mIL-2 (10
U/mL), 4C12 (10 µg/mL), or purified TL1A-Ig (0.1 µg/mL) in triplicates for each condition. ***P <
0.001 versus appropriate control. Significance was determined by 1-way ANOVA with Tukey post-test.
Error bars indicate mean + SEM. NV refers to not visible.
We failed to detect basal levels of TNFRSF25 expressed by all T cell subsets using
TL1A-Ig or biotinylated TL1A-Ig (data not shown) that maybe due to the low expression
53 of TNFRSF25 on resting T cells. Anti-CD3 antibody activated T cells including CD4+
Tconv and Treg cells and CD8+ T cells express elevated levels of TNFRSF25 compared
to resting T cells as detected by TL1A-Ig (Figure 24b) (62). In vitro incubation of Tconv
cells with TL1A-Ig, but not 4C12, costimulated proliferation of Tconv cells in an antiCD3 antibody and IL-2 dependent fashion (Figure 24c). The fusion protein, unlike 4C12,
also stimulated proliferation of purified natural CD4+FoxP3+ Treg cells in vitro in the
presence of low 10 U/ml IL-2, even without the addition of anti-CD3 (Figure 24d). The
sorting of live CD4+FoxP3+ Treg was made possible by utilizing FoxP3-reporter (FIR)
mice (175) expressing a red fluorescent protein (RFP) under the FoxP3 promoter.
Activation of naïve CD4+ Tcells by anti-CD3 or specific antigen in the presence
of TGF-β, retinoic acid (RA) and IL-2 results in the induction of FoxP3 in a substantial
proportion of Tconv cells resulting in iTregs. Foxp3 induction is partially suppressed by
the TNFRSF25-agonist 4C12 and more completely by TL1A-Ig (Figure 25a) similar to
OX40L (119, 120, 176). Additionally, TL1A-Ig also mediated proliferation of iTregs that
were generated from culturing CD4+FoxP3- Tconv with plate-bound anti-CD3, IL2,
a
acid
%FoxP3/Total CD4+
100
80
(RA),
and
TGF-β
b
No Agonist
4C12
TL1A-Ig
150
CPM (103)
retinoic
60
40
100
(Figure
25b).
***
Unstimulated
IL-2 only
IL-2 + 4C12
IL-2 + TL1A-Ig
ns
50
20
0
Polyclonal
iTregs
0
OT-II
iTregs
Figure 25. TL1A-Ig blocks induction and induces proliferation of iTregs (a) For iTreg induction,
FIR Tconv were cultured with plate-bound anti-CD3, TGF-β, RA, mIL-2 and 4C12 or TL1A-Ig and
+
OTII Tconv were cultured as above plus 1:2 APCs and OVA. Cultures were analyzed for FoxP3 cells
+
+
in the CD4 gate. (b) Polyclonal CD4 FIR iTregs were generated as described in (a) and cultured in
proliferation assays for 72 hrs. One representative analysis of 3 independent experiments is shown.
***P < 0.001 versus IL-2 only. Significance was determined by 1-way ANOVA with Tukey post-test.
54 Since TNFRSF25 mediated Treg proliferation is dependent on TCR engagement
of MHC class II as reported by Dr. Schreiber et al. (130), we wanted to determine why
freshly isolated CD4+FoxP3+ Treg proliferate in vitro without the addition of anti-CD3.
Since both Tconv and Treg express MHC II (Figure 26a), our first hypothesis was that
TCR expressed on highly purified Treg cell surface, recognize self-antigen that is
presented by MHC II expressed on other cocultured Tregs. However, addition of
blocking MHC II antibody did not abrogate TL1A-Ig mediated Treg expansion in the
absence
of
anti-CD3
a
(Figure
26b).
b
CPM (103)
50
40
30
20
Unstimulated
Only IL-2
IL-2+TL1A-Ig
IL-2+TL1A-Ig+Iso Control
IL-2+TL1A-Ig+MHC II blocking Ab
ns
10
0
Figure 26. MHCII blocking antibody does not abrogate TL1A-Ig mediated Treg proliferation in
vitro. (a)Total CD4+ cells and T cell depleted APCs were magnetically separated and stained with
FITC isotype control or anti-MHC II antibody. MHC II+ cells were detected in FIR- Tconv or FIR+
Treg cells pregated on CD4+ cells using FLOW cytometry. (b) Flow cytometry sorted CD4+FIR+ Treg
cells were cultured in proliferation assays in the indicated conditions. Cells were treated with isotype
antibody or blocking anti-MHC II for 45 minutes at 4°C before IL-2 and TL1A-Ig were added. Cultures
3
in were pulsed with H-thymidine for the last 6 hours of 72 hr incubation and incorporated isotope was
measured by liquid scintillation counting.1way ANOVA and Tukey's Multiple Comparison Test.
Our next hypothesis was that residual TCR signaling from recognizing selfantigen in vivo enabled TNFRSF25 mediated proliferation of freshly isolated Tregs in
vitro. In an attempt to decrease/completely abrogate TCR signaling, Tregs were “rested”
in 100U/mL IL-2 for 72 hours prior to culturing them in proliferation assays. This
method resulted in a significant decrease in proliferation of cells that received only IL-2
and TL1A-Ig as compared to cultures that received anti-CD3, IL-2 and TL1A-Ig (Figure
27a). Supporting our second hypothesis, the addition of cyclosporine to cultures inhibited
55 TL1A-Ig triggered proliferation of Tregs (Figure 27b) suggesting that freshly isolated
Treg maintain residual TCR signals from binding to self-antigens or commensal antigens
normally present in vivo. Others have reported that freshly isolated Tregs have a higher
level of phosphorylation of the TCR ζ-chain compared to Tconv cells, which is a
150
CPM (103)
a
consequence
100
of
IL-2
**
IL-2 and anti-CD3
anti-CD3 and TL1A-Ig
IL-2 and TL1A-Ig
IL2+anti-CD3+TL1A-Ig
TCR
b
80
CPM (103)
biochemical
50
60
signaling
(177).
IL-2 only
IL-2+TL1A-Ig
IL-2 + TL1A-Ig+ CsA
40
20
0
0.1
0
1
10
CsA (µg/mL)
100
Figure 27. Treg cells maintain residual TCR signaling from self-peptide recognition in vivo. (a)
“Resting” Tregs were made by FACs sorting CD4+FoxP3+ Tregs from FIR splenocytes and culturing
them with 100 U/mL IL-2 for 72 hrs. “Resting" Tregs were harvested, washed and cultured in
proliferation assays in the indicated conditions. (b) FACs sorted CD4+FIR+ Treg cells were cultured in
proliferation assays in the indicated conditions. 2 ug/mL anti-CD3 (2C11), 100 ng/mL TL1A-Ig, and/or
10 U/mL IL-2 were used where indicated in (a) and (b). Cultures were pulsed with thymidine for the
last 6-8 hrs of 72 hr incubation and thymidine uptake measured using scintillion counter. 1way
ANOVA and Tukey's Multiple Comparison Test. **P < 0.01.
2.3 In vivo functional activity of TL1A-Ig
2.3.1 Half-lives of TL1A-Ig and 4C12
Before determining functional effects in vivo, we determined the half-life of both
TNFRSF25 agonists injecting mice with 4C12 or TL1A-Ig and analyzing timed serum
samples by ELISA specific for Armenian hamster IgG or TL1A (Figure 28).
Serum concentration
(% of initial)
256
128
64
32
16
8
4
2
1
0
Figure 28. Half-lives of TL1A-ig and 4C12
are 13.5 hr and 4 d, respectively. (a) The
time-related serum concentrations of 4C12 or
TL1A-Ig in C57BL/6 mice (n=3 or more) was
determined after a single i.p injection of 100
ug protein. Protein concentration was
measured in blood samples collected at
indicated time points by a sandwich ELISA
specific for 4C12 or TL1A.
4C12
TL1A-Ig
2
4
6
8
Time post injection (d)
56 B6 mice were injected i.p with 100 µg 4C12 or TL1A-Ig in 200 µl PBS on day 0. Blood
samples were collected at time points 3 hr, 6 hr, 12 hr, and 1-8 days. After each
collection, the sample was allowed to clot by leaving it undisturbed at room temperature
for 30 minutes. The clot was removed by centrifuging the sample at 1,000 g for 30
minutes in a refrigerated centrifuge. The resulting supernatant (serum) was stored at 20°C for further analysis. A sandwich ELISA utilizing primary antibodies specific to
Armenian hamster IgG and TL1A was performed to measure protein concentration at
each time point. The observed half-life is 13.5 hours for TL1A-Ig and 4 days for the
4C12 antibody. The shorter half-life of TL1A-Ig requires daily injection but allows easy
adjustment of desired serum levels.
2.3.2 TL1A-Ig induces rapid proliferation of CD4+FoxP3+ Treg cells in vivo
We previously reported that TNFRSF25 stimulation by 4C12 rapidly expands preexisting Tregs in vivo (130). By the use of naïve FoxP3-reporter mice (FIR), we were
able to continuously monitor the frequency and phenotype of the Treg population in
peripheral blood following one i.p dose of 50 or 100 µg TL1A-Ig (Figure 29a).
20
100 µg IgG
15
10 µg TL1A-Ig
50 µg TL1A-Ig
100 µg TL1A-Ig
b
10
5
0
0
5
60
%FoxP3+/Total CD4+
%FoxP3+/Total CD4+
a
Time (d)
50 µg TL1A-Ig i.p
100 µg TL1A-Ig i.p
100 µg IgG i.v
50 µg TL1A-Ig i.v
100 µg TL1A-Ig i.v
40
30
20
10
0
10
100 µg IgG i.p
50
0
5
10
15
20
Time(d)
Figure 29. TL1A-Ig stimulates rapid proliferation of CD4+FoxP3+ Treg cells in vivo. (a) The
kinetics and dose-dependent expansion of Treg cells in peripheral blood was determined after a
single i.p injection of titrating doses of TL1A-Ig (n=3) or IgG (n=3) in FIR mice. Mice were bled
on indicated days and the percentage of peripheral Tregs relative to total CD4+ cell was determined
by flow cytometry. (b) Comparison of in vivo expansion of Tregs determined as (a) after 3
consecutive i.v or i.p injections of IgG or TL1A-Ig. Data representative of 2 or more independent
experiments, with 2 or more mice per group. All data are mean + SEM.
57 Due to the short half-life, a single dose of TL1A-Ig did not induce a 3-fold expansion of
Tregs in the total CD4+ T cell population in the blood as observed with one 10 µg dose of
4C12. Maximal Treg expansion with TL1A-Ig was dependent on 3 consecutive doses of
fusion protein with no difference between 50 or 100 µg TL1A-Ig i.p to see maximal
responses (Figure 29b). The site of injection did not play a role in this expansion, as
demonstrated by equivalent Treg expansion following TL1A-Ig injections either i.p. or
i.v (Figure 29b). In vivo expanded TL1A-Ig-Tregs persisted in the peripheral blood
examined for 2 weeks, slowly contracting to levels observed in unstimulated mice.
To determine whether in vivo TNFRSF25 mediated Treg expansion is dependent
on TCR signaling, we adoptively transferred purified CD4+ T cells harvested from FIR
mice into MHC-II-deficient (CD74-/-) or CD4-/- mice. CD74-/- mice lack MHC IIantigen presentation and as a consequence also lack CD4+ T cells, while CD4-/- mice can
present antigen via MHC II to adoptively transferred CD4+ cells including CD4+ Tregs.
%FoxP3/Total CD4+
15
Ig G
T L 1 A -Ig
10
5
0
CD74-/- CD4-/Spleen
CD74-/- CD4-/Lymph nodes
Figure 30. TNFRSF25 mediated Treg expansion is
dependent on MHC class II. (a) Ten million CD4+
cells were highly purified by FACS sorting from FIR
mice and adoptively transferred into CD74-/- or CD4-/mice. After three days (Day 0), recipient mice were
treated with 100 ug of TL1A-Ig or IgG followed by 2
consecutive doses on days 1 and 2. The percentage of
FoxP3+ cells was analyzed in the spleen and pooled
lymph nodes on day 6. Data representative of 2
independent experiments, with 2 or more mice per
group. All data are mean + SEM.
Two days after transfer (day 0), mice were treated from days 0 – 2 with 100 µg TL1A-Ig
or isotype control IgG (Figure 30). Analysis on day 6 revealed no TL1A-Ig mediated
CD4+FoxP3+ Treg expansion in MHC II deficient mice. In contrast, TL1A-Ig mediated
potent Treg expansion in CD4-/- mice that are able to present MHC II antigens. Treg
expanded even in the absence of extraneous TL1A-Ig suggesting TCR signaling
58 primarily at intestinal mucosal sites (Figure 30). These experiments indicate that MHC II
is required for costimulation of Treg proliferation by TNFRSF25. These data imply that
TCR signaling is a pre-requisite for TNFRSF25 costimulation, which is similar to the
requirements that we observed for TNFRSF25 signaling for in vitro Tconv and Treg
proliferation assays and the known requirement for TNFRSF25 signaling in Tconv (41,
152).
2.3.3 Long-term administration of TL1A-Ig maintains elevated Treg levels
Daily administration of TL1A-Ig was able to sustain high Treg levels for over 20
days (Figure 31a) in the peripheral blood. Memory markers were also continuously
monitored during the 20-day period that revealed an increase in effector memory Tconv
cells (TEM) (CD62LlowCD44high) (Figure 30b), while central memory (TCM) markers
(CD62LhighCD44high) were upregulated on gated CD4+FoxP3+Treg cell population
as
50
b
IgG
***
%FoxP3+/Total CD4+
compared
TL1A-Ig
**
40
**
30
**
**
20
*
10
0
0
5
10 15 20 25 30
to
mice
injected
c
30
IgG
TL1A-Ig
20
10
0
with
%CD62LhighCD44high/Total Treg
a
30c)
%CD62LlowCD44high/Total Tconv
(Figure
0
5
Time(d)
10
15
Time(d)
20
25
60
control
IgG.
IgG
TL1A-Ig
40
20
0
0
5
10
15
20
25
Time(d)
Figure 31. Daily injections of TL1A-Ig lead to sustained Treg expansion in vivo for over 20 days.
(a) Treg expansion was monitored in the peripheral blood while administering daily i.p injections of
100 ug TL1A-Ig (n=4) or IgG (n=2) starting on day 0 and ending on day 20. Expression of central and
effector memory markers CD62L and CD44 were determined on (b) FoxP3 – Tconv and (c) FoxP3+
Treg cells pre-gated on CD3+CD4+ cells. Statistical analysis was performed by unpaired two-tailed
Student’s t-test (a). All data are mean + SEM. *P< 0.05, **P < 0.01, ***P < 0.001 versus control.
59 H&E staining of formalin fixed tissue sections from the heart, kidney, lungs, liver,
and intestine harvested on day 5 from 3-day TL1A-Ig treated mice (data not shown) or
analyzed on day 23 from 21-day TL1A-Ig treated mice did not indicate any cellular
infiltration
or
tissue
inflammation
(Figure
32).
TL1A-Ig
IgG
Heart
200x
Kidney
200x
Liver
200x
Lung
200x
Small
Intestine
100x
Large
Intestine
100x
Figure 32: H&E staining of tissue sections from mice injected daily with TL1A-Ig. B6 mice were
injected i.p with 100 µg IgG or TL1A-Ig daily from day 0 to day 20 and the heart, liver, kidney, lungs,
and intestine were harvested on day 23. Indicated tissue sections were H&E stained, 100-200x.
60 2.3.4 Characterization of T cell subsets in TL1A-Ig treated mice.
In the course of examining the effect of TL1A-Ig on Tregs in vivo, we observed
slightly enlarged spleens and lymph nodes in TL1A-Ig treated mice as compared to agematched IgG treated control mice (Figure 33a). The total cellularity increased slightly in
the spleen, while we observed a significant increase in the total cell number harvested
from inguinal, mesenteric, axillary and brachial pooled lymph nodes (LN) on day four
after the first dose of TL1A-Ig. Flow cytometric analysis revealed significantly increased
numbers of CD4+ T cells in the LNs and to a lesser degree in the spleen, while CD8 T
cell yields were only slightly higher in LN and normal in the spleen (Figure 33b).
Lymph nodes
c
Spleen
80
100
IgG
TL1A-Ig
60
40
20
0
Tconv
Treg
*"
Ig G
T L 1 A -Ig
Ig G
T L 1 A -Ig
Total CD3 CD4 CD8
cells
Lymph nodes
% Ki67+of
T cell subset
% Ki67+of
T cell subset
100
Ig G
Ig G
T L 1 A -Ig
Ig G
T L 1 A -Ig
Ig G
T L 1 A -Ig
Total CD3 CD4 CD8
cells
***"
20
0
T L 1 A -Ig
0
**"
40
T L 1 A -Ig
50
****"
60
Ig G
100
80
T L 1 A -Ig
6
N o . C e lls ( 1 0 )
150
Ig G
TL1A-Ig
6
IgG
Spleen
b
N o . C e lls ( 1 0 )
a
80
60
40
20
0
CD8
IgG
TL1A-Ig
Tconv
Treg
CD8
Figure 33. Characterization of T cell subsets in TL1A-Ig treated mice. (a) Spleen and pooled LNs
(inguinal, mesenteric, axillary and brachial pooled LN) harvested on day 4 from FIR mice that were
+
injected i.p with 100 µg IgG or TL1A-Ig on days 0,1, and 2. (b) Absolute numbers of total cells, CD3
+
+
T cells, CD4 T cells, and CD8 T cells from spleen and pooled LNs from 8-12 weeks old age-matched
IgG and TL1A-Ig treated mice as described in (a). The line represents the average absolute number
from mice analyzed in 7 independent experiments. (c) Increased numbers of T cell subsets in TL1A-Ig
+
+
treated mice as depicted by the frequency of proliferating (Ki67 ) Tconv, Treg, and CD8 T cells in the
spleen and pooled LNs. Statistical analysis was performed by unpaired two-tailed Student’s t-test. .
*P< 0.05, **P < 0.01, ***P<0.001 ****P < 0.0001.
61 The percentage of proliferating antigen Ki-67 expressing CD4+FoxP3+ Treg increased
from 20% to 80% in the spleen and LNs. Only about 5% of CD4+FoxP3- Tconv cells and
CD8+ T cells in IgG treated mice were Ki67+ and this ratio increased to 25% and 17%
after TL1A-Ig treatment (Figure 33c). TL1A-Ig treated mice had normal frequencies of
B cells, macrophages, dendritic cells (DC), natural killer cells (NK), and NKT cells
(Table 2).
Table 2: Leukocyte subsets with similar frequencies in IgG-treated
and TL1A-Ig treated mice
Cell Type
IgG
TL1A-Ig
B cells (CD19+)
58%
56%
Macrophages (F4/80+)
2%
3%
Dendritic cells (CD11c high)
5%
6%
NK cells (NK1.1+CD3-)
11%
16%
2%
2%
NKT cells
(NK1.1+CD3+)
Total splenocytes were harvested from IgGtreated and TL1A-Ig-treated mice as in Figure
33. Listed frequency is the percentage of alive
gated cells per total cells analyzed by flow
cytometry.
The frequency of memory CD4+ T cells (TM), including FoxP3-CD62LhighCD44high
central memory CD4+ cells (TCM) and FoxP3-CD62LlowCD44high effector memory CD4+
cells
(TEM),
doubled
IgG
in
spleen
CD62L
5%
6%
12%
LN
(Figure
IgG
TL1A-Ig
Spleen
78%
and
34).
