Download Tracking antigen specific T cell dynamics in vivo

M. Carrie Miceli
Regulatory T Cells
May18, 2005
• Reviving Suppression, Harvey Cantor, Nature
Immunology, April 2004
• A well adapted regulatory contrivance:regulatory
T cell development and the forkhead family
transcription factor Foxp3. Fontenot and
Rudensky, Nature Immunology March 2005
• How does the immune system prevent self
reactivity while maintaining reactivity to
invaders/non-self?
– Clonal deletion/inactivation of auto-reactive
cells
– Regulatory T cells keep potentially pathogenic
self reactive T cells in check through
“suppressive” mechanisms
CD25+CD4 regulatory T cells
play a role in maintaining
peripheral T cell tolerance
• Taboo of T “suppressor” cells
– CD8+
– Complex regulatory networks mapping to I-J (within
the MHC)
– Molecular cloning of TCR and MHC disappointed
• CD4+ Tregs gain respect
– CD4+; CD25+;CTLA-4+;CD45RBlo;GITRhi
– We need better markers, still heterogeneity
– Foxp 3 dependent
Journal of Immunology 1995 155:1151
lymph
nodes
from a
2 month
old balb c
Autoimmune disease
Induced by adoptive
transfer of lymphocyte
populations
depleted by antibody and
complement
CTLA-4 is expressed constitutively on
CD25+CD45RBlow CD4+ cells (but not a
specific marker).
F. powrie J exp Med
. (a–c) CTLA-4 staining on permeabilized
(a)CD45RBhighCD4+,(b)CD25+CD45RBlo
wCD4+, and © CD25-CD45RBlowCD4+
splenocytes. SSC, side scatter. (d–f) CTLA-4
expression on the progeny of (e)
CD25+CD45RBlowCD4+ and (d and f)
CD45RBhighCD4+ cells after transfer into
immunodeficient mice. C57BL/6
recombination activating gene (RAG)2-/mice received CD45RBhighCD4+ cells from
C57BL/6 mice (CD45.2) (d) alone or (e and
f) in combination with 105
CD25+CD45RBlowCD4+ cells
from C57BL/6.SJL.CD45 congenic mice
(CD45.1). 4 wk after transfer, cells from the
mesenteric lymph nodes were stained for
CTLA-4 and congenic marker expression
b=CD4+CD25+
CD4+CD25+ (CD45low) inhibit autoimmune
colitis. Anti–CTLA-4 treatment abrogates the
function of regulatory T cells.
Open control Ig
Closed CTLA4. Adoptive
transfer of CD4
T cell into RAG
deficient mice
Re-evaluation of CTLA-4
autoimmune phenotype
• Could CTLA4 deficiency result in defective Treg
function and thus autoimminty (rather than role of
CTLA-4 on T cell autonomous regulation)?
• CTLA phenotype rescued by complementing
CTLA-4 -/- BM with wild type BM (mixed
chimera).
• CTLA-4 deficient bone marrow can make
CD25+CD4+ (function vs development)
CD4+CD25+ Immunoregulatory T Cells
Suppress Polyclonal T Cell Activation In Vitro
by Inhibiting Interleukin 2 Production
Angela M. Thornton and Ethan M. Shevach J.
Exp. Med. 1998 188: 287-296.
Treg cells look like half activated
anergic cells; IL-2 rescues
Fig. 1. Characterization of
CD4+CD25+ cells. (A) Total lymph
node cells (left) or purified
CD4+CD25+ cells (right) from
BALB/c mice were stained with PEconjugated anti-CD4 and biotinconjugated anti-CD25 followed by
FITC-conjugated streptavidin. (B)