TL1A-Ig
Lymph nodes
53%
5%
77%
11%
31%
17%
2%
73%
5%
4%
11%
11%
CD44
Figure 34. TL1A-Ig treatment increases expression of memory markers on Tconvs. Representative
+
flow cytometry plots and compilation of percentage of memory markers on CD4 FIR Tconv cells
harvested from spleen and pooled LN from IgG and TL1A-Ig injected mice. 8-12 week old mice; the
data are compiled percent averages of 5-7 mice per group from 2 independent experiments.
62 There was no significant difference in the expression of the activation marker CD69 on
either CD4+ TM cell subsets in the spleens and LN of IgG or TL1A-Ig treated mice (data
not shown). No differences in memory/activation markers were detected in the CD8+ T
cell subset between the IgG and TL1A-Ig treated mice (data not shown).
Since the intensity of an immune response is associated with the balance of Tconv
to Treg, the Tconv:Treg and TM:Treg ratios were determined (Table 3). Together, these
data suggest that treatment with TL1A-Ig in the absence of exogenous antigen resulted in
the proliferation of FoxP3- CD4+ T conventional cells that may have previously
encountered environmental and/or commensal antigens. As mentioned before, H&E
staining of tissue sections from the heart, kidney, lungs, liver, and intestine harvested
from 3-day (data not shown) or 21-day (Figure 32) TL1A-Ig treated mice did not
indicate
any
cellular
infiltration
or
tissue
inflammation. Table 3.
TL1A-Ig decreases Tconv: Treg ratio and (TCM + TEM):Treg
IgG
TL1A-Ig
Tconv per spleen (no.)
8,048,067
7,201,228
TCM per spleen (no.)
299,608
575,196
TEM per spleen (no.)
1,074,847
2,369,402
Treg per spleen (no.)
1,350,559
5,121,313
Tconv:Treg ratio
(TCM+TEM):Treg ratio
6.0
1.0
1.4
0.6
Total splenocytes were harvested as in
Figure 32. Cell numbers were calculated
by multiplying the number of cells
obtained in a single-cell suspension of
the spleen by the percentage of lymphoid
gated cells per total cells analyzed by
flow cytometry by the percentage of total
Tconv, TCM (CD4+FoxP3-CD62LhiCD44hi),
TEM (CD4+FoxP3-CD62LloCD44hi ), or
Tregs within the lymphoid gated cell
population.
To determine the phenotype of TL1A-Ig expanded Tregs, we analyzed
CD4+FoxP3+ cells from the spleen and lymph nodes harvested from TL1A-Ig or IgG
control treated mice. TL1A-Ig treatment significantly increased the frequency of CD25int
Treg population (Figure 35a) such that both CD25hi and CD25int Treg populations have
similar frequencies (51% and 49% of the CD4+FoxP3+ gate). The expansion of the
63 CD25int Treg cells was not entirely unexpected since similar observations were reported
after
4C12
a
IgG
administration
TL1A-Ig
10%
b
c
IgG
d 53%
(130).
71%
8%
Ki67+CD25int
Ki67+CD25hi
CD4
IgG
TL1A-Ig
TL1A-Ig
14%
23%
Ki67-
100
% of total
CD4+FoxP3+ Treg
FoxP3
21%
80
60
40
20
49%
63%
-Ig
1A
32%
Ig
51%
TL
68%
CD25
CD25
G
0
Ki67
FSC
hi
Figure 35. TL1A-Ig treatment leads to Treg expansion in vivo by inducing proliferation of CD25
int
and CD25 Tregs . (a) FIR mice were treated with IgG or TL1A-Ig on days 0,1, and 2 and splenocytes
were harvested on day 4 and analyzed by flow cytometry. Representative plots are shown. (b)
+
+
+
hi
Representative dot plots were pre-gated on CD4 FIR Tregs and analyzed for Ki67 on CD25 and
int
CD25 Tregs. Percentages indicate the proportion of each phenotype of the total fraction of
+
+
hi
int
CD4 FoxP3 Tregs. (c) Proportion of Ki67+ and Ki67– cells among CD25 and CD25 Tregs as in b.
8-12 week old mice; the data are compiled percent averages of 5-7 mice per group analyzed in 2
independent experiments.
However, Schreiber et al. reported that majority of 4C12-expanded Treg comprised of
CD25int cells. Flow cytometric analysis revealed approximately 3-fold increase in the
expression of Ki-67 in both the CD25hi and CD25int Treg populations in TL1A-Ig treated
mice compared to IgG controls (Figure 35b,c). An increase in αEβ7 integrin (CD103) and
killer cell lectin-like receptor G1 (KLRG1) indicating activation was observed in FoxP3+
Tregs in spleen and LNs of TL1A-Ig treated mice (Figure 36a) (178-180), while the
expression was unchanged in Tconv and CD8+ T cell subsets (data not shown). No
differences were observed in the expression of CD127, CTLA-4, OX40, GITR, and
64 CD39 on FoxP3+ Treg between IgG and TL1A-Ig treated mice. We also found an
increase in the frequency of splenic and LN FoxP3+ Treg with increased expression of Tcell memory markers in the TL1A-Ig treated mice as compared to IgG treated controls
(Figure 36b).
IgG
a
KLRG1
b
CD103
6%
2%
IgG
15%
19%
TL1A-Ig
Spleen
34%
13%
28%
26%
8%
20%
28%
44%
21%
7%
28%
32%
28%
41%
CD62L
TL1A-Ig
Lymph nodes
Spleen
Lymph nodes
6%
25%
13%
31%
17%
40%
CD44
Figure 36. TL1A-Ig treatment enhanced activation and memory formation of Treg cells. (a)
+
+
Representative histogram plots (spleen) for KLRG1 and CD103 expression on CD4 FoxP3 Tregs in
the spleen and pooled LN. Black: Spleen; Red: Pooled LN (b) Representative flow cytometric profiles
+
+
and compilation of the percentage of memory markers as a percentage of CD4 FoxP3 Treg cells in
spleen and pooled LN (8-12 week old mice; the data are compiled percent averages of 5-7 mice per
group analyzed in 2 independent experiments).
2.3.5 FoxP3+ Tregs from TL1A-Ig treated mice are highly suppressive ex vivo
To determine whether TL1A-Ig-expanded Tregs retain suppressive activity, we
purified CD4+FoxP3+ Tregs from FIR mice on day 4 after i.p injections of either TL1AIg or IgG isotype control antibody on days 0, 1 and 2. These Treg cells were then
cultured in proliferation assays in the presence of APCs and CD4+CD25- transgenic
Tconv expressing a dominant negative mutant of TNFRSF25 (DN-TNFRSF25). DN
TNFRSF25 Tconv are not costimulated in the presence of anti-CD3 and TL1A-Ig
65 (Figure 37a) in vitro. The Tregs from TL1A-Ig treated mice suppressed proliferation of
CD4+CD25– cells to a greater degree than the Tregs from IgG treated mice (Figure
37b). To determine whether addition of TL1A-Ig during the suppression assay affects the
suppressive activity of Tregs, similar assays were performed in the presence of TL1A-Ig
and IgG1. The presence of TL1A-Ig partially recovered DN TNFRSF25 Tconv
proliferation whether the Tregs were obtained from TL1A-Ig (Figure 37c) or IgG isotype
control–treated mice (data not shown). Like 4C12, these data show that inhibition of
Treg-suppressive activity by TL1A-Ig mediated TNFRSF25 signaling occurred in
conditions in which only Tregs express a functional TNFRSF25, which shows that this
effect is the result of signaling by TNFRSF25 on Tregs and not Tconvs. b
0
11::1
1
Unstimu- α-CD3
lated
No Treg
d
250
ns
TL1A-Ig-Treg + IgG
TL1A-Ig-Treg + TL1A-Ig
200
150
100
50
Treg:Tconv
IgG Treg
IgG Treg +TL1A-Ig
TL1A-Ig Treg
TL1A-Ig Treg+TL1A-Ig
200
150
100
50
16
1:
0
1:
16
1:
8
1:
4
1
2
8
1:
4
2
1:
0
Treg:Tconv
1:
No Treg
1:
1:
1
0
1:
250
TL1A-Ig α-CD3+
TL1A-Ig
CPM (103)
c
CPM (103)
50 50
0
0
CPM (103)
100 100
1:
1:4
4
1:
8
1:
1: 8
16
1:
16
20
150 150
1:
2
40
IgG-Treg
TL1A-Ig-Treg
200 200
1:
60
250 250
CPM (103)
WT Tconv
DN TNFRSF25 Tconv
CPM (103)
CPM (103)
80
CPM (103)
a
Figure 37. Suppressive activity of in vivo expanded Tregs. (a) WT and DN-TNFRSF25 CD4+CD25Tconv were magnetically sorted and cultured in proliferation assays in the indicated conditions for 72
hrs. (b,c) Tregs were FACs sorted from TL1A-Ig and IgG injected mice (i.p days 0, 1 and 2) on day 4
and subjected to standard in vitro suppression assays using DN-TNFRSF25 CD4+CD25- cells as
Tconv. (d) Tregs were FACs sorted from TL1A-Ig and IgG injected mice (100 ug i.p daily D0-D20 ) on
day 23 and cultured in a suppression assay using wt CD4+CD25- cells as Tconv. (a-d) Cultures were
stimulated with soluble anti-CD3 (0.5 µg/mL) and 1:1 wt APCs in the absence or presence of titrating
numbers of IgG or TL1A-Ig-Treg. (a-d) IgG or TL1A-Ig was added to the suppression assay (0.1
3
µg/mL) where indicated. (a-d) H-thymidine was added for the last 6 hr of the 72 hr incubation period
and incorporated isotope was measured by liquid scintillation counting. Data are mean ± SEM of
triplicates.
66 Notably, Tregs expanded in vivo with daily injections of TL1A-Ig for 21-days and then
subjected to in vitro suppression assays were highly suppressive for proliferation of w.t
CD4+CD25- Tconv under conditions in which the TL1A-Ig was not present (Figure
37d). The addition of TL1A-Ig to the suppression assays also partly restored the
proliferative capacity of the Tconvs.
2.3.6 Depletion of gut commensal bacteria by antibiotics treatment
To test the hypothesis that elimination of gut commensal bacteria will abrogate
TNFRSF25 mediated activation and proliferation of memory CD4+ T cells, mice were
treated with an antibiotics cocktail for a month with subsequent TL1A-Ig administration.
Briefly, mice were provided ampicillin (1 g/L), vancomycin (500 mg/L), neomycin
sulfate (1 g/L), and metronidazole (1 g/l, Sigma) in drinking water from day -25 to day 5.
On days 0-3 mice were treated with 3 consecutive doses of 100 µg of control IgG or
TL1A-Ig
a
and
sacrificed
Antibiotics cocktail in drinking water
Day -25
0
and
b
analyzed
on
day
5
(Figure
38a).
Antibiotic-treated
5
IgG or TL1A-Ig
Analysis:
Flow
Cytometry
Figure 38. Antibiotics treatment
protocol results in ceca dilation. (a)
Schematic of the experimental design
(b) Representative (TL1A-Ig treated
mouse) gross anatomy of antibiotictreated mice later treated with IgG and
TL1A-Ig and analyzed on day 5.
The gross anatomy of IgG and TL1A-Ig treated mice was very similar and
revealed a dilated ceca due to antibiotics treatment (181) (Figure 38b). The frequency of
proliferation antigen Ki-67 expressing Tconv, Treg, and CD8+ T cells was higher in the
pooled LNs and spleens of TL1A-Ig treated mice as compared to IgG treated mice
(Figure 39a) similar to the observations with experiments done without treating mice
with antibiotics (Figure 33c).
67 b
80
60
IgG
TL1A-Ig
Spleen
IgG
TL1A-Ig
40
20
0
80
60
Tconv Tregs
CD8
Lymph nodes
IgG
TL1A-Ig
40
20
0
Tconv Tregs
CD62L
%Ki67+
of T cell subset
%Ki67+
of T cell subset
Lymph nodes
Spleen
a
CD8
CD44
Figure 39. Antibiotics treatment does not abrogate TL1A-Ig mediated proliferation and
activation of memory T cells. (a) Increased numbers of T cell subsets in TL1A-Ig treated mice as
+
+
depicted by the frequency of proliferating (Ki67 ) Tconv, Treg, and CD8 T cells in the spleen and
pooled LNs. (b) Representative flow cytometry plots and compilation of percentage of memory markers
on CD4+FIR- Tconv cells harvested from spleen and pooled LN from IgG and TL1A-Ig injected mice
treated as Fig. 38a. Data are compiled percent averages of 2-3 mice per group analyzed.
Notably, treatment with antibiotics prior to costimulation with fusion protein
increased the frequency of both TCM and TEM CD4+ T cell populations in the total CD4+ T
cell population as compared to mice that were treated with IgG (Figure 39b). This
outcome suggests that removal of microbiota in the gut does not influence TL1A-Ig
mediated costimulation of memory CD4+ T cells. Increase in activation markers KLRG1
and CD103 (Figure 40a) and T cell memory markers (Figure 40b) on the FoxP3+ Treg
population was observed in TL1A-Ig treated mice as compared to controls.
68 a
b
IgG
KLRG1
5.6%
1.6%
CD103
17.2%
36.5
11.7%
22.1%
TL1A-Ig
45.6%
15.1%
5.0%
22.4%
37.9%
11.8%
29.1%
25.8%
37.3%
Figure 40. Antibiotics treatment does not abrogate TL1A-Ig mediated Treg activation and
formation of memory Tregs . (a) Representative histogram plots (spleen) for KLRG1 and CD103
expression on CD4+FoxP3+ Tregs in the spleen and pooled LN. Black: Spleen; Red: Pooled LN. (b)
Representative flow cytometric profiles and compilation of the percentage of memory markers as a
percentage of CD4+FoxP3+ Treg cells in spleen and pooled LN. Data are compiled percent averages of
2-3 mice per group analyzed.
2.3.7 TNFRSF25 costimulation and induction of ovalbumin specific Tregs in vivo
Since our previous in vitro data suggested that stimulation through TNFRSF25
abrogates induction of iTregs from Tconv cells in the presence of cognate antigen, RA,
and TGF-β, we wanted to determine if the same occurs in vivo. This protocol required the
use of splenocytes from transgenic mice with MHC Class-II restricted T-cell receptors
specific for ovalbumin (OTII mice) as a source for CD4+FoxP3- naïve T cells. Briefly,
CD4+FIR-OTII (from FIR-OTII mice) and CD4+FIR+FoxP3+ nTreg (from FIR mice)
were adoptively transferred i.v into congenic CD45.1 SJL mice on day -2. On day 0, all
mice were treated i.p with ova/alum in combination with 100 µg IgG or TL1A-Ig on days
0-3 (Figure 41a).
69 a
CD62L&
OVA/ALUM
%CD62L-CD44-/
Total OTII
300
1.5
15
200
1.0
10
100
0.3
1.0
0.2
50
0.5
2
1
0.1
Ig
Ig
G
Ig
A-
TL
1
Ig
G
0
TL
1
Ig
Ig
G
ATL
1
Ig
A-
Ig
G
TL
1
0.0
A-
0
0.0
Ig
50
100
40
90
30
80
20
70
10
60
0
50
Ig
3
A-
Ig
TL
1
0.5
0.4
1.5
50
A-
Ig
G
Ig
ATL
1
150
2.0
Ig
G
A-
Ig
G
TL
1
AIg
TL
1
Ig
G
Ig
0
0.0
60
0
A-
0.0
0
70
10
Ig
G
5
TL
1
0.5
Ig
G
mLN
80
TL
1
100
0.5
spleen
90
30
20
1.0
%EM OTII/Total OTII
100
40
2.0
1.5
%CM OTII/Total OTII
Ig
%Naïve OTII/Total OTII
A-
OTII Teff: iTreg
CD44&
Ig
2.5
Sacrifice
100 ug IgG or TL1A-Ig
A-
%iTreg/total OTII
EM&
Ig
G
b
///&
TL
1
1.5 x 106 OTII-FIR0.5 x 106 nTreg-FIR+
5
Ig
G
0
CM&
TL
1
-2
Naive&
Figure 41. TL1A-Ig does not abrogate the induction of Tregs in vivo. (a) Schematic of the
experimental design and markers on effector memory (EM) and central memory (CM) cells (b) IgG
and TL1A-Ig treated mice were sacrificed on day 5 and harvested mLNs, pLNs (data not shown),
and spleens were analyzed for the frequencies of ova-specific OTII Tregs (gated on CD45.1TCRα+β+CD4+FoxP3+) and ova-specific OTII Tconv (pre-gated CD45.1-TCRα+β+CD4+FoxP3-)
memory subsets in the total OTII compartment. OTII specific Teff:iTreg ratios were also
calculated. Data are mean ± SEM with each dot representing a value from one mouse.
All mice were sacrificed on day 5, single cell suspensions were made from harvested
mLNs, pLNs (data not shown), and spleens, and cells were analyzed for ova-specific
OTII Tregs (gated on CD45.1-TCRα+β+CD4+FoxP3+) and ova-specific OTII Tconv
(CD45.1-TCRα+β+CD4+FoxP3-) on memory subsets as defined in Figure 41a.
Interestingly, TNFRSF25 costimulation given at the same time as cognate antigen (OVA)
does not abrogate the induction of iTreg (Figure 41b). In the spleen, the % iTreg in the
total OTII cell population was 1.0% in IgG treated mice, which significantly increased to
1.7% in the TL1A-Ig treated mice. TNFRSF25 stimulation by TL1A-Ig resulted in the
reduction of the ovalbumin-specific Teff to Treg ratio in the spleen from 100 in control
70 IgG treated mice to 50 in TL1A-Ig-treated mice. These data indicate that in the presence
of cognate antigen, TL1A-Ig mediated TNFRSF25 expansion of Tregs may effectively
suppress an antigen-specific effector response as indicated by the skewing of the antigen
specific Teff:Treg towards a regulatory phenotype and away from effector dominance.
Although induction of iTregs was observed in the mLN, there was no difference in the
%iTreg of total OTII cells or the ova-specific Teff:Treg ratio between IgG and TL1A-Ig
treated mice. The percentage of TEM cells in the total OTII cell population increased in
the spleens and mLN of TL1A-Ig treated mice.
2.3.8 Suppression of allergic lung inflammation by TL1A-Ig expanded Tregs
We have shown previously that in vivo stimulation of TNFRSF25 by the agonistic
antibody 4C12 expands Tregs in ovalbumin-sensitized mice resulting in suppression of
allergic lung inflammation upon exposure to aerosolized ovalbumin (130). We sought to
determine whether the activity of TL1A-Ig is sufficiently specific to Treg in the acute
allergic lung inflammation model to prevent airway pathology. A modified therapeutic
protocol
was
Day
developed
5
OVA/ALUM i.p.
based
on
the
10
findings
15
TL1A-Ig or OVA/PBS
Aerosol
IgG i.p.
of
4C12
(Figure
42).
20
Analysis:
BALF
Histology
Flow Cytometry
Figure 42. Therapeutic protocol for the acute model of allergic lung inflammation.
Naïve FIR mice were primed on day 0 and boosted on day 5 with ovalbumin adsorbed to
ALUM adjuvant. Starting on day 11, ovalbumin-sensitized mice were injected with either
TL1A-Ig or mIgG isotype control i.p for three consecutive days. Mice treated with
71 TL1A-Ig showed significant expansion of CD4+FoxP3+ Tregs in the peripheral blood,
with a peak approximately 4-5 days after the first administration with TL1A-Ig or IgG
(Figure 43a). On day 16, all mice were exposed to aerosolized ovalbumin in saline for 1
hour, and three days later, day 19, analyzed to evaluate the Tregs in the lungs and
spleens. The frequency of FoxP3+ Tregs was significantly increased in single-cell
suspensions of lung tissue and spleens of mice treated with TL1A-Ig (Figure 43b).
b
Lung
60
40
20
g
-I
A
1
L
T
4
3
2
***
1
g
-I
G
A
g
A
-I
G
T
1
L
L
e
1
ro
Ig
l.
0
so
g
A
G
l.
ro
e
-a
n
o
N
0
Lung
5
T c o n v :T re g
50
-I
G
*
100
L
Ig
so
g
A
1
L
T
150
1
Ig
T
d
o
N
N
o
n
-a
e
ro
so
l.
0
20
-I
G
l.
Ig
so
ro
e
-a
n
o
N
4
ns
50
N o . T re g c e lls (1 0 )
100
40
0
Lung
-a
150
***
60
n
4
N o . T c o n v c e lls (1 0 )
Lung
80
0
11 12 13 14 15
D a y s a fte r O V A /a lu m
c
T o ta l C D 4
***
80
% FoxP 3+/
25
T o ta l C D 4
% FoxP 3+/
50
0
Spleen
100
100
T
Ig G
T L 1 A -Ig
Ig
Blood
75
T o ta l C D 4 +
% FoxP 3+/
a
Figure 43. TL1A-Ig mediated Treg expansion in the peripheral blood, lungs and spleens. (a)
+
+
Peripheral blood was collected and analyzed for FoxP3 cells gated in CD4 T cells from OVA/alum
immunized mice treated with either control IgG or TL1A-Ig on days 11, 12, and 13. Black arrows
indicate injection of IgG or TL1A-Ig. (b) Total lung cells and splenocytes were harvested and analyzed
+
+
for the frequency of FoxP3 Treg cells in the CD4 T cell compartment on day 19. (c) The total
+
numbers of CD4 FoxP3 Tconv and CD4+FoxP3+ Tregs in the lung tissue. (d) TL1A-Ig decreases
Tconv/Treg cell number ratio in the lung tissue. Data are mean ± SEM of 2 independent experiments
with each dot representing a value from one mouse (n=6-8). *P < 0.05 ***P < 0.001, 1-way ANOVA
with Tukey post-test.
Treg analysis in the lung tissue revealed that the frequency of Treg cells was 66% of all
CD4+ T cells in TL1A-Ig treated mice as compared to 25% in control IgG-treated mice.
The number of CD4+FoxP3- Tconv cells within the lungs was similar between control
72 IgG and TL1A-Ig treated mice, while in TL1A-Ig treated mice the number of Tregs was
significantly increased (Figure 43c). Several studies have reported that the balance
between Tconv to Tregs is a better determinant of disease pathogenesis rather than the
absolute number of Treg cells (182, 183), hence the Tconv:Treg ratio was calculated in
the lung tissue (Figure 43d). The average Tconv:Treg ratio in the lung tissue decreased
from 2.8 to 0.5 in TL1A-Ig treated mice.
Analysis of BALF recovered from TL1A-Ig mice sacrificed on day 19 resulted in
observations similar to treatment with anti-TNFRSF25 antibody, 4C12 (130).
IgG
control-treated mice showed significant eosinophilia in the total cells recovered from
BALF as compared to TL1A-Ig treated mice, both in the percentage and absolute number
of eosinophils recovered (70.4% of BALF and 400x103 cells, Figure 44). In mice that
were treated with TL1A-Ig, analysis of BALF showed a significant decrease in both the
percentage and number of eosinophils recovered, as compared to IgG control-treated
(17.2%
60x103
and
cells,
***
25
0 .5
*
e
-a
g
-I
G
A
1
L
ro
A
1
L
T
o
N
N
o
n
n
T
l.
so
g
-I
G
Ig
so
ro
e
1 .0
0 .0
l.
0
-a
1 .5
6
in B A L F (1 0 )
T o ta l e o s in o p h ils
75
50
44).
BALF
100
in B A L F
% e o s in o p h ils
BALF
Figure
Ig
mice
Figure 44. TL1A-Ig treatment reduces eosinophils in the BALF. Bronchiolar lavage fluid was
harvested 3 days(day 19) after aerosolization with OVA/PBS, The percentage (left) and absolute number
(right) of eosinophil are shown. *P < 0.05, ***P < 0.001, 1-way ANOVA with Tukey post-test. n=6-8
To determine whether the therapeutic expansion of Tregs by TL1A-Ig prior to
allergen aerosol affects histopathology, formalin-fixed lung sections from control and
73 TL1A-Ig treated mice were obtained for histology on day 19. H&E and PAS staining of
lung tissue revealed reduced lymphocyte infiltration and airway mucus secretion in
TL1A-Ig-treated animals as compared to OVA sensitized/aerosolized IgG-treated mice
(Figure 45a). PAS staining is a marker for mucus production in goblet cells.
Quantification of images from PAS-stained slides using ImageJ software showed a large
number of PAS positive cells within the bronchiolar epithelium in control IgG treated
mice as compared to TL1A-Ig treated mice that showed significant reduction in the
number
a
of
positive
cells
(Figure
45b).
b
IgG
TL1A-Ig
100
H&E 200x
No. PAS+ Cells
Saline Aerosol
PAS
***
80
60
40
20
nv
PAS
200x
O
O VA
VA /
/P PB PBS
BS S
+
+
I
TL gG
1A
-Ig
0
Figure 45. TL1A-Ig treatment reduces lymphocyte infiltration and airway mucus secretion in the
lungs (a) Images from formalin fixed and paraffin embedded lung sections from OVA
sensitized/aerosolized mice treated with IgG or TL1A-Ig and analyzed on day 19. Representative H&E
and PAS image at 200x for each treatment group. (b) Images from PAS stained lung sections were
quantified using Image J software as described in Methods. The number of PAS positive cells per
bronchiole in IgG and TL1A-Ig treated mice is shown. 2 or more representative images were quantified
from each treatment group.
2.3.9 Prolongation of allogeneic skin transplant by TL1A-Ig expanded Tregs
Recently much effort has been focused on targeting TNF members as therapeutic
agents in transplantation. Dr. Wolf et al. reported that TNFRSF25 mediated expansion of
endogenous Treg population by 4C12 prior to transplantation results in prolongation of
cardiac allograft survival in a mouse model of fully major histocompatibility complexmismatched ectopic heart transplants (146). We were then interested in testing the
74 efficacy of TL1A-Ig in a stringent murine allogeneic skin transplantation model.
Although several immunosuppressive therapies are available, acute rejection is prevalent
in many patients that receive composite tissue allografts (CTAs). The skin is usually the
first, if not the only tissue, to reject, making it the most difficult component of a CTA.
To test if pre-treatment with TNFRSF25 agonists can prolong survival of a full mismatch
skin transplant, recipient B6 mice were injected with one 10 ug dose of 4C12 (day -4) or
daily 100 ug dose of TL1A-Ig (day -4 until day of graft rejection) (Figure 46a).
a Day
-4
0
5
15
10
Hamster IgG or 4C12
mIgG or TL1A-Ig
Remove bandage
Analyze for Treg frequency in blood
Allogeneic skin transplant Balb/c!B6
100
Percent survival
b P=0.04&
hamster IgG
4C12
mouse IgG
mTL1A-Ig
80
60
*P=0.04&
*P=0.04&
40
20
0
0
5
10
15
20
Graft survival (Days)
Figure 46. TNFRSF25 agonists prolong allogeneic skin grafts. (a) Protocol for the allogeneic
skin transplant model. FIR (C57BL/6 background) mice were injected with control antibody or
TNFRSF25 agonists (20 µg hamster IgG or 4C12 or 100 µg mIgG or TL1A-Ig i.p) on days indicated.
In vivo Treg levels were determined on day 0 and allogeneic (Balb/c) ear skin was grafted onto the
right thorax of control and TNFRSF25 agonist treated recipients. Bandages were removed on day 6
and skin grafts were monitored for rejection. TL1A-Ig was administered daily until the day of
rejection. Grafts were scored as rejected when more than 75% of the grafted tissue area had been lost.
(b) Survival curves of mice undergoing allogeneic skin transplants .
On day 0 (day of transplantation), mice were bled and the frequency of endogenous Treg
in the 4C12 and TL1A-Ig treated mice was confirmed to be 30-35% of the total CD4+ cell
population. The median graft survival for 4C12 and TL1A-Ig treated mice, determined by
visually observing donor skin necrosis, was 14 and 13 days, respectively (Figure 46b).
75 This median survival was significantly longer than median survival of 8 days for IgG
controls (p=0.04). Daily TL1A-Ig treated mice maintained an elevated endogenous Treg
population on day 9 post-transplantation (data not shown), however, the larger Treg
population did not prolong graft survival as compared to 4C12 treated mice.
CHAPTER 3: Discussion
The importance of Tregs in the control of several disease settings, including
autoimmunity, asthma, allergy, and transplantation, is very well established. Tregs have
the capacity to actively block immune responses, inflammation, and tissue destruction by
suppressing the function of various cell types including CD4+ effector T cells, B cell
antibody production and affinity maturation, CD8+ CTL granule release, and APC
function and maturation state (184-186). Several pharmacological and cellular therapies
have been investigated that shift the overall balance in the immune system to favor Treg
mediated immune regulation. Some of the drugs used and being developed promote Treg
development and survival in vivo, while others target the cells themselves, using
therapeutic approaches to isolate and expand polyclonal or antigen specific Tregs for
immunotherapy. These approaches have important implications in the potential use of
drug therapy that promote Treg in patients with autoimmunity or allergy and adoptive
transfer of Tregs to correct an immunological balance or promote survival of future
transplanted tissues. TL1A-Ig mediated in vivo Treg expansion and maintenance of the
immunosuppressive functional cell subset with daily injections circumvents the need to
expand Tregs in vitro for cellular therapy, an approach that may be problematic due to
practical limitations in the isolation and expansion of sufficient numbers of these cells for
clinical use.
The majority of therapeutic approaches to enhance immune regulation have
focused on generalized immunosuppression rather than targeting Treg specific markers
with heterologous antibodies (non-human derived products) that result in undesirable side
effects such as serum sickness and depletion of multiple cell types resulting in prolonged
76 77 lymphophenia. Efforts have been focused on designing monoclonal antibodies targeting
cell specific markers including studies of humanized mAbs directed at the CD3-ε chain of
the TCR complex. Anti-CD3 treatment altered the Teff/Treg ratio balance through a
combination of activation induced cell death of Teff cells, selective inactivation of Th1
cells (187, 188), while sparing nTregs and TGF-β dependent iTregs (189). However,
treatment with such targeting reagents leads to depletion of most lymphocytes that results
in uncontrolled global immunosuppression. Our laboratory’s previous study showed that
in vivo administration of TNFRSF25-agonist 4C12 induced the proliferation of preexisting Tregs, and similar observations with the TL1A-Ig fusion protein support the
hypothesis that specific TNFRSF25 signaling mediated the expansion of Tregs to
therapeutic levels, and this phenomenon is not a consequence of unexpected off-target
effects of 4C12. However, the non-antigenic fusion protein has an advantage over
heterologous 4C12 since repeated administration of TL1A-Ig maintains elevated Treg
levels in vivo. Unlike 4C12, hexameric TL1A-Ig may be able to crosslink multiple
TNFRSF25 receptor complexes that enhances TNFRSF25 signaling allowing the fusion
protein to induce Treg proliferation in vitro.
A summary of comparative studies of 4C12 and TL1A-Ig indicates that both
TNFRSF25 agonists have varying outcomes in vitro and in vivo (Table 3). Similarly,
OX40 agonist antibody, OX86, has also been reported to expand Tregs in naïve mice
(190) yet fails to induce Treg proliferation in vitro (124). Studies have suggested that
TNF agonist antibodies and ligands can stimulate different components of the signaling
pathway (166) or totally different signaling pathways (165), hence there are limitations of
studying
physiological
pathways
using
receptor
agonist
antibodies.
78 Table 4: Summary of comparative studies of 4C12 and TL1A-Ig
Similarities
Differences
In vitro
In vivo
1. 
Block de novo biogenesis of induced Tregs
1. 
2. 
Induce caspase activation and apoptosis in
TNFRSF25-transfected cells
Mediate in vivo expansion of Tregs such that
they become 30-35% percent of the total
CD4+ T cells
2. 
Do not expand Tregs in MHC II deficient
mice, indicating requirement of TCR
activation prior to TNFRSF25 costimulation
3. 
In vivo expanded Tregs are highly suppressive
for Tconvs proliferation ex vivo
4. 
Therapeutic pre-expansion of Tregs in vivo
p r o t e c t s m i c e a g a i n s t a l l e rg i c l u n g
inflammation
1. 
TL1A-Ig in combination with IL-2 induces 1. 
the proliferation of polyclonal and induced
Tregs.
Unlike 4C12, daily injections of TL1A-Ig
maintains elevated levels of Tregs for at least
20 days.
2. 
TL1A-Ig in combination with anti-CD3 and 2. 
IL-2 induces the proliferation of Tconvs.
3. 
4C12 fails to induce proliferation of CD4+
T cells.
Unlike 4C12, TL1A-Ig administration
significantly increases cellularity and absolute
numbers of CD3+, CD4+ and CD8+ cells in
lymph nodes.
3. 
Unlike 4C12, TL1A-Ig treatment increases
expression of activation markers and memory
markers on both Tconvs and Tregs in the
spleen and lymph nodes.
The explanation may be because of the qualitative differences between the agonist
antibody and the natural ligand. The ligand aggregation model and the crystal structure of
TL1A (191), suggests that ligand aggregates or clusters three receptor fragments,
resulting in strong stimulation that activates intracellular effector molecules. Based on
structural studies, the receptor-binding interface appears to involve the inner surface of
the groove formed between two adjacent TNFRSF25 subunits. The ligand interacts with
the receptor in a symmetrical fashion, where the receptor runs parallel to the ligand with
the face-to-face interaction surfaces are distributed evenly along the upper and lower
parts of the ligand (171, 172) whereas, the agonist antibody is expected to aggregate as
many as two adjacent receptors back-to-back resulting in a different 3-dimensional
interaction. According to our hydrodynamic analysis, the TL1A-Ig construct is a dimer of
79 TL1A-trimers. Each trimer will be able to interact with each subunit of TNFRSF25 and
crosslink two receptor complexes. This difference in binding sites and ability to crosslink
may account for some of the differences observed between TL1A-Ig and 4C12 in vitro
and in vivo. Both TNFRSF25 agonists induced apoptosis in transfected tumor cells
overexpressing TNFRSF25. TL1A-Ig, however, is effective at a 100-fold lower
concentration, suggesting higher affinity and/or effects of cross-linking.
To evaluate the requirements of TL1A-Ig mediated proliferation of CD4+T cells
in vitro, highly purified CD4+CD25- Tconv or CD4+FoxP3+ Treg cells were cultured in
several different conditions. We demonstrated that 4C12 is a poor costimulator of antiCD3 activated CD4+ Tconv in vitro, while TL1A-Ig is quite effective with soluble antiCD3. As reported previously, 4C12 does not costimulate proliferation of Treg in vitro
while TL1A-Ig is quite potent, however strictly in dependence of added IL-2 and TL1AIg. In contrast, both the agonist antibody to GITR and Fc-GITRL fusion protein (14)
function as costimulatory molecules for T-cell activation in vitro since the GITRL
associates and activates GITR as a dimer (192) and not a trimer. An interesting extension
of TNFRSF25 studies is to determine if the addition of 4C12 crosslinking antibodies
results in proliferation of both Tconv and Treg in vitro. Interestingly, TL1A-Ig mediated
Treg proliferation occurred in the absence of soluble anti-CD3 and was abrogated with
the addition of cyclosporine A. Moreover, “resting” Tregs proliferate more robustly in the
presence of soluble anti-CD3, IL-2, and TL1A-Ig as compared to IL-2 and TL1A-Ig only.
These observations suggest that prior TCR signaling by self-antigens was maintained in
Treg cells that enabled subsequent TNFRSF25 mediated proliferation in vitro. An
alternative approach to test this hypothesis is to determine whether the proliferation of
80 freshly isolated antigen-specific CD4+Foxp3+ OTII Tregs is dependent on the presence of
OVA in addition to IL-2 and TL1A-Ig in culture.
In vivo we find that both 4C12 and TL1A-Ig potently costimulate CD4+FoxP3+
Treg expansion with similar kinetics. As suggested by the increase in the ratio of the
CD4+FoxP3+CD25hi Treg to CD4+FoxP3+CD25int, the majority of Ki67+ cells were
CD4+FoxP3+CD25int in mice treated with TL1A-Ig. However, TL1A-Ig mediated
expansion of CD25int cells (49% of CD4+FoxP3+ Treg gate) was not as robust as reported
with 4C12 treatment (75% of CD4+FoxP3+ Treg gate) (130). Unlike 4C12, approximately
3-fold increase in Ki67 expression was observed in both CD25hi and CD25int Treg
populations with TL1A-Ig treatment. TL1A-Ig and 4C12 treatment resulted in a smaller
portion, 14% and 27% of total CD4+FoxP3+ Treg cells respectively, that did not stain for
Ki67. It is unclear whether the relative boost in CD25hi Treg cells proliferation following
TL1A-Ig treatment as compared to 4C12 treatment was due to the availability of IL-2
produced by proliferating Tconv, which was not observed with 4C12 administration.
To evaluate whether in vivo TNFRSF25-induced Treg proliferation was
dependent upon TCR engagement with MHC class II, total CD4+ T cells (comprising of
10% FoxP3+ Treg) were adoptively transferred into MHC II deficient mice and treated
with TL1A-Ig. In vitro and in vivo abrogation of fusion protein mediated Treg
proliferation due to disrupted signaling downstream of TCR (with cyclosporine) or the
absence of MHC II implicates that TCR recognition of self-antigen is a prerequisite to
TNFRSF25 costimulation.
Our observations using both TNFRSF25 agonists, TL1A-Ig and 4C12,
81 complement data reporting that TNFRSF25 signaling mediates activation and
proliferation of both opposing cell types, Tconv and Treg, in the presence of cognate
antigen (41, 106, 130, 152). Self-peptide activated Treg cells were costimulated in vivo
by 4C12 in the absence of foreign antigen, while treatment with TL1A-Ig simultaneously
expanded a population of CD4+ T memory cells (TM). Meylen et al. reported that
transgenic overexpression of TL1A on T cells results in the spontaneous development of
inflammatory small bowel pathology. To determine whether the costimulation and
intestinal inflammation triggered by TL1A is dependent on T cells recognition of
microflora or other environmental mucosal antigens, they restricted the T cell repertoire
of TL1A transgenic mice to a monoclonal specificity by back-crossing the CD2-TL1A
transgenic line (transgenic overexpression of TL1A on T cells) to the OT-II ovalbumin
specific TCR transgene. Transgenic expression of TL1A in this background was not able
to increase the percentage of memory T cells and revealed a significant amelioration of
TL1A-dependent intestinal changes (128). These observations and our data indicating
costimulation of TM cells after TL1A-Ig injections in vivo support two hypothesis: 1)
TL1A-Ig mediates a stronger signal through TNFRSF25 as compared to 4C12, inducing
TM costimulation with suboptimal TCR activation provided by low concentration of
microbial/environmental antigens. Our in vitro Tconv proliferation assays also report that
TL1A-Ig, and not 4C12, induces the proliferation of Tconv that is dependent on soluble
anti-CD3. 2) TL1A-Ig treatment results in a larger and more immunosuppressive Treg
population that interferes with TM expansion and function. The absolute numbers of Treg,
total Tconv, TCM and TEM in response to TL1A-Ig stimulation in mice clearly reveal that
Treg expand more extensively than Tconv resulting in a reversal in the (TCM+TEM):Treg
82 and Tconv:Treg ratio in favor of Treg. Although memory CD4+ T cells proliferate due to
TL1A-Ig, this effect is more than off-set by the larger Treg proliferation and expansion
resulting in an overall immunosuppressive effect of TL1A-Ig administration in vivo as
shown in the acute lung inflammation model. Additionally, we did not observe any
physical signs of inflammation, such as weight loss, diarrhea, or dermatitis in naive mice
that were treated with TL1A-Ig with 3 daily consecutive doses or daily for a 20-day
period. Histopathology also failed to detect cellular infiltration or inflammation in any
organ. In studies investigating suppressive activity in vitro, Tregs harvested from TL1AIg injected mice significantly abrogated the proliferation of Tconv cells. An increase in
αEβ7 integrin (CD103) and killer cell lectin-like receptor G1 (KLRG1) indicating
activation, as well as, T-cell memory markers, were also observed in FoxP3+ Tregs in the
spleen and LNs of TL1A-Ig treated mice (178-180).
To test the hypothesis that elimination of gut commensal bacteria will abrogate
TNFRSF25 mediated activation and proliferation of memory CD4+ T cells, mice were
treated with an antibiotics cocktail prior to treatment with TL1A-Ig and subsequent
analysis of memory CD4+ T cells in the spleen and lymph nodes. Interestingly, Bollyky et
al. reported that they were able to culture limited numbers of viable fecal bacteria four
weeks after the initiation of antibiotics (193). The persistence of memory CD4+ T cell
costimulation with TL1A-Ig administration may be explained by activation provided by
the remaining commensal bacteria in the gut that are sufficient for subsequent TNFSF25
costimulation. Furthermore, there has been extensive debate about the role of persisting
antigen required for the maintenance of CD4+ T cell memory (194, 195) and it is now
generally accepted that memory CD4+ T cell maintenance is antigen-independent. Hence
83 it is possible that depletion of commensal antigen does not affect the costimulation ability
of pre-existing CD4+ memory T cells. An interesting extension of these studies would be
the treatment of OTII Rag -/- transgenic mice with TL1A-Ig and analyzing the absolute
counts and activation status of ova-specific CD4+ OTII cells. We would expect the
expansion of self-peptide activated natural Treg without costimulation of CD4+ Tconv
cells.
In the iTreg induction studies, we sought to address whether costimulation of
TNFRSF25 abrogates the generation of iTregs. In vitro, activation of naïve CD4+ T cells
by anti-CD3 or specific antigen in the presence of TGF-β, retinoic acid (RA) and IL-2
results in the induction of FoxP3 in a substantial proportion of cells. Our studies indicate
that Foxp3 induction is partially suppressed by the TNFRSF25-agonist 4C12 and more
completely by TL1A-Ig similar to OX40L (119, 120, 176). We also examined the role of
TNFRSF25 costimulation in the induction of Treg in vivo. In these studies antigen
specific CD4+FoxP3- Tconv cells were adoptively transferred into recipient mice that
were subsequently treated with OVA/ALUM and TL1A-Ig or IgG. Surprisingly, there
was induction of iTreg in mice treated with TL1A-Ig although the cognate antigen and
TNFRSF25 costimulation were provided at the same time. Moreover, the frequency of
splenic iTreg in the total CD4+ T cell population was higher in TL1A-Ig treated mice
compared to IgG treated controls suggesting that TNFRSF25 costimulation also induced
proliferation of newly generated iTregs. The discrepancy between in vitro and in vivo
observations may be explained by the 2-day hiatus between the last dose of TL1A-Ig (day
2) and analysis (day 5) that could have resulted in TL1A-Ig depletion in the Tconv cell
microenvironment permitting TGF-β dependent iTreg induction and proliferation. An
84 extension of investigating requirements for TNFRSF25 abrogation of Treg induction is to
determine an in vivo treatment schedule that will replicate conditions maintained in vitro.
The murine asthma studies, performed in collaboration with Dr. Matthew Tsai,
indicate that both agonists, 4C12 (130) and TL1A-Ig, work similarly in the presence of
foreign antigen. In allergic lung inflammation, an inflammatory disease that is mainly
driven by CD4+ T cells (144, 145), treatment with either TNFRSF25 agonists prior to
airway antigen challenge (ova aerosolization) induced the preferential accumulation of
Tregs, but not Tconvs, within the lungs and reduced eosinophilia and mucus production
in the bronchoalveolar space.
There was no significant difference in the absolute
numbers of Tconv population in the lungs of ovalbumin-sensitized mice treated with
TL1A-Ig or IgG and analyzed 3 days after aerosolization. These observations are
consistent with 4C12 treated mice compared to hamster isotype control treated IgG mice
(130), suggesting that TL1A-Ig, like 4C12, administered during non-inflammatory setting
specifically induces a 4.6-fold induction of Treg cells without increasing the absolute
number of Tconv cells in the lungs, resulting in a lower Tconv:Treg ratio predictive of
reduced disease pathogenesis (182, 183). Since the mice were exposed to OVA antigen
prior to and post treatment with TL1A-Ig, we were expecting that TCR signals provided
by OVA antigen will permit TNFRSF25 mediated Tconv costimulation. However, that
was not the case. The preferential expansion of Tregs and not Tconv may be accountable
by the following possibilities: 1) Induction of antigen-specific Tregs after OVA/ALUM
priming can (directly or via dendritic cells) interfere with subsequent Tconv proliferation.
2) OVA antigen was completely cleared or at present at very low concentration that may
85 be insufficient for Tconv costimulation, while polyclonal Tregs are continuously TCRactivated with self-peptide. 3) Tregs maintain TCR-activation/signaling from prior
stimulation for a longer period of time as compared to Tconv, hence giving them a
“head” start when costimulated by TL1A-Ig subsequently. This possibility is similar to
possibility 2 since it suggests a lower TCR-activation threshold for TNFRSF25
costimulation. Possibility 3 is supported by our polyclonal in vitro Treg assays showing
that TL1A-Ig mediated proliferation of Treg cells is only dependent on IL-2 and TL1A-Ig
and is abrogated with cyclosporine suggesting residual TCR-activation from in vivo selfantigen exposure.
Currently we have ongoing experiments to investigate the affect of TL1A-Ig on
antigen specific CD4+ T cell memory response and tolerance induction. We hypothesize
that ova-sensitized mice treated with TL1A-Ig will have a reduced/non-existing ovaspecific Tconv memory response, resulting in an ova-specific Tconv:Treg ratio favoring a
regulatory over inflammatory phenotype. Briefly, highly FACs purified CD45.2 OT-II
cells were adoptively transferred i.v into congeneically marked CD45.1 mice on day -2.
On days 0 and 5 mice were immunized with OVA/ALUM followed by treatment with
control IgG or TL1A-Ig on days 12, 13 and 14. Mice were allowed to rest until day 40 on
which they were immunized with OVA/ALUM. On day 45, mice were sacrificed and the
peripheral lymph nodes, mesenteric lymph nodes, and spleens were harvested for
analysis. Although the transferred transgenic OTII cells were detectable in the peripheral
blood on day 5 after the second treatment with OVA/ALUM, we failed to detect OTII
cells on the final day of analysis (day 45). We will therefore utilize magnetic bead
86 separation instead of FACs sorting to isolate the CD45.2 OTII to increase number and
quality of cell transferred into recipients.
In the murine model of allogeneic skin transplantation, we sought to determine
whether the in vivo expansion of Treg by administration of TNFRSF25 agonist, 4C12 or
TL1A-Ig, prior to transplantation prolongs the survival of an allogeneic skin transplant.
Recent studies in our lab reveal that TNFRSF25 mediated expansion (by 4C12) of
endogenous Treg population prior to transplantation results in prolongation of cardiac
allograft survival in a mouse model of fully major histocompatibility complexmismatched ectopic heart transplants (146). In the allogeneic skin transplant model,
TL1A-Ig and 4C12 mediated expansion of Tregs was associated with a significant
prolongation of median graft survival from 8 days to 15 days. Daily administration of
TL1A-Ig post transplantation maintained elevated level of Treg in vivo, however, they
did not extend the graft survival as compared to 4C12 treated mice. There is increasing
evidence suggesting that lack of vascularization of skin allografts at the time of their
placement is responsible for increased immunogenicity of these grafts by favoring skin
dendritic cell trafficking via lymphatic vessels that migrate to draining lymph nodes and
activate T cells, while preventing some regulatory immunity requiring blood circulation.
It is possible that alloantigen specific effector T cells compete with TNFRSF25-expanded
Treg to the graft site and augment inflammation caused by an earlier innate immune
response. It is likely that combination therapy using TL1A-Ig with reagents that block
effector T cell activation and proliferation, such as rapamycin, may provide therapeutic
benefit in allogeneic skin transplantation.
The functional efficiency of TL1A-Ig in murine models can readily be translated
87 for human disease by constructing human TL1A-Ig in an analogous fashion. Rigorous
experimental verification to that effect is ongoing in mice with a human immune system
to determine whether human TL1A-Ig is sufficiently Treg-specific in vivo to warrant
further clinical development. Since these studies require a large quantity of TL1A-Ig, we
have established the mammalian cell lines using the GS gene as a selection and
amplification marker. Bebbington et al. (170) first reported the use of this selectable
marker in myeloma cells in which transfectants, expressing GS and a chimeric antibody,
were selected by culturing in a glutamine-free medium. Subsequently, vector
amplification was selected by adding glutamine synthetase inhibitor, MSX. Other popular
cell lines used for the production of antibodies and recombinant proteins include NSO
(mouse myeloma cells), CHO (chinese hamster ovarian cells), and HEK-293 (human
embryonic kidney cells). We have created this technology in our laboratory using CHO
cells and selected high producing clones that can accumulate up to 4.4g/L of TL1A-Ig in
a hollow-fiber cartridge system. Because TNFRSF25 signaling has the unique ability to
expand and transiently inhibit Treg suppressive function, we are also investigating the
potential use of this reagent in modulating immune responses in disease models of
transplantation, autoimmune disease, chronic infection and cancer to benefit clinical
outcome.
CHAPTER 4: Material and Methods
4.1 Mice
Wild-type C57BL/6 mice were purchased from Jackson Laboratories. The FoxP3 reporter
mice on C57BL/6 background (FIR mice) were bred in our animal facility (provided by
R. Flavell, Yale University, New haven, Connecticut, USA; ref (175)). Congeneic
CD45.1 SJL, CD4-/-, CD74 -/-, OTII, and Balb/c mice were also bred in our animal
facility. Mice were used at 6-12 weeks of age and were maintained in pathogen-free
conditions at the University of Miami animal facilities. The University of Miami Animal
Care and Use Committee approved all the animal procedures used in this study.
4.2 Antibodies and reagents
Intracellular
Ki67
and
FoxP3
stainings
were
performed
using
mAb
and
fixation/permeabilization reagents from eBioscience (San Diego, CA). IgG isotype
controls were purchased from BioLegend (San Diego, CA). All commercial antibodies
used for flow cytometry staining were purchased from eBioscience, BD BiosciencesPharmingen (San Jose, VA), Biolegend, and Jackson ImmunoResearch (West Grove,
PA). Recombinant mouse IL-2 was purchased from R & D systems (Minneapolis, MN).
Armenian hamster hybridoma producing antibody to mouse TNFRSF25 (agonist 4C12)
was maintained as described previously (106). 4C12 (anti-TNFRSF25 agonist) and L4G6
(anti-TL1A blocking mAb) were produced in hollow fiber bioreactors (Fibercell
Systems) and purified from serum-free supernatants on protein G column using PBS for
elution. Cyclosporin-A was purchased from Calbiochem/EMD.
88 89 4.3 Plasmid Construction
The mTL1A cDNA was purchased from OriGene. Varying lengths of the mTL1A
were PCR amplified using primers that were located upstream and downstream of the
cloning sites. All constructs had downstream primer 5’CATGCGGCCGCAATA
TCGTTCTT-3’ paired with a unique upstream primer for each construct: TL1A-Ig full
(65-252 aa): 5’CATGAATTCCGGGTCCCCGGAAAAGACTG-3’, TL1A-Ig -3aa(68252 aa): 5’- CATGAATTCGGAAAAGACTGTATGCTTCG-3’, TL1A-Ig -5aa (70-252
aa): 5’- CATGAATTCGACTGTATGCTTCGGGCCAT-3’, and mTL1A-Ig -11aa (76252 aa): 5’CATGAATTCATAACAGAAGAGAGATCTGAGCC TTCACC 3’. The
mouse IgG kappa chain leader sequence was ligated to the 5’end of the cDNA encoding
the Fc portion of murine IgG1, containing the hinge, CH2, and CH3 regions. IgG1 had
been mutated to replace the three cysteines of the hinge portion to serines (169), with the
aim of reducing the antibody-dependent cellular cytotoxicity (ADCC) that can occur due
to binding of IgG1 to Fcγ receptors (196). Each of the four TL1A PCR amplified
products mentioned above were ligated to the 3’end of the cDNA encoding the Fc mIgG1
fragment, resulting in four final constructs containing the leader sequence, hinge-CH2CH3, and TL1A ECD. The final TL1A-Ig cDNA was cloned into the pBMGs-neo
expression plasmid (pBMGs-neo-TL1A-Ig). The correct primary structure was verified
by sequencing for each construct.
The -3aa ECD TL1A-Ig (aa 68-252) cDNA and murine glutamine synthetase
(mGS) cDNA were inserted into two independent expression cassettes of the plasmid
pVitro2-hygro (Invivogen) using the PCR strategy. Briefly, SGRA1 and NHE1 restriction
sites were added to the flanking regions of TL1A-Ig-3aa in the pBMGs-neo-TL1A-Ig
90 expression plasmid. TL1A-Ig was cut, and ligated into the second multiple cloning site of
pVitro2-mGS, which had the mGS cDNA inserted in the first multiple cloning site
(pVitro2-GS-TL1A-Ig expression plasmid). The mGS cDNA was purchased from
OriGene, and the open reading frame (ORF) was PCR amplified with primers containing
the MLU1/SAL1 restriction sites.
4.4 Cells and Culture Conditions
3T3-NIH cells and CHO-K1 cells were purchased from the American Type Culture
Collection (Manasas, VA). Untransfected cells were cultured in Iscove's Modified
Dulbecco's Medium (IMDM) supplemented with 10% bovin serum (BS) (3T3-NIH cells)
or 10% fetal bovine serum (FBS) (CHO cells) and 0.5 mg/mL gentamicin (Invitrogen).
4.5 Transfection and selection
Lipofectimine reagent was used to transfect the pBMGs-neo-TL1A-Ig expression
plasmid into 3T3-NIH cells according to the vendor’s protocol (Invitrogen). The doublecassette pVitro2-GS-TL1A-Ig expression plasmid expressing TL1A-Ig and mGS was
first transfected into adherent CHO-K1 cells (cultured in 10% FBS IMDM) using
electroporation. The following conditions were applied while using the Gene Pulser
apparatus for transfection: 0.4 cm cuvette gap, 0.75 kV, 25 µF and 0.5 msec time
constant. After electroporation the cells were cultured in 10% FBS IMDM media and
equally distributed into a 6-well plate (Costar). At 48 hours, the cells were re-transfected
with the same plasmid using standard procedures for the Effectene reagent (Qiagen) and
subjected to selection pressure when cultured in 10%FBS IMDM supplemented with 1
mg/mL hygromycin. Transfectants were single-cell cloned in 96-well round-bottomed
plates (Costar) and subjected to hygromycin selection to generate supernatant samples for
91 analysis by mouse IgG ELISA. The highest producing clone (secreting 40 µg/mL TL1AIg/1 x 106 cells/24 hrs) was expanded to confluency and subjected to sequential
adaptation from 10% FBS supplemented IMDM to serum free OptiCHO media
supplemented with 8 mM L-glutamine, and GS and HT supplements (Invitrogen). Fiveseparate CHO clones were established in this manner and subjected to further selection
when cultured in serum free OptiCHO media lacking L-Glutamine. Another mIgG
ELISA was used to pick the highest producing clone, which was chosen for the third
round of selection under varying concentrations of MSX (25 µM, 100 µM, 1 mM and 2
mM). The surviving clone at 1 mM MSX was passaged and ELISA samples were
collected as described previously. A single clone expressing TL1A-Ig at about 200
µg/mL/1 x 106 cells/24 hrs was chosen for generation of protein.
4.6 Production and Purification Conditions
The highest yielding TL1A-Ig CHO cells were placed into hollow fiber cell culture
cartridges (Fibercell Systems) and cultured in serum free and glutamine free media
containing GS and HT supplements. About 50% of the cells were harvested with 20 mL
of TL1A-Ig supernatants every 3 days, on which the cartridge was replenished with 1L of
fresh media. Cell viability was maintained at 50% viable cells/mL in the cartridge,
allowing us to maintain culture for many months of continuous production. IgG directed
ELISA was used to quantify TL1A-Ig in the harvested media, which was stored at -20°C
until purification. The secreted fusion proteins were purified by the traditional protein A
affinity chromatography method using a basic elution buffer (0.1 M diethylamine; 11.5
pH). In initial comparative studies, a TL1A-Ig preparation was purified to homogeneity
on a protein G column. The bound protein was eluted with an acidic elution buffer (0.1 M
92 glycine; 2.5 pH). In another TL1A-Ig preparation, solid ammonium sulfate was added to
the to the TL1A-Ig supernatant to achieve a 55% ammonium sulfate and the solution was
spun overnight at 4° C. The solution was centrifuged and the precipitate containing
TL1A-Ig was resuspended in binding buffer (1.6 M Glycine, 3.2 M NaCl, pH 9.0) and
dialyzed against binding buffer overnight. The solution from the dialysis tube was, spun,
filtered and applied on a protein A column for purification.
4.7 ELISA quantification assays
Ninety-six well plates were coated with capture antibody anti-mIgG1 (100 µl/well; 10
µg/mL) for 1 hour at 37°C, followed by blocking (10% FBS in PBS), and 3 washes with
washing buffer (PBS containing 0.1% Tween-20). 100 µl of standard (mIgG1), TL1A-Ig,
or blank, was added to each well (with serial dilutions) and incubated for 2 hours. After
washing, 100 µl of secondary antibody horseradish peroxidase (HRP) conjugated antimIgG was added to each well (1:1000 dilution in PBS) and incubated for 1 hour. After 5
washes, 100 µl of ABTs substrate was added to each well, incubated for 10 – 20 minutes,
and analyzed using a Benchmark Plus microplate spectrophotometer at 405 OD. This
protocol was used to pick the highest TL1A-Ig producing cell clones and to analyze the
production of TL1A-Ig in the CHO-cell cartridges. For 4C12 and TL1A-Ig half-life
experiments, serum samples were analyzed by ELISA using an anti-Armenian hamster
IgG (10 µg/mL) and an anti-mTL1A specific capture antibody (L4G6, 10 µg/mL),
respectively. Specie and isotype matched HRP anti-armenian hamster IgG or HRP antimouse IgG was used as a secondary detection antibody (Jackson Research).
93 4.8 Coomassie Blue stain and Western blot analysis
TL1A-Ig was denatured by boiling for 5 min in the absence or presence of 2-ME,
Laemmli sample buffer (2% SDS, 2.5% glycerol, 15 mM Tris (pH 6.8)) and separated by
electrophoresis on a 4-15% SDS-PAGE gel. For the detection of protein bands, the gel
was incubated with a Coomassie Blue stain solution followed by washing with destaining
buffer (50% v/v methanol and 10% v/v acetic acid). For western-blotting analysis, the
SDS-PAGE gel was transferred to a nitrocellulose membrane. The membrane was
washed with TTBS (50 mM Tris, 150 mM NaCl, 0.05% Tween 20 (pH 7.6)) and the
TL1A-Ig fusion proteins were detected with an anti-TL1A Ab (0.5 µg/mL; Jackson
ImmunoResearch) or an anti-mIgG Ab (1:1000 dilution;Jackson ImmunoResearch),
HRP-coupled secondary Ab (1:1000 dilution; Jackson ImmunoResearch), and the
SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific). The
glycosylation studies were performed according to the protocol provided with the
Enzymatic Deglycosylation Kit GK80110 (Prozyme). Briefly, four samples of reduced
purified TL1A-Ig (2 µg) were incubated in the absence or presence of 1 µl each of Nglycanase, Sialidase A, and O-glycanase in the appropriate tube. The first control
replicate received no enzymes, the second tube received only N-glycanase, the third tube
received only N-glycanase, and the fourth tube received all the 3 enzymes. The tubes
were incubated for 3 hours at 37°C. Samples were loaded on an SDS-PAGE gel,
subjected to electrophoresis, and stained with Coomassie Blue.
4.9 Gel filtration and dot blot analysis
TL1A-Ig (purified or in supernatant) was analyzed by size exclusion chromatography on
a Superdex 200 gel filtration column equilibrated with Tris buffer (10 mM Tris, 150 mM
94 NaCl, 1mM EDTA). The fractions were collected at a flow rate of 0.5 mL/min (200
µl/fraction). The column was calibrated with a mixture of standards including bovine
thyroglobulin (670 kDa), aldolase (148 kDa) and OVA (43 kDa) (GE Healthcare). The
apparent molecular weights were calculated based on the elution volumes of the
standards. Dot blot analysis was done to determine the molecular weights of dilute
TL1A-Ig (secreted by 3T3 cells) in supernatants that contained serum as well as other
proteins. After gel filtration of 500 µl of TL1A-Ig supernantant, 10 µl of each fraction
was applied as a dot on a nitrocellulose membrane through circular holes on an apparatus.
Membranes were blocked with 5% non-fat dry milk, washed with TTBS, and incubated
with HRP anti-mouse IgG antibody (1:1000 in PBS) to detect membrane bound TL1A-Ig.
4.10 In vitro binding assays to mouse TNFRSF25 expressing cells
P815 cells (1x106 cells) transfected with TNFRSF25 were incubated with TL1A-Ig,
L4G6 treated TL1A-Ig, 4C12 or isotype control IgG (0.5 µg/million cells) at 4°C for 30
mins, followed by washing with FACs (1% BSA and 0.1% sodium azide in PBS) buffer.
Cells were stained with fluorochrome conjugated anti-mIgG (BioLegend) at 4°C for 30
mins, washed with FACs buffer, and analyzed using flow cytometry for TNFRSF25
positive cells. Total splenocytes or CD4+ or CD8+ T cells were isolated from
FoxP3+FIR mice or B6 mice for experiments testing for binding of TL1A-Ig and 4C12 to
endogenous TNFRSF25 on T cells. CD4+ T cells and CD8+ T cells were isolated using
positive selection beads (Miltenyi Biotec). For activation, cells were plated at 2 x 106
cells/mL in a 6-well plate pre-treated with 1 ug/mL of anti-CD3 antibody. The plate was
incubated at 37°C for 4 days. Cells were harvested, washed, and incubated for 15 min at
4°C with 1 µg /106 cells of Fc blocking antibody (eBioscience). Recovered cells were
95 incubated with 1 µg/106 cells TL1A-Ig, 4C12 or isotype control. The cells were washed
and double stained with fluorochrome conjugated anti-CD4 or anti-CD8 (1 µg /106 cells)
and anti-IgG (1 µg /106 cells). Finally, CD4+FIR- Tconvs, CD4+FIR+ Tregs and CD8+ T
cells were analyzed using flow cytometry for cell bound 4C12 or TL1A-Ig.
4.11 In vitro caspase assays
The enzyme activity of natural caspases was detected using the fluorimetric
homogeneous caspase assay according to the manufacturer’s protocol (Roche
Diagnostics). Briefly, TNFRSF25-P815 cells were incubated with titrating concentrations
of IgG, TL1A-Ig (purified or in supernatant), TL1A-Ig pretreated with L4G6, or 4C12 for
5 h at 37°C in a 96-well plate (total volume 100 µl). Subsequently, substrate solution was
added to the wells and the plates were incubated for 1.5 h. This treatment lysed the cells
and allowed caspases to cleave the substrate to free rhodamine 110 (R110). Free R110
was determined fluorimetrically at λmax=521 nm (Wallac Victor 1420). The developed
fluorochrome was directly proportional to the activity of caspases.
4.12 In vitro T cell culture
For proliferation assays, CD4+FIR+ Tregs were highly purified (>98% purity) from CD4+
enriched FIR splenocytes using a FACs aria cell sorter (BD Biosciences) and CD4+CD25Tconv cells were purified by using the CD4+CD25+ Regulatory T Cell Isolation Kit
(Miltenyi Biotec) to obtain a Treg-free Tconv population. Tregs or Tconv (5 x 104/well
in triplicates) were plated in 96-well round bottomed plates and treated in presence or
absence of: anti-CD3 (2 µg/mL; 2C11), mIL-2 (10 U/mL), 4C12 (10 µg/mL), or purified
TL1A-Ig (0.1 µg/mL). In some Treg proliferation assays, FACs sorted Treg cells were
pre-incubated with MHC II blocking antibody (10 µg/mL) for 45 min before culturing
96 them. In another experiment, FACs sorted Tregs were cultured in 100 U/mL IL-2 for 72
hours to make “resting” Tregs before adding them into proliferations assays. Cultures
were incubated for 72 hours at 37°C and pulsed with 3H-thymidine (1 µCi/well, Perkin
Elmer) for the last 6 hours. The incorporated isotope was measured by liquid scintillation
counting (Micro Beta TriLux counter; Perkin Elmer). For iTreg induction experiments,
Tconv cells were FACs sorted from FIR mice (CD4+FIR-) and OT-II transgenic mice
(FACs sorted for CD4+CD25lowGITRlow). For induction of polyclonal iTregs, FIR Tconv
were seeded in 24-well flat bottomed plate pre-treated with plate-bound anti-CD3 (2
µg/mL), in the presence of TGF-β (5 ng/mL), IL-2 (100 U/mL); retinoic acid (100 nM)
plus or minus 4C12 or TL1A-Ig (10 µg/mL). For OT-II iTreg, OTII Tconv were cultured
with 1:2 APCs, ovalbumin protein (10 nM), TGF-β (5 ng/mL), IL-2 (100 U/mL); retinoic
acid (100 nM) in the absence or presence of 10 ug/ml 4C12 or TL1A-Ig. Cultures were
incubated for 72 hours, washed, and analyzed for FoxP3+ cells in the CD4+ gate.
4.13 In vitro suppression assays
TNFRSF25 DN CD4+CD25- cells (5 × 104) were plated in 96-well round-bottomed plates
and activated with 0.5 µg soluble anti-CD3 (2C11) antibody in the presence of APCs (1:1
ratio) and titrating numbers of CD4+FIR+ Tregs isolated from IgG treated or TL1A-Ig
treated mice. Control IgG or TL1A-Ig was added at a concentration of 0.1 µg/ml in some
experiments. In some experiments wt CD4+CD25- T cells were used as responders.
Cultures were incubated for 72 hours and pulsed with 3H-thymidine (1 µCi/well; Perkin
Elmer) for the last 6 hours. Incorporated isotope was measured by liquid scintillation
counting (Micro Beta TriLux counter; Perkin Elmer).
97 4.14 Antibiotics
Mice were treated with ampicillin (1 g/L), vancomycin (500 mg/L), neomycin sulfate (1
g/L), and metronidazole (1 g/l, Sigma) in drinking water from day -25 to day 5 to remove
gut microbiota. On days 0-3 mice were injected i.p with 100 µg isotype control IgG or
TL1A-Ig in 100 µl PBS. Mice were sacrificed on day 5 to harvest spleens and lymph
nodes for further analysis.
4.15 Induction of allergic lung inflammation
Mice were sensitized by i.p. injection of 66 µg OVA (crystallized chicken egg albumin,
grade V; Sigma-Aldrich) adsorbed to 6.6 mg alum (aluminum potassium sulfate; SigmaAldrich) in 200 µl PBS on day 0, with an i.p. boost on day 5. On days 11, 12 and 13 mice
were injected i.p. 100 µg TL1A-Ig or mouse IgG (Jackson ImmunoResearch Laboratories
Inc.) in 200 µl PBS. On day 16, mice were aerosol challenged with 0.5% OVA (SigmaAldrich) in PBS for 1 hour using a BANG nebulizer (CH Technologies) into a JaegerNYU Nose-Only Directed-Flow Inhalation Exposure System (CH Technologies). On day
19, mice were sacrificed, lungs were perfused with PBS, and bronchoalveolar lavages
were obtained. Lung lobes and spleens were harvested for flow cytometry analysis, and
lung lobes were also analyzed for lung histology as described previously (106).
Quantification of periodic acid-Schiff–stained (PAS-stained) lung sections was
performed using MacBiophotonics Image J software by color deconvolution (using the H
PAS vector) followed by thresholding of images (color “2,” set to 95) and counted using
the nucleus counter (limits set between 400 and 7,000).
98 4.16 Skin transplantation
Recipient FIR-B6 mice were pre-injected i.p with one 20 µg dose of hamster-IgG or
4C12, or daily 100 µg doses of mouse IgG or TL1A-Ig 4 days prior to skin
transplantation (days -4 to 0). On day of transplantation (day 0), 4C12 and TL1A-Ig
treated mice were bled and the CD4+FoxP3+ Treg frequency was analyzed to ensure
expansion of Treg to at least 30% of the total CD4+ T cell gate. A 1 cm skin incision was
made at the operative site on the right thorax of FIR-B6 recipients. Full thickness skin
grafts (1-2 cm in diameter) were obtained from the ears of Balb/c donor mice, trimmed to
fit graft bed, and transplanted onto the right thorax of recipient FIR-B6 mice. The
recipient mouse was wrapped with a sterile bandage for 6-7 days after transplant. On day
6-7, the bandage was removed to assess graft survival. TL1A-Ig treated mice were
injected with 100 µg TL1A-Ig i.p daily until graft rejection, which was scored as rejected
when more than 75% of the grafted tissue area had been lost.
4.17 Tumor inoculation and immunization
EG7 cells were isolated from log-phase cultures and washed in PBS. One million cells
were then injected s.c. in the hind-flank of mice in 100 µL PBS. One million
nonirradiated EG7-gp96-Ig cells were injected i.p. in 100 µL PBS or in combination with
20 µg 4C12 or 100 µg TL1A-Ig on days of vaccination (day 7,10,13,16,19, and 22). In
some experiments, TL1A-Ig was injected on day 7, 8, 9, 10 and subsequently on days of
EG7-gp96-Ig vaccination.
4.18 Flow cytometry analysis and FACs sorting
Single cell suspensions were made from peripheral blood, spleens, and lymph nodes. One
to three million cells were used to stain with different antibody combinations. Flow
99 cytometry analysis was performed on a BD FACs Fortessa instrument with further
analysis done with the FlowJo software. Negative enrichment of CD4+ T cells was done
using the EasySep Mouse CD4+ T cell Pre-enrichment Kit from Stem Cell Technologies
followed by staining for the appropriate markers before sorting for live cells using a
FACSAria cell sorter (BD) after .
4.19 Cell adoptive transfer experiments
Ten million CD4+ cells (comprised of approximately 10% FoxP3+ Treg cells) were
highly purified by FACS sorting from FIR mice and adoptively transferred into CD74-/or CD4-/- mice. After three days (day 0), recipient mice were treated with 100 ug of
mTL1A-Ig or IgG followed by 2 consecutive doses on days 1 and 2. The percentage of
FoxP3+ cells was analyzed in the spleen and pooled lymph nodes on day 6. In OTII
adoptive transfer experiments, CD4+CD25lowGITRlow cells were FACs sorted from OTII
mice. One million cells were adoptively transferred i.v into CD41.5 SJL mice on day -2.
On day 0, mice were immunized with i.p injection of OVA/ALUM in 200 µl PBS. Mice
were treated with 100 µg isotype control IgG or TL1A-Ig on day 0, 1, 2 and sacrificed for
analysis on day 5. In OT-1 expansion experiments, FIR mice were injected i.v on day -2
with 1 million OT-1 (purified using positive selection for CD8+ receptor) followed by an
i.p injection of gp96-Ig vaccine cells on day 0. TL1A-Ig or mIgG1 (100 ug) in 100 µl
PBS was injected ip on days 1 and 3 after vaccination. All mice were sacrificed on day 5
and analyzed for OT-1 frequency in the CD8+ gate in the peritoneal cavity and spleen.
4.20 Histology
The heart, kidney, liver, lung, small and large intestine were harvested from mice that
were treated with 100 µg i.p IgG or TL1A-Ig daily on days 0-2 or days 0-20 and fixed in
100 10% neutral buffered formalin. The fixed samples were sent for Hematoxylin and Eosin
staining to the Comparative Pathology Core at the University of Miami. In the acute
asthma studies, lungs were removed from mice after the bronchial lavage procedure,
fixed and submitted to the Comparative Pathology Core were they were embedded,
sectioned, and stained with H&E and Periodic Acid-Schiff.
4.21 Statistics
All data was graphed and analyzed using GraphPad Prism 5. Paired comparisons were
performed using Student's t test, multiple analysis was performed using a one-way
ANOVA, P values are indicated as necessary.
Appendix 1: Combination therapy of gp96-Ig vaccine and TNFRSF25 agonists to
enhance anti-tumor immunity
Although several reports have examined the role of TNFRSF25 regulation of
CD4+ T cell subsets, including Tregs, not much is known about the function of
TNFRSF25 in modulating CD8+ T cell responses. Recently a group reported that
TNFRSF25 triggering in vivo using soluble TL1A induces the proliferation and
accumulation of antigen-specific CD8+ T cells and their differentiation into CTLs (197).
We next determined whether TL1A-Ig costimulates OVA-specific CD8+ T cells in the
presence of cognate antigen (OVA) secreted from the gp96-Ig vaccine. We have
previously reported that replacing the KDEL ER retention signal of heat shock protein
gp96 with the Fc portion of IgG and transfecting the cDNA into cell lines, including
tumor cells, results in the expression of gp96-Ig fusion protein chaperoning endogenous
antigens (or tumor antigens) into the extracellular matrix that effectively stimulates cross
presentation to CTLs (198). This protocol required the use of splenocytes from transgenic
mice with MHC Class-I restricted T-cell receptors specific for ovalbumin (OT-I mice) as
a source for CD8+ T cells (OT-I cells) (Figure 47).
IgG or TL1A-Ig
i.p
Day
0
5
1 x 106 GFP-OTI 1 x 106 gp96-Ig
i.v into FIR mice vaccine cells i.p
Analysis: PEC
and spleen flow
cytometry
Figure 47. Protocol for TL1A-Ig and
vaccine treatment for OTI cell expansion.
FIR mice were injected i.v on day -2 with 1
million OT-1 followed by an i.p injection of
gp96-Ig vaccine cells on day 0. TL1A-Ig or
mIgG1 (100 ug) was injected ip on days 1
and 3 after vaccination. All mice were
sacrificed on day 5 for analysis.
Briefly, we adoptively transferred ova-specific OT-I cells into recipient mice on day -2,
followed by an injection of 1 million 3T3-NIH-gp96-Ig vaccine cells on day 0 and i.p
injection of IgG or TL1A-Ig on days 1 and 3. All mice were sacrificed on day 5 and the
101 102 frequency of OT-I and Treg cell populations were analyzed in the spleen and lymph
nodes of TL1A-Ig or IgG treated controls. The OT-I frequency in the CD8+ T cell gate
was significantly higher in the spleens of TL1A-Ig mice as compared to controls.
Concurrent significant Treg expansion was also observed in the spleens and LNs of
TL1A-Ig
treated
mice
48a-b).
b
50
PEC
Spleen
*
40
%FoxP3+/total CD4+
%GFP-OTI/total CD8+
a
(Figure
30
20
ns
10
0
IgG
TL1A-Ig
IgG
TL1A-Ig
50
PEC
Spleen
40
**
***
30
20
10
0
IgG
TL1A-Ig
IgG
TL1A-Ig
Figure 48. TL1A-Ig boosts clonal expansion of CD8+ T cells and Tregs when combined with
gp96-Ig vaccine. (a) OT-1 frequency in the CD8+ gate in the peritoneal cavity and spleen. (b)
CD4+FoxP3+ Tregs were also analyzed in the CD4 gate using flow cytometry. Significance was
determined by Student’s t test. PEC: peritoneal cavity.
Although these results seem to conflict with the studies described earlier on the effect of
TNFRSF25 on Treg cells, they are not necessarily mutually exclusive. The enhancement
of cell proliferation and survival by TNFRSF25 has been described in studies of
pathogenic effector CD4+ and CD8+ T cells (105, 128, 129, 152, 162, 167, 197, 199,
200), as well as immunosuppressive Treg cells (130).
TNFRSF25 agonists in combination with gp96-Ig vaccine and tumor rejection
It is generally recognized that CD8+ cytotoxic T cells (CTLs) play a crucial role in
inhibiting and killing tumor cells that hinders tumor growth. However, the extent of their
contribution to anti-cancer immunity is dampened by immunosuppressive mechanisms
such as recruitment of Tregs and secretion of immunosuppressive cytokines in the tumor
microenvironment that tumors utilize to evade the immune system. A study suggests that
signaling of GITR results in Treg-resistant CD8+ T cells (201). Hence we hypothesized
103 that TNFRSF25 signaling in the presence of vaccination results in a CTL response that is
15
Only vaccine
Rejected: 2/3
10
5
0
0 5 10 15 20 25 30
Days post tumor inoculations
b
20
imposed
Vaccine + 4C12
Rejected: 3/5
15
10
5
** * * * *
0
0 5 10 15 20 25 30
Days post tumor inoculations
Tumor Diameter (mm)
20
suppression
Tumor Diameter (mm)
to
Tumor Diameter (mm)
a
Tumor Diameter (mm)
resistant
by
Tregs.
20 Vaccine + TL1A-Ig
15 Rejected: 1/5
10
5
** * * * *
0
0 5 10 15 20 25 30
Days post tumor inoculations
20 Vaccine + TL1A-Ig
15 Rejected: 5/10
10
5
**** * * * *
0
0 5 10 15 20 25 30
Days post tumor inoculations
Figure 49. TNFRSF25 agonist TL1A-Ig does not enhance gp96-Ig mediated rejection of
established tumors. WT B6 mice were injected s.c in left flank with 1 million EG7 tumor cells.
Tumors were established for 5 days and (a) vaccine (arrow) was administered i.p alone or in
combination with 4C12 or TL1A-Ig (orange stars) on days 7,10,13,16,19,& 22. (b) One group
received 4 consecutive doses of TL1A-Ig on days 7,8,9,&10 in addition to the regimen described
in (a).
Previous studies by Dr. Schreiber reported that frequent vaccination with EG7-gp96-Ig
resulted in a thirty percent tumor rejection rate. His studies indicate that combination
therapy with gp96-Ig vaccine and 4C12 (agonist hamster anti- TNFRSF25) mediated
stimulation of TNFRSF25 increased tumor rejection rate to about 80%. We next wanted
to determine if TL1A-Ig induces similar anti-tumor activity. Briefly, 1 million tumor cells
(EG7) were transplanted (s.c) in the flank of mice. The tumor was allowed to establish
for 7 days, at which time it is fully vascularized. From day 7 on all mice were vaccinated
6 times with 1 million live EG7-gp96-Ig i.p. every three days (arrows). IgG, 4C12 (20
µg) or TL1A-Ig (100 µg) was injected i.p on vaccine days (Figure 49a). Tumor size was
measured daily. Sixty percent tumor rejection was observed in the group that received
104 vaccine only and vaccine plus 4C12, while only 20% tumor rejection was observed in the
group that received TL1A-Ig. Tumor dormancy was observed in one animal in the TL1AIg group. One group of mice received 2 extra doses of TL1A-Ig between vaccine doses 1
and 2 (Figure 49b), increasing the tumor rejection rate to 50%. These experiments
indicate that neither TNFRSF25 agonists, 4C12 or TL1A-Ig, enhance the tumor rejection
observed with vaccine only. However, experiments with different schedule dosing and
with more mice in each treatment group need to be performed in order to have conclusive
results.
References
1.
Kenneth Murphy, P. T., Mark Walport. 2008. Janeway's Immunobiology. Garland
Science.
2.
Ruffell, B., D. G. DeNardo, N. I. Affara, and L. M. Coussens. 2010. Lymphocytes
in cancer development: polarization towards pro-tumor immunity. Cytokine
Growth Factor Rev 21:3-10.
3.
Annunziato, F., and S. Romagnani. 2009. Heterogeneity of human effector CD4+
T cells. Arthritis Res Ther 11:257.
4.
Carding, S. R., and P. J. Egan. 2002. Gammadelta T cells: functional plasticity
and heterogeneity. Nat Rev Immunol 2:336-345.
5.
Zhu, J., and W. E. Paul. 2008. CD4 T cells: fates, functions, and faults. Blood
112:1557-1569.
6.
Djuretic, I. M., D. Levanon, V. Negreanu, Y. Groner, A. Rao, and K. M. Ansel.
2007. Transcription factors T-bet and Runx3 cooperate to activate Ifng and
silence Il4 in T helper type 1 cells. Nat Immunol 8:145-153.
7.
Hwang, E. S., S. J. Szabo, P. L. Schwartzberg, and L. H. Glimcher. 2005. T
helper cell fate specified by kinase-mediated interaction of T-bet with GATA-3.
Science 307:430-433.
8.
Lazarevic, V., X. Chen, J. H. Shim, E. S. Hwang, E. Jang, A. N. Bolm, M. Oukka,
V. K. Kuchroo, and L. H. Glimcher. 2011. T-bet represses T(H)17 differentiation
by preventing Runx1-mediated activation of the gene encoding RORgammat. Nat
Immunol 12:96-104.
9.
Oestreich, K. J., A. C. Huang, and A. S. Weinmann. 2011. The lineage-defining
factors T-bet and Bcl-6 collaborate to regulate Th1 gene expression patterns. J
Exp Med 208:1001-1013.
10.
Zhu, J., H. Yamane, J. Cote-Sierra, L. Guo, and W. E. Paul. 2006. GATA-3
promotes Th2 responses through three different mechanisms: induction of Th2
cytokine production, selective growth of Th2 cells and inhibition of Th1 cellspecific factors. Cell Res 16:3-10.
11.
Zhu, J., and W. E. Paul. 2010. Heterogeneity and plasticity of T helper cells. Cell
Res 20:4-12.
12.
Lohr, J., B. Knoechel, and A. K. Abbas. 2006. Regulatory T cells in the periphery.
Immunol Rev 212:149-162.
105 106 13.
Zou, W. 2006. Regulatory T cells, tumour immunity and immunotherapy. Nat Rev
Immunol 6:295-307.
14.
Brusko, T. M., A. L. Putnam, and J. A. Bluestone. 2008. Human regulatory T
cells: role in autoimmune disease and therapeutic opportunities. Immunol Rev
223:371-390.
15.
Annunziato, F., L. Cosmi, F. Liotta, E. Lazzeri, R. Manetti, V. Vanini, P.
Romagnani, E. Maggi, and S. Romagnani. 2002. Phenotype, localization, and
mechanism of suppression of CD4(+)CD25(+) human thymocytes. J Exp Med
196:379-387.
16.
Sanjabi, S., L. A. Zenewicz, M. Kamanaka, and R. A. Flavell. 2009. Antiinflammatory and pro-inflammatory roles of TGF-beta, IL-10, and IL-22 in
immunity and autoimmunity. Curr Opin Pharmacol 9:447-453.
17.
Niedbala, W., X. Q. Wei, B. Cai, A. J. Hueber, B. P. Leung, I. B. McInnes, and F.
Y. Liew. 2007. IL-35 is a novel cytokine with therapeutic effects against
collagen-induced arthritis through the expansion of regulatory T cells and
suppression of Th17 cells. Eur J Immunol 37:3021-3029.
18.
Shevach, E. M. 2009. Mechanisms of foxp3+ T regulatory cell-mediated
suppression. Immunity 30:636-645.
19.
Haribhai, D., W. Lin, L. M. Relland, N. Truong, C. B. Williams, and T. A.
Chatila. 2007. Regulatory T cells dynamically control the primary immune
response to foreign antigen. J Immunol 178:2961-2972.
20.
Betts, R. J., N. Prabhu, A. W. Ho, F. C. Lew, P. E. Hutchinson, O. Rotzschke, P.
A. Macary, and D. M. Kemeny. 2012. Influenza A virus infection results in a
robust, antigen-responsive, and widely disseminated Foxp3+ regulatory T cell
response. J Virol 86:2817-2825.
21.
Littman, D. R., and A. Y. Rudensky. 2010. Th17 and regulatory T cells in
mediating and restraining inflammation. Cell 140:845-858.
22.
Vignali, D. A., L. W. Collison, and C. J. Workman. 2008. How regulatory T cells
work. Nat Rev Immunol 8:523-532.
23.
Liu, Y., P. Zhang, J. Li, A. B. Kulkarni, S. Perruche, and W. Chen. 2008. A
critical function for TGF-beta signaling in the development of natural
CD4+CD25+Foxp3+ regulatory T cells. Nat Immunol 9:632-640.
24.
Saxena, A., G. Martin-Blondel, L. T. Mars, and R. S. Liblau. 2011. Role of CD8
T cell subsets in the pathogenesis of multiple sclerosis. FEBS Lett 585:3758-3763.
107 25.
Sad, S., R. Marcotte, and T. R. Mosmann. 1995. Cytokine-induced differentiation
of precursor mouse CD8+ T cells into cytotoxic CD8+ T cells secreting Th1 or
Th2 cytokines. Immunity 2:271-279.
26.
Huber, M., S. Heink, H. Grothe, A. Guralnik, K. Reinhard, K. Elflein, T. Hunig,
H. W. Mittrucker, A. Brustle, T. Kamradt, and M. Lohoff. 2009. A Th17-like
developmental process leads to CD8(+) Tc17 cells with reduced cytotoxic
activity. Eur J Immunol 39:1716-1725.
27.
Chavez-Galan, L., M. C. Arenas-Del Angel, E. Zenteno, R. Chavez, and R.
Lascurain. 2009. Cell death mechanisms induced by cytotoxic lymphocytes. Cell
Mol Immunol 6:15-25.
28.
Slifka, M. K., F. Rodriguez, and J. L. Whitton. 1999. Rapid on/off cycling of
cytokine production by virus-specific CD8+ T cells. Nature 401:76-79.
29.
Trandem, K., J. Zhao, E. Fleming, and S. Perlman. 2011. Highly activated
cytotoxic CD8 T cells express protective IL-10 at the peak of coronavirus-induced
encephalitis. J Immunol 186:3642-3652.
30.
Sun, J., R. Madan, C. L. Karp, and T. J. Braciale. 2009. Effector T cells control
lung inflammation during acute influenza virus infection by producing IL-10. Nat
Med 15:277-284.
31.
Palmer, E. M., B. C. Holbrook, S. Arimilli, G. D. Parks, and M. A. AlexanderMiller. 2010. IFNgamma-producing, virus-specific CD8+ effector cells acquire
the ability to produce IL-10 as a result of entry into the infected lung
environment. Virology 404:225-230.
32.
Zhang, N., and M. J. Bevan. 2011. CD8(+) T cells: foot soldiers of the immune
system. Immunity 35:161-168.
33.
Carswell, E. A., L. J. Old, R. L. Kassel, S. Green, N. Fiore, and B. Williamson.
1975. An endotoxin-induced serum factor that causes necrosis of tumors. Proc
Natl Acad Sci U S A 72:3666-3670.
34.
Granger, G. A., S. J. Shacks, T. W. Williams, and W. P. Kolb. 1969. Lymphocyte
in vitro cytotoxicity: specific release of lymphotoxin-like materials from
tuberculin-sensitive lymphoid cells. Nature 221:1155-1157.
35.
Pennica, D., G. E. Nedwin, J. S. Hayflick, P. H. Seeburg, R. Derynck, M. A.
Palladino, W. J. Kohr, B. B. Aggarwal, and D. V. Goeddel. 1984. Human tumour
necrosis factor: precursor structure, expression and homology to lymphotoxin.
Nature 312:724-729.
108 36.
Gray, P. W., B. B. Aggarwal, C. V. Benton, T. S. Bringman, W. J. Henzel, J. A.
Jarrett, D. W. Leung, B. Moffat, P. Ng, L. P. Svedersky, and et al. 1984. Cloning
and expression of cDNA for human lymphotoxin, a lymphokine with tumour
necrosis activity. Nature 312:721-724.
37.
Hehlgans, T., and K. Pfeffer. 2005. The intriguing biology of the tumour necrosis
factor/tumour necrosis factor receptor superfamily: players, rules and the games.
Immunology 115:1-20.
38.
Croft, M. 2009. The role of TNF superfamily members in T-cell function and
diseases. Nat Rev Immunol 9:271-285.
39.
Aggarwal, B. B. 2003. Signalling pathways of the TNF superfamily: a doubleedged sword. Nat Rev Immunol 3:745-756.
40.
Croft, M., W. Duan, H. Choi, S. Y. Eun, S. Madireddi, and A. Mehta. 2012. TNF
superfamily in inflammatory disease: translating basic insights. Trends Immunol
33:144-152.
41.
Migone, T. S., J. Zhang, X. Luo, L. Zhuang, C. Chen, B. Hu, J. S. Hong, J. W.
Perry, S. F. Chen, J. X. Zhou, Y. H. Cho, S. Ullrich, P. Kanakaraj, J. Carrell, E.
Boyd, H. S. Olsen, G. Hu, L. Pukac, D. Liu, J. Ni, S. Kim, R. Gentz, P. Feng, P.
A. Moore, S. M. Ruben, and P. Wei. 2002. TL1A is a TNF-like ligand for DR3
and TR6/DcR3 and functions as a T cell costimulator. Immunity 16:479-492.
42.
Prehn, J. L., L. S. Thomas, C. J. Landers, Q. T. Yu, K. S. Michelsen, and S. R.
Targan. 2007. The T cell costimulator TL1A is induced by FcgammaR signaling
in human monocytes and dendritic cells. J Immunol 178:4033-4038.
43.
Fesik, S. W. 2000. Insights into programmed cell death through structural
biology. Cell 103:273-282.
44.
Peschon, J. J., J. L. Slack, P. Reddy, K. L. Stocking, S. W. Sunnarborg, D. C. Lee,
W. E. Russell, B. J. Castner, R. S. Johnson, J. N. Fitzner, R. W. Boyce, N.
Nelson, C. J. Kozlosky, M. F. Wolfson, C. T. Rauch, D. P. Cerretti, R. J. Paxton,
C. J. March, and R. A. Black. 1998. An essential role for ectodomain shedding in
mammalian development. Science 282:1281-1284.
45.
Croft, M. 2003. Co-stimulatory members of the TNFR family: keys to effective
T-cell immunity? Nat Rev Immunol 3:609-620.
46.
Idriss, H. T., and J. H. Naismith. 2000. TNF alpha and the TNF receptor
superfamily: structure-function relationship(s). Microsc Res Tech 50:184-195.
Eck, M. J., and S. R. Sprang. 1989. The structure of tumor necrosis factor-alpha at
2.6 A resolution. Implications for receptor binding. J Biol Chem 264:1759517605.
47.
109 48.
Locksley, R. M., N. Killeen, and M. J. Lenardo. 2001. The TNF and TNF receptor
superfamilies: integrating mammalian biology. Cell 104:487-501.
49.
Bodmer, J. L., P. Schneider, and J. Tschopp. 2002. The molecular architecture of
the TNF superfamily. Trends Biochem Sci 27:19-26.
50.
Smith, C. A., T. Farrah, and R. G. Goodwin. 1994. The TNF receptor superfamily
of cellular and viral proteins: activation, costimulation, and death. Cell 76:959962.
51.
Chan, F. K., H. J. Chun, L. Zheng, R. M. Siegel, K. L. Bui, and M. J. Lenardo.
2000. A domain in TNF receptors that mediates ligand-independent receptor
assembly and signaling. Science 288:2351-2354.
52.
Siegel, R. M., F. K. Chan, H. J. Chun, and M. J. Lenardo. 2000. The multifaceted
role of Fas signaling in immune cell homeostasis and autoimmunity. Nat Immunol
1:469-474.
53.
Schneider, P., N. Holler, J. L. Bodmer, M. Hahne, K. Frei, A. Fontana, and J.
Tschopp. 1998. Conversion of membrane-bound Fas(CD95) ligand to its soluble
form is associated with downregulation of its proapoptotic activity and loss of
liver toxicity. J Exp Med 187:1205-1213.
54.
Grell, M., E. Douni, H. Wajant, M. Lohden, M. Clauss, B. Maxeiner, S.
Georgopoulos, W. Lesslauer, G. Kollias, K. Pfizenmaier, and P. Scheurich. 1995.
The transmembrane form of tumor necrosis factor is the prime activating ligand of
the 80 kDa tumor necrosis factor receptor. Cell 83:793-802.
55.
Tanaka, M., T. Itai, M. Adachi, and S. Nagata. 1998. Downregulation of Fas
ligand by shedding. Nat Med 4:31-36.
56.
Haswell, L. E., M. J. Glennie, and A. Al-Shamkhani. 2001. Analysis of the
oligomeric requirement for signaling by CD40 using soluble multimeric forms of
its ligand, CD154. Eur J Immunol 31:3094-3100.
57.
Wilson, N. S., V. Dixit, and A. Ashkenazi. 2009. Death receptor signal
transducers: nodes of coordination in immune signaling networks. Nat Immunol
10:348-355.
58.
Kischkel, F. C., D. A. Lawrence, A. Chuntharapai, P. Schow, K. J. Kim, and A.
Ashkenazi. 2000. Apo2L/TRAIL-dependent recruitment of endogenous FADD
and caspase-8 to death receptors 4 and 5. Immunity 12:611-620.
59.
Nagata, S. 1999. Fas ligand-induced apoptosis. Annu Rev Genet 33:29-55.
110 60.
Li, H., H. Zhu, C. J. Xu, and J. Yuan. 1998. Cleavage of BID by caspase 8
mediates the mitochondrial damage in the Fas pathway of apoptosis. Cell 94:491501.
61.
Gupta, S., A. Agrawal, S. Agrawal, H. Su, and S. Gollapudi. 2006. A paradox of
immunodeficiency and inflammation in human aging: lessons learned from
apoptosis. Immun Ageing 3:5.
62.
Chinnaiyan, A. M., K. O'Rourke, G. L. Yu, R. H. Lyons, M. Garg, D. R. Duan, L.
Xing, R. Gentz, J. Ni, and V. M. Dixit. 1996. Signal transduction by DR3, a death
domain-containing receptor related to TNFR-1 and CD95. Science 274:990-992.
63.
Kitson, J., T. Raven, Y. P. Jiang, D. V. Goeddel, K. M. Giles, K. T. Pun, C. J.
Grinham, R. Brown, and S. N. Farrow. 1996. A death-domain-containing receptor
that mediates apoptosis. Nature 384:372-375.
64.
Bodmer, J. L., K. Burns, P. Schneider, K. Hofmann, V. Steiner, M. Thome, T.
Bornand, M. Hahne, M. Schroter, K. Becker, A. Wilson, L. E. French, J. L.
Browning, H. R. MacDonald, and J. Tschopp. 1997. TRAMP, a novel apoptosismediating receptor with sequence homology to tumor necrosis factor receptor 1
and Fas(Apo-1/CD95). Immunity 6:79-88.
65.
Wen, L., L. Zhuang, X. Luo, and P. Wei. 2003. TL1A-induced NF-kappaB
activation and c-IAP2 production prevent DR3-mediated apoptosis in TF-1 cells.
J Biol Chem 278:39251-39258.
66.
Wang, L., F. Du, and X. Wang. 2008. TNF-alpha induces two distinct caspase-8
activation pathways. Cell 133:693-703.
67.
Schutze, S., V. Tchikov, and W. Schneider-Brachert. 2008. Regulation of TNFR1
and CD95 signalling by receptor compartmentalization. Nat Rev Mol Cell Biol
9:655-662.
68.
Dempsey, P. W., S. E. Doyle, J. Q. He, and G. Cheng. 2003. The signaling
adaptors and pathways activated by TNF superfamily. Cytokine Growth Factor
Rev 14:193-209.
69.
Dadgostar, H., B. Zarnegar, A. Hoffmann, X. F. Qin, U. Truong, G. Rao, D.
Baltimore, and G. Cheng. 2002. Cooperation of multiple signaling pathways in
CD40-regulated gene expression in B lymphocytes. Proc Natl Acad Sci U S A
99:1497-1502.
70.
Vonderheide, R. H. 2007. Prospect of targeting the CD40 pathway for cancer
therapy. Clin Cancer Res 13:1083-1088.
111 71.
Fu, Y. X., and D. D. Chaplin. 1999. Development and maturation of secondary
lymphoid tissues. Annu Rev Immunol 17:399-433.
72.
Dougall, W. C., M. Glaccum, K. Charrier, K. Rohrbach, K. Brasel, T. De Smedt,
E. Daro, J. Smith, M. E. Tometsko, C. R. Maliszewski, A. Armstrong, V. Shen, S.
Bain, D. Cosman, D. Anderson, P. J. Morrissey, J. J. Peschon, and J. Schuh. 1999.
RANK is essential for osteoclast and lymph node development. Genes Dev
13:2412-2424.
73.
Kong, Y. Y., U. Feige, I. Sarosi, B. Bolon, A. Tafuri, S. Morony, C. Capparelli, J.
Li, R. Elliott, S. McCabe, T. Wong, G. Campagnuolo, E. Moran, E. R. Bogoch,
G. Van, L. T. Nguyen, P. S. Ohashi, D. L. Lacey, E. Fish, W. J. Boyle, and J. M.
Penninger. 1999. Activated T cells regulate bone loss and joint destruction in
adjuvant arthritis through osteoprotegerin ligand. Nature 402:304-309.
74.
Kim, D., R. E. Mebius, J. D. MacMicking, S. Jung, T. Cupedo, Y. Castellanos, J.
Rho, B. R. Wong, R. Josien, N. Kim, P. D. Rennert, and Y. Choi. 2000.
Regulation of peripheral lymph node genesis by the tumor necrosis factor family
member TRANCE. J Exp Med 192:1467-1478.
75.
Guckelberger, O., J. M. Langrehr, W. O. Bechstein, R. Neuhaus, R. Luesebrink,
H. P. Lemmens, B. Kratschmer, S. Jonas, M. Knoop, and P. Neuhaus. 1996. Does
the choice of primary immunosuppression influence the prevalence of
cardiovascular risk factors after liver transplantation? Transplant Proc 28:31733174.
76.
Cuff, C. A., R. Sacca, and N. H. Ruddle. 1999. Differential induction of adhesion
molecule and chemokine expression by LTalpha3 and LTalphabeta in
inflammation elucidates potential mechanisms of mesenteric and peripheral
lymph node development. J Immunol 162:5965-5972.
77.
Luther, S. A., T. Lopez, W. Bai, D. Hanahan, and J. G. Cyster. 2000. BLC
expression in pancreatic islets causes B cell recruitment and lymphotoxindependent lymphoid neogenesis. Immunity 12:471-481.
78.
Banks, T. A., B. T. Rouse, M. K. Kerley, P. J. Blair, V. L. Godfrey, N. A. Kuklin,
D. M. Bouley, J. Thomas, S. Kanangat, and M. L. Mucenski. 1995. Lymphotoxinalpha-deficient mice. Effects on secondary lymphoid organ development and
humoral immune responsiveness. J Immunol 155:1685-1693.
79.
Alimzhanov, M. B., D. V. Kuprash, M. H. Kosco-Vilbois, A. Luz, R. L.
Turetskaya, A. Tarakhovsky, K. Rajewsky, S. A. Nedospasov, and K. Pfeffer.
1997. Abnormal development of secondary lymphoid tissues in lymphotoxin betadeficient mice. Proc Natl Acad Sci U S A 94:9302-9307.
112 80.
Pfeffer, K., T. Matsuyama, T. M. Kundig, A. Wakeham, K. Kishihara, A.
Shahinian, K. Wiegmann, P. S. Ohashi, M. Kronke, and T. W. Mak. 1993. Mice
deficient for the 55 kd tumor necrosis factor receptor are resistant to endotoxic
shock, yet succumb to L. monocytogenes infection. Cell 73:457-467.
81.
Ehlers, S., C. Holscher, S. Scheu, C. Tertilt, T. Hehlgans, J. Suwinski, R. Endres,
and K. Pfeffer. 2003. The lymphotoxin beta receptor is critically involved in
controlling infections with the intracellular pathogens Mycobacterium
tuberculosis and Listeria monocytogenes. J Immunol 170:5210-5218.
82.
Garrone, P., E. M. Neidhardt, E. Garcia, L. Galibert, C. van Kooten, and J.
Banchereau. 1995. Fas ligation induces apoptosis of CD40-activated human B
lymphocytes. J Exp Med 182:1265-1273.
83.
Chan, K. F., M. R. Siegel, and J. M. Lenardo. 2000. Signaling by the TNF
receptor superfamily and T cell homeostasis. Immunity 13:419-422.
Nagata, S., and P. Golstein. 1995. The Fas death factor. Science 267:1449-1456.
84.
85.
Lenardo, M., K. M. Chan, F. Hornung, H. McFarland, R. Siegel, J. Wang, and L.
Zheng. 1999. Mature T lymphocyte apoptosis--immune regulation in a dynamic
and unpredictable antigenic environment. Annu Rev Immunol 17:221-253.
86.
Rieux-Laucat, F., F. Le Deist, C. Hivroz, I. A. Roberts, K. M. Debatin, A.
Fischer, and J. P. de Villartay. 1995. Mutations in Fas associated with human
lymphoproliferative syndrome and autoimmunity. Science 268:1347-1349.
87.
Jacob, C. O., G. D. Lewis, and H. O. McDevitt. 1991. MHC class II-associated
variation in the production of tumor necrosis factor in mice and humans:
relevance to the pathogenesis of autoimmune diseases. Immunol Res 10:156-168.
88.
Scheu, S., J. Alferink, T. Potzel, W. Barchet, U. Kalinke, and K. Pfeffer. 2002.
Targeted disruption of LIGHT causes defects in costimulatory T cell activation
and reveals cooperation with lymphotoxin beta in mesenteric lymph node genesis.
J Exp Med 195:1613-1624.
89.
Tamada, K., K. Shimozaki, A. I. Chapoval, G. Zhu, G. Sica, D. Flies, T. Boone,
H. Hsu, Y. X. Fu, S. Nagata, J. Ni, and L. Chen. 2000. Modulation of T-cellmediated immunity in tumor and graft-versus-host disease models through the
LIGHT co-stimulatory pathway. Nat Med 6:283-289.
90.
Oshima, H., H. Nakano, C. Nohara, T. Kobata, A. Nakajima, N. A. Jenkins, D. J.
Gilbert, N. G. Copeland, T. Muto, H. Yagita, and K. Okumura. 1998.
Characterization of murine CD70 by molecular cloning and mAb. Int Immunol
10:517-526.
113 91.
Agematsu, K., T. Kobata, K. Sugita, G. J. Freeman, M. P. Beckmann, S. F.
Schlossman, and C. Morimoto. 1994. Role of CD27 in T cell immune response.
Analysis by recombinant soluble CD27. J Immunol 153:1421-1429.
92.
Hintzen, R. Q., S. M. Lens, K. Lammers, H. Kuiper, M. P. Beckmann, and R. A.
van Lier. 1995. Engagement of CD27 with its ligand CD70 provides a second
signal for T cell activation. J Immunol 154:2612-2623.
93.
Hendriks, J., L. A. Gravestein, K. Tesselaar, R. A. van Lier, T. N. Schumacher,
and J. Borst. 2000. CD27 is required for generation and long-term maintenance of
T cell immunity. Nat Immunol 1:433-440.
94.
Rogers, P. R., J. Song, I. Gramaglia, N. Killeen, and M. Croft. 2001. OX40
promotes Bcl-xL and Bcl-2 expression and is essential for long-term survival of
CD4 T cells. Immunity 15:445-455.
95.
Brocker, T., A. Gulbranson-Judge, S. Flynn, M. Riedinger, C. Raykundalia, and
P. Lane. 1999. CD4 T cell traffic control: in vivo evidence that ligation of OX40
on CD4 T cells by OX40-ligand expressed on dendritic cells leads to the
accumulation of CD4 T cells in B follicles. Eur J Immunol 29:1610-1616.
96.
Murata, K., M. Nose, L. C. Ndhlovu, T. Sato, K. Sugamura, and N. Ishii. 2002.
Constitutive OX40/OX40 ligand interaction induces autoimmune-like diseases. J
Immunol 169:4628-4636.
Gramaglia, I., A. Jember, S. D. Pippig, A. D. Weinberg, N. Killeen, and M. Croft.
2000. The OX40 costimulatory receptor determines the development of CD4
memory by regulating primary clonal expansion. J Immunol 165:3043-3050.
97.
98.
99.
De Smedt, T., J. Smith, P. Baum, W. Fanslow, E. Butz, and C. Maliszewski.
2002. Ox40 costimulation enhances the development of T cell responses induced
by dendritic cells in vivo. J Immunol 168:661-670.
Xiao, Z., K. A. Casey, S. C. Jameson, J. M. Curtsinger, and M. F. Mescher. 2009.
Programming for CD8 T cell memory development requires IL-12 or type I IFN.
J Immunol 182:2786-2794.
100.
Le Bon, A., N. Etchart, C. Rossmann, M. Ashton, S. Hou, D. Gewert, P. Borrow,
and D. F. Tough. 2003. Cross-priming of CD8+ T cells stimulated by virusinduced type I interferon. Nat Immunol 4:1009-1015.
101.
Kelly, J. M., P. K. Darcy, J. L. Markby, D. I. Godfrey, K. Takeda, H. Yagita, and
M. J. Smyth. 2002. Induction of tumor-specific T cell memory by NK cellmediated tumor rejection. Nat Immunol 3:83-90.
102.
Liu, C., Y. Lou, G. Lizee, H. Qin, S. Liu, B. Rabinovich, G. J. Kim, Y. H. Wang,
Y. Ye, A. G. Sikora, W. W. Overwijk, Y. J. Liu, G. Wang, and P. Hwu. 2008.
114 Plasmacytoid dendritic cells induce NK cell-dependent, tumor antigen-specific T
cell cross-priming and tumor regression in mice. The Journal of Clinical
Investigation 118:1165-1175.
103.
Zingoni, A., T. Sornasse, B. G. Cocks, Y. Tanaka, A. Santoni, and L. L. Lanier.
2004. Cross-talk between activated human NK cells and CD4+ T cells via OX40OX40 ligand interactions. J Immunol 173:3716-3724.
104.
Wilcox, R. A., K. Tamada, S. E. Strome, and L. Chen. 2002. Signaling through
NK cell-associated CD137 promotes both helper function for CD8+ cytolytic T
cells and responsiveness to IL-2 but not cytolytic activity. J Immunol 169:42304236.
105.
Papadakis, K. A., J. L. Prehn, C. Landers, Q. Han, X. Luo, S. C. Cha, P. Wei, and
S. R. Targan. 2004. TL1A synergizes with IL-12 and IL-18 to enhance IFNgamma production in human T cells and NK cells. J Immunol 172:7002-7007.
106.
Fang, L., B. Adkins, V. Deyev, and E. R. Podack. 2008. Essential role of TNF
receptor superfamily 25 (TNFRSF25) in the development of allergic lung
inflammation. J Exp Med 205:1037-1048.
107.
Elgueta, R., M. J. Benson, V. C. de Vries, A. Wasiuk, Y. Guo, and R. J. Noelle.
2009. Molecular mechanism and function of CD40/CD40L engagement in the
immune system. Immunol Rev 229:152-172.
108.
Kawabe, T., T. Naka, K. Yoshida, T. Tanaka, H. Fujiwara, S. Suematsu, N.
Yoshida, T. Kishimoto, and H. Kikutani. 1994. The immune responses in CD40deficient mice: impaired immunoglobulin class switching and germinal center
formation. Immunity 1:167-178.
109.
Castigli, E., F. W. Alt, L. Davidson, A. Bottaro, E. Mizoguchi, A. K. Bhan, and
R. S. Geha. 1994. CD40-deficient mice generated by recombination-activating
gene-2-deficient blastocyst complementation. Proc Natl Acad Sci U S A
91:12135-12139.
110.
Schiemann, B., J. L. Gommerman, K. Vora, T. G. Cachero, S. Shulga-Morskaya,
M. Dobles, E. Frew, and M. L. Scott. 2001. An essential role for BAFF in the
normal development of B cells through a BCMA-independent pathway. Science
293:2111-2114.
111.
Miller, D. J., and C. E. Hayes. 1991. Phenotypic and genetic characterization of a
unique B lymphocyte deficiency in strain A/WySnJ mice. Eur J Immunol
21:1123-1130.
115 112.
Thompson, J. S., S. A. Bixler, F. Qian, K. Vora, M. L. Scott, T. G. Cachero, C.
Hession, P. Schneider, I. D. Sizing, C. Mullen, K. Strauch, M. Zafari, C. D.
Benjamin, J. Tschopp, J. L. Browning, and C. Ambrose. 2001. BAFF-R, a newly
identified TNF receptor that specifically interacts with BAFF. Science 293:21082111.
113.
Kashiwakura, J., H. Yokoi, H. Saito, and Y. Okayama. 2004. T cell proliferation
by direct cross-talk between OX40 ligand on human mast cells and OX40 on
human T cells: comparison of gene expression profiles between human tonsillar
and lung-cultured mast cells. J Immunol 173:5247-5257.
114.
Nakae, S., H. Suto, M. Iikura, M. Kakurai, J. D. Sedgwick, M. Tsai, and S. J.
Galli. 2006. Mast cells enhance T cell activation: importance of mast cell
costimulatory molecules and secreted TNF. J Immunol 176:2238-2248.
115.
Nishimoto, H., S. W. Lee, H. Hong, K. G. Potter, M. Maeda-Yamamoto, T.
Kinoshita, Y. Kawakami, R. S. Mittler, B. S. Kwon, C. F. Ware, M. Croft, and T.
Kawakami. 2005. Costimulation of mast cells by 4-1BB, a member of the tumor
necrosis factor receptor superfamily, with the high-affinity IgE receptor. Blood
106:4241-4248.
116.
Baumann, R., S. Yousefi, D. Simon, S. Russmann, C. Mueller, and H. U. Simon.
2004. Functional expression of CD134 by neutrophils. Eur J Immunol 34:22682275.
117.
Kim, M. Y., F. M. Gaspal, H. E. Wiggett, F. M. McConnell, A. GulbransonJudge, C. Raykundalia, L. S. Walker, M. D. Goodall, and P. J. Lane. 2003.
CD4(+)CD3(-) accessory cells costimulate primed CD4 T cells through OX40
and CD30 at sites where T cells collaborate with B cells. Immunity 18:643-654.
118.
Kim, M. Y., K. M. Toellner, A. White, F. M. McConnell, F. M. Gaspal, S. M.
Parnell, E. Jenkinson, G. Anderson, and P. J. Lane. 2006. Neonatal and adult
CD4+ CD3- cells share similar gene expression profile, and neonatal cells upregulate OX40 ligand in response to TL1A (TNFSF15). J Immunol 177:30743081.
119.
So, T., and M. Croft. 2007. Cutting edge: OX40 inhibits TGF-beta- and antigendriven conversion of naive CD4 T cells into CD25+Foxp3+ T cells. J Immunol
179:1427-1430.
120.
Vu, M. D., X. Xiao, W. Gao, N. Degauque, M. Chen, A. Kroemer, N. Killeen, N.
Ishii, and X. C. Li. 2007. OX40 costimulation turns off Foxp3+ Tregs. Blood
110:2501-2510.
116 121.
Chen, M., X. Xiao, G. Demirci, and X. C. Li. 2008. OX40 controls islet allograft
tolerance in CD154 deficient mice by regulating FOXP3+ Tregs. Transplantation
85:1659-1662.
122.
Takeda, I., S. Ine, N. Killeen, L. C. Ndhlovu, K. Murata, S. Satomi, K. Sugamura,
and N. Ishii. 2004. Distinct roles for the OX40-OX40 ligand interaction in
regulatory and nonregulatory T cells. J Immunol 172:3580-3589.
123.
Choi, B. K., J. S. Bae, E. M. Choi, W. J. Kang, S. Sakaguchi, D. S. Vinay, and B.
S. Kwon. 2004. 4-1BB-dependent inhibition of immunosuppression by activated
CD4+CD25+ T cells. J Leukoc Biol 75:785-791.
124.
Valzasina, B., C. Guiducci, H. Dislich, N. Killeen, A. D. Weinberg, and M. P.
Colombo. 2005. Triggering of OX40 (CD134) on CD4(+)CD25+ T cells blocks
their inhibitory activity: a novel regulatory role for OX40 and its comparison with
GITR. Blood 105:2845-2851.
125.
Kroemer, A., X. Xiao, M. D. Vu, W. Gao, K. Minamimura, M. Chen, T. Maki,
and X. C. Li. 2007. OX40 controls functionally different T cell subsets and their
resistance to depletion therapy. J Immunol 179:5584-5591.
126.
Piconese, S., B. Valzasina, and M. P. Colombo. 2008. OX40 triggering blocks
suppression by regulatory T cells and facilitates tumor rejection. J Exp Med
205:825-839.
127.
Robertson, S. J., R. J. Messer, A. B. Carmody, R. S. Mittler, C. Burlak, and K. J.
Hasenkrug. 2008. CD137 costimulation of CD8+ T cells confers resistance to
suppression by virus-induced regulatory T cells. J Immunol 180:5267-5274.
128.
Meylan, F., Y. J. Song, I. Fuss, S. Villarreal, E. Kahle, I. J. Malm, K. Acharya, H.
L. Ramos, L. Lo, M. M. Mentink-Kane, T. A. Wynn, T. S. Migone, W. Strober,
and R. M. Siegel. 2011. The TNF-family cytokine TL1A drives IL-13-dependent
small intestinal inflammation. Mucosal Immunol 4:172-185.
129.
Taraban, V. Y., T. J. Slebioda, J. E. Willoughby, S. L. Buchan, S. James, B.
Sheth, N. R. Smyth, G. J. Thomas, E. C. Wang, and A. Al-Shamkhani. 2011.
Sustained TL1A expression modulates effector and regulatory T-cell responses
and drives intestinal goblet cell hyperplasia. Mucosal Immunol 4:186-196.
130.
Schreiber, T. H., D. Wolf, M. S. Tsai, J. Chirinos, V. V. Deyev, L. Gonzalez, T.
R. Malek, R. B. Levy, and E. R. Podack. 2010. Therapeutic Treg expansion in
mice by TNFRSF25 prevents allergic lung inflammation. The Journal of Clinical
Investigation 120:3629-3640.
117 131.
Moore, R. J., D. M. Owens, G. Stamp, C. Arnott, F. Burke, N. East, H.
Holdsworth, L. Turner, B. Rollins, M. Pasparakis, G. Kollias, and F. Balkwill.
1999. Mice deficient in tumor necrosis factor-alpha are resistant to skin
carcinogenesis. Nat Med 5:828-831.
132.
Carvalho, R. F., A. K. Ulfgren, M. Engstrom, E. Klint, and G. Nilsson. 2009.
CD153 in rheumatoid arthritis: detection of a soluble form in serum and synovial
fluid, and expression by mast cells in the rheumatic synovium. J Rheumatol
36:501-507.
133.
Gerli, R., C. Pitzalis, O. Bistoni, B. Falini, V. Costantini, A. Russano, and C.
Lunardi. 2000. CD30+ T cells in rheumatoid synovitis: mechanisms of
recruitment and functional role. J Immunol 164:4399-4407.
134.
Giacomelli, R., A. Passacantando, R. Perricone, I. Parzanese, M. Rascente, G.
Minisola, and G. Tonietti. 2001. T lymphocytes in the synovial fluid of patients
with active rheumatoid arthritis display CD134-OX40 surface antigen. Clin Exp
Rheumatol 19:317-320.
135.
Tan, S. M., D. Xu, V. Roschke, J. W. Perry, D. G. Arkfeld, G. R. Ehresmann, T.
S. Migone, D. M. Hilbert, and W. Stohl. 2003. Local production of B lymphocyte
stimulator protein and APRIL in arthritic joints of patients with inflammatory
arthritis. Arthritis Rheum 48:982-992.
136.
Cheema, G. S., V. Roschke, D. M. Hilbert, and W. Stohl. 2001. Elevated serum B
lymphocyte stimulator levels in patients with systemic immune-based rheumatic
diseases. Arthritis Rheum 44:1313-1319.
137.
Seyler, T. M., Y. W. Park, S. Takemura, R. J. Bram, P. J. Kurtin, J. J. Goronzy,
and C. M. Weyand. 2005. BLyS and APRIL in rheumatoid arthritis. The Journal
of Clinical Investigation 115:3083-3092.
Siddiqui, S., V. Mistry, C. Doe, S. Stinson, M. Foster, and C. Brightling. 2010.
Airway wall expression of OX40/OX40L and interleukin-4 in asthma. Chest
137:797-804.
138.
139.
Oflazoglu, E., I. S. Grewal, and H. Gerber. 2009. Targeting CD30/CD30L in
oncology and autoimmune and inflammatory diseases. Advances in experimental
medicine and biology 647:174-185.
140.
Hastie, A. T., W. C. Moore, D. A. Meyers, P. L. Vestal, H. Li, S. P. Peters, and E.
R. Bleecker. 2010. Analyses of asthma severity phenotypes and inflammatory
proteins in subjects stratified by sputum granulocytes. J Allergy Clin Immunol
125:1028-1036 e1013.
141.
Straus, S. E., E. S. Jaffe, J. M. Puck, J. K. Dale, K. B. Elkon, A. Rosen-Wolff, A.
M. Peters, M. C. Sneller, C. W. Hallahan, J. Wang, R. E. Fischer, C. M. Jackson,
118 A. Y. Lin, C. Baumler, E. Siegert, A. Marx, A. K. Vaishnaw, T. Grodzicky, T. A.
Fleisher, and M. J. Lenardo. 2001. The development of lymphomas in families
with autoimmune lymphoproliferative syndrome with germline Fas mutations and
defective lymphocyte apoptosis. Blood 98:194-200.
142.
Weinberg, A. D., D. N. Bourdette, T. J. Sullivan, M. Lemon, J. J. Wallin, R.
Maziarz, M. Davey, F. Palida, W. Godfrey, E. Engleman, R. J. Fulton, H. Offner,
and A. A. Vandenbark. 1996. Selective depletion of myelin-reactive T cells with
the anti-OX-40 antibody ameliorates autoimmune encephalomyelitis. Nat Med
2:183-189.
143.
Boot, E. P., G. A. Koning, G. Storm, J. P. Wagenaar-Hilbers, W. van Eden, L. A.
Everse, and M. H. Wauben. 2005. CD134 as target for specific drug delivery to
auto-aggressive CD4+ T cells in adjuvant arthritis. Arthritis Res Ther 7:R604615.
144.
Doherty, T. A., P. Soroosh, D. H. Broide, and M. Croft. 2009. CD4+ cells are
required for chronic eosinophilic lung inflammation but not airway remodeling.
Am J Physiol Lung Cell Mol Physiol 296:L229-235.
145.
Chapoval, S. P., E. V. Marietta, M. K. Smart, and C. S. David. 2001.
Requirements for allergen-induced airway inflammation and hyperreactivity in
CD4-deficient and CD4-sufficient HLA-DQ transgenic mice. J Allergy Clin
Immunol 108:764-771.
146.
Wolf, D., T. H. Schreiber, P. Tryphonopoulos, S. Li, A. G. Tzakis, P. Ruiz, and E.
R. Podack. 2012. Tregs Expanded In Vivo by TNFRSF25 Agonists Promote
Cardiac Allograft Survival. Transplantation 94:569-574.
147.
PB, J. R., T. H. Schreiber, N. K. Rajasagi, A. Suryawanshi, S. Mulik, T. VeigaParga, T. Niki, M. Hirashima, E. R. Podack, and B. T. Rouse. 2012. TNFRSF25
Agonistic Antibody and Galectin-9 Combination Therapy Controls Herpes
Simplex Virus-Induced Immunoinflammatory Lesions. J Virol 86:10606-10620.
148.
Zhang, N., A. J. Sanders, L. Ye, H. G. Kynaston, and W. G. Jiang. 2010.
Expression of vascular endothelial growth inhibitor (VEGI) in human urothelial
cancer of the bladder and its effects on the adhesion and migration of bladder
cancer cells in vitro. Anticancer Res 30:87-95.
149.
Zhai, Y., J. Yu, L. Iruela-Arispe, W. Q. Huang, Z. Wang, A. J. Hayes, J. Lu, G.
Jiang, L. Rojas, M. E. Lippman, J. Ni, G. L. Yu, and L. Y. Li. 1999. Inhibition of
angiogenesis and breast cancer xenograft tumor growth by VEGI, a novel
cytokine of the TNF superfamily. Int J Cancer 82:131-136.
119 150.
Zhai, Y., J. Ni, G. W. Jiang, J. Lu, L. Xing, C. Lincoln, K. C. Carter, F. Janat, D.
Kozak, S. Xu, L. Rojas, B. B. Aggarwal, S. Ruben, L. Y. Li, R. Gentz, and G. L.
Yu. 1999. VEGI, a novel cytokine of the tumor necrosis factor family, is an
angiogenesis inhibitor that suppresses the growth of colon carcinomas in vivo.
Faseb J 13:181-189.
151.
Yue, T. L., J. Ni, A. M. Romanic, J. L. Gu, P. Keller, C. Wang, S. Kumar, G. L.
Yu, T. K. Hart, X. Wang, Z. Xia, W. E. DeWolf, Jr., and G. Z. Feuerstein. 1999.
TL1, a novel tumor necrosis factor-like cytokine, induces apoptosis in endothelial
cells. Involvement of activation of stress protein kinases (stress-activated protein
kinase and p38 mitogen-activated protein kinase) and caspase-3-like protease. J
Biol Chem 274:1479-1486.
152.
Meylan, F., T. S. Davidson, E. Kahle, M. Kinder, K. Acharya, D. Jankovic, V.
Bundoc, M. Hodges, E. M. Shevach, A. Keane-Myers, E. C. Wang, and R. M.
Siegel. 2008. The TNF-family receptor DR3 is essential for diverse T cellmediated inflammatory diseases. Immunity 29:79-89.
153.
Wang, E. C., J. Kitson, A. Thern, J. Williamson, S. N. Farrow, and M. J. Owen.
2001. Genomic structure, expression, and chromosome mapping of the mouse
homologue for the WSL-1 (DR3, Apo3, TRAMP, LARD, TR3, TNFRSF12)
gene. Immunogenetics 53:59-63.
154.
Screaton, G. R., X. N. Xu, A. L. Olsen, A. E. Cowper, R. Tan, A. J. McMichael,
and J. I. Bell. 1997. LARD: a new lymphoid-specific death domain containing
receptor regulated by alternative pre-mRNA splicing. Proc Natl Acad Sci U S A
94:4615-4619.
155.
Tan, K. B., J. Harrop, M. Reddy, P. Young, J. Terrett, J. Emery, G. Moore, and A.
Truneh. 1997. Characterization of a novel TNF-like ligand and recently described
TNF ligand and TNF receptor superfamily genes and their constitutive and
inducible expression in hematopoietic and non-hematopoietic cells. Gene 204:3546.
156.
Warzocha, K., P. Ribeiro, C. Charlot, N. Renard, B. Coiffier, and G. Salles. 1998.
A new death receptor 3 isoform: expression in human lymphoid cell lines and
non-Hodgkin's lymphomas. Biochem Biophys Res Commun 242:376-379.
157.
Kang, Y. J., W. J. Kim, H. U. Bae, D. I. Kim, Y. B. Park, J. E. Park, B. S. Kwon,
and W. H. Lee. 2005. Involvement of TL1A and DR3 in induction of proinflammatory cytokines and matrix metalloproteinase-9 in atherogenesis.
Cytokine 29:229-235.
158.
Haritunians, T., K. D. Taylor, S. R. Targan, M. Dubinsky, A. Ippoliti, S. Kwon,
X. Guo, G. Y. Melmed, D. Berel, E. Mengesha, B. M. Psaty, N. L. Glazer, E. A.
120 Vasiliauskas, J. I. Rotter, P. R. Fleshner, and D. P. McGovern. 2010. Genetic
predictors of medically refractory ulcerative colitis. Inflamm Bowel Dis 16:18301840.
159.
Tremelling, M., C. Berzuini, D. Massey, F. Bredin, C. Price, C. Dawson, S. A.
Bingham, and M. Parkes. 2008. Contribution of TNFSF15 gene variants to
Crohn's disease susceptibility confirmed in UK population. Inflamm Bowel Dis
14:733-737.
160.
Latiano, A., O. Palmieri, T. Latiano, G. Corritore, F. Bossa, G. Martino, G.
Biscaglia, D. Scimeca, M. R. Valvano, M. Pastore, A. Marseglia, R. D'Inca, A.
Andriulli, and V. Annese. 2011. Investigation of multiple susceptibility loci for
inflammatory bowel disease in an Italian cohort of patients. PLoS One 6:e22688.
161.
Shih, D. Q., R. Barrett, X. Zhang, N. Yeager, H. W. Koon, P. Phaosawasdi, Y.
Song, B. Ko, M. H. Wong, K. S. Michelsen, G. Martins, C. Pothoulakis, and S. R.
Targan. 2011. Constitutive TL1A (TNFSF15) expression on lymphoid or myeloid
cells leads to mild intestinal inflammation and fibrosis. PLoS One 6:e16090.
162.
Pappu, B. P., A. Borodovsky, T. S. Zheng, X. Yang, P. Wu, X. Dong, S. Weng, B.
Browning, M. L. Scott, L. Ma, L. Su, Q. Tian, P. Schneider, R. A. Flavell, C.
Dong, and L. C. Burkly. 2008. TL1A-DR3 interaction regulates Th17 cell
function and Th17-mediated autoimmune disease. J Exp Med 205:1049-1062.
163.
Bull, M. J., A. S. Williams, Z. Mecklenburgh, C. J. Calder, J. P. Twohig, C.
Elford, B. A. Evans, T. F. Rowley, T. J. Slebioda, V. Y. Taraban, A. AlShamkhani, and E. C. Wang. 2008. The Death Receptor 3-TNF-like protein 1A
pathway drives adverse bone pathology in inflammatory arthritis. J Exp Med
205:2457-2464.
164.
Zhang, J., X. Wang, H. Fahmi, S. Wojcik, J. Fikes, Y. Yu, J. Wu, and H. Luo.
2009. Role of TL1A in the pathogenesis of rheumatoid arthritis. J Immunol
183:5350-5357.
165.
Thilenius, A. R., K. Braun, and J. H. Russell. 1997. Agonist antibody and Fas
ligand mediate different sensitivity to death in the signaling pathways of Fas and
cytoplasmic mutants. Eur J Immunol 27:1108-1114.
166.
Totpal, K., R. LaPushin, T. Kohno, B. G. Darnay, and B. B. Aggarwal. 1994.
TNF and its receptor antibody agonist differ in mediation of cellular responses. J
Immunol 153:2248-2257.
167.
Barrett, R., X. Zhang, H. W. Koon, M. Vu, J. Y. Chang, N. Yeager, M. A.
Nguyen, K. S. Michelsen, D. Berel, C. Pothoulakis, S. R. Targan, and D. Q. Shih.
121 2012. Constitutive TL1A expression under colitogenic conditions modulates the
severity and location of gut mucosal inflammation and induces fibrostenosis. Am
J Pathol 180:636-649.
168.
Schreiber, T. H., S. Q. Khan, and E. R. Podack. 2011. Response to Taraban,
Ferdinand, and Al-Shamkhani. The Journal of Clinical Investigation 121:465465.
169.
Strbo, N., K. Yamazaki, K. Lee, D. Rukavina, and E. R. Podack. 2002. Heat
shock fusion protein gp96-Ig mediates strong CD8 CTL expansion in vivo. Am J
Reprod Immunol 48:220-225.
170.
Bebbington, C. R., G. Renner, S. Thomson, D. King, D. Abrams, and G. T.
Yarranton. 1992. High-level expression of a recombinant antibody from myeloma
cells using a glutamine synthetase gene as an amplifiable selectable marker.
Biotechnology (N Y) 10:169-175.
171.
Zhan, C., Q. Yan, Y. Patskovsky, Z. Li, R. Toro, A. Meyer, H. Cheng, M.
Brenowitz, S. G. Nathenson, and S. C. Almo. 2009. Biochemical and structural
characterization of the human TL1A ectodomain. Biochemistry 48:7636-7645.
172.
Zhan, C., Y. Patskovsky, Q. Yan, Z. Li, U. Ramagopal, H. Cheng, M. Brenowitz,
X. Hui, S. G. Nathenson, and S. C. Almo. 2011. Decoy strategies: the structure of
TL1A:DcR3 complex. Structure 19:162-171.
Jin, T., F. Guo, S. Kim, A. Howard, and Y. Z. Zhang. 2007. X-ray crystal
structure of TNF ligand family member TL1A at 2.1A. Biochem Biophys Res
Commun 364:1-6.
173.
174.
Jin, T., S. Kim, F. Guo, A. Howard, and Y. Z. Zhang. 2007. Purification and
crystallization of recombinant human TNF-like ligand TL1A. Cytokine 40:115122.
175.
Wan, Y. Y., and R. A. Flavell. 2005. Identifying Foxp3-expressing suppressor T
cells with a bicistronic reporter. Proc Natl Acad Sci U S A 102:5126-5131.
176.
Ito, T., Y. H. Wang, O. Duramad, S. Hanabuchi, O. A. Perng, M. Gilliet, F. X.
Qin, and Y. J. Liu. 2006. OX40 ligand shuts down IL-10-producing regulatory T
cells. Proc Natl Acad Sci U S A 103:13138-13143.
177.
Andersson, J., I. Stefanova, G. L. Stephens, and E. M. Shevach. 2007.
CD4+CD25+ regulatory T cells are activated in vivo by recognition of self. Int
Immunol 19:557-566.
178.
Leithauser, F., T. Meinhardt-Krajina, K. Fink, B. Wotschke, P. Moller, and J.
Reimann. 2006. Foxp3-expressing CD103+ regulatory T cells accumulate in
122 dendritic cell aggregates of the colonic mucosa in murine transfer colitis. Am J
Pathol 168:1898-1909.
179.
Zhao, D., C. Zhang, T. Yi, C. L. Lin, I. Todorov, F. Kandeel, S. Forman, and D.
Zeng. 2008. In vivo-activated CD103+CD4+ regulatory T cells ameliorate
ongoing chronic graft-versus-host disease. Blood 112:2129-2138.
180.
Beyersdorf, N., X. Ding, J. K. Tietze, and T. Hanke. 2007. Characterization of
mouse CD4 T cell subsets defined by expression of KLRG1. Eur J Immunol
37:3445-3454.
181.
Yoshiya, K., P. H. Lapchak, T. H. Thai, L. Kannan, P. Rani, J. J. Dalle Lucca, and
G. C. Tsokos. 2011. Depletion of gut commensal bacteria attenuates intestinal
ischemia/reperfusion injury. Am J Physiol Gastrointest Liver Physiol 301:G10201030.
Monteiro, J. P., J. Farache, A. C. Mercadante, J. A. Mignaco, M. Bonamino, and
A. Bonomo. 2008. Pathogenic effector T cell enrichment overcomes regulatory T
cell control and generates autoimmune gastritis. J Immunol 181:5895-5903.
182.
183.
184.
Tang, Q., J. Y. Adams, C. Penaranda, K. Melli, E. Piaggio, E. Sgouroudis, C. A.
Piccirillo, B. L. Salomon, and J. A. Bluestone. 2008. Central role of defective
interleukin-2 production in the triggering of islet autoimmune destruction.
Immunity 28:687-697.
Taams, L. S., E. P. Boot, W. van Eden, and M. H. Wauben. 2000. 'Anergic' T
cells modulate the T-cell activating capacity of antigen-presenting cells. J
Autoimmun 14:335-341.
185.
Eddahri, F., G. Oldenhove, S. Denanglaire, J. Urbain, O. Leo, and F. Andris.
2006. CD4+ CD25+ regulatory T cells control the magnitude of T-dependent
humoral immune responses to exogenous antigens. Eur J Immunol 36:855-863.
186.
Mempel, T. R., M. J. Pittet, K. Khazaie, W. Weninger, R. Weissleder, H. von
Boehmer, and U. H. von Andrian. 2006. Regulatory T cells reversibly suppress
cytotoxic T cell function independent of effector differentiation. Immunity
25:129-141.
187.
Smith, J. A., J. Y. Tso, M. R. Clark, M. S. Cole, and J. A. Bluestone. 1997.
Nonmitogenic anti-CD3 monoclonal antibodies deliver a partial T cell receptor
signal and induce clonal anergy. J Exp Med 185:1413-1422.
188.
Smith, J. A., Q. Tang, and J. A. Bluestone. 1998. Partial TCR signals delivered by
FcR-nonbinding anti-CD3 monoclonal antibodies differentially regulate
individual Th subsets. J Immunol 160:4841-4849.
189.
Belghith, M., J. A. Bluestone, S. Barriot, J. Megret, J. F. Bach, and L. Chatenoud.
2003. TGF-beta-dependent mechanisms mediate restoration of self-tolerance
123 induced by antibodies to CD3 in overt autoimmune diabetes. Nat Med 9:12021208.
190.
Xiao, X., W. Gong, G. Demirci, W. Liu, S. Spoerl, X. Chu, D. K. Bishop, L. A.
Turka, and X. C. Li. 2012. New insights on OX40 in the control of T cell
immunity and immune tolerance in vivo. J Immunol 188:892-901.
191.
Bazzoni, F., and B. Beutler. 1996. The tumor necrosis factor ligand and receptor
families. N Engl J Med 334:1717-1725.
192.
Zhou, Z., Y. Tone, X. Song, K. Furuuchi, J. D. Lear, H. Waldmann, M. Tone, M.
I. Greene, and R. Murali. 2008. Structural basis for ligand-mediated mouse GITR
activation. Proc Natl Acad Sci U S A 105:641-645.
193.
Bollyky, P. L., J. B. Bice, I. R. Sweet, B. A. Falk, J. A. Gebe, A. E. Clark, V. H.
Gersuk, A. Aderem, T. R. Hawn, and G. T. Nepom. 2009. The toll-like receptor
signaling molecule Myd88 contributes to pancreatic beta-cell homeostasis in
response to injury. PLoS One 4:e5063.
194.
Bruno, L., J. Kirberg, and H. von Boehmer. 1995. On the cellular basis of
immunological T cell memory. Immunity 2:37-43.
Garcia, S., J. DiSanto, and B. Stockinger. 1999. Following the development of a
CD4 T cell response in vivo: from activation to memory formation. Immunity
11:163-171.
195.
196.
Gillies, S. D., and J. S. Wesolowski. 1990. Antigen binding and biological
activities of engineered mutant chimeric antibodies with human tumor
specificities. Hum Antibodies Hybridomas 1:47-54.
197.
Slebioda, T. J., T. F. Rowley, J. R. Ferdinand, J. E. Willoughby, S. L. Buchan, V.
Y. Taraban, and A. Al-Shamkhani. 2011. Triggering of TNFRSF25 promotes
CD8(+) T-cell responses and anti-tumor immunity. Eur J Immunol 41:2606-2611.
198.
Yamazaki, K., T. Nguyen, and E. R. Podack. 1999. Cutting edge: tumor secreted
heat shock-fusion protein elicits CD8 cells for rejection. J Immunol 163:51785182.
199.
Prehn, J. L., S. Mehdizadeh, C. J. Landers, X. Luo, S. C. Cha, P. Wei, and S. R.
Targan. 2004. Potential role for TL1A, the new TNF-family member and potent
costimulator of IFN-gamma, in mucosal inflammation. Clin Immunol 112:66-77.
200.
Twohig, J. P., M. Marsden, S. M. Cuff, J. R. Ferdinand, A. M. Gallimore, W. V.
Perks, A. Al-Shamkhani, I. R. Humphreys, and E. C. Wang. 2012. The death
receptor 3/TL1A pathway is essential for efficient development of antiviral
CD4(+) and CD8(+) T-cell immunity. Faseb J 26:3575-3586.
201.
124 Nishikawa, H., T. Kato, M. Hirayama, Y. Orito, E. Sato, N. Harada, S. Gnjatic, L.
J. Old, and H. Shiku. 2008. Regulatory T cell-resistant CD8+ T cells induced by
glucocorticoid-induced tumor necrosis factor receptor signaling. Cancer Res
68:5948-5954.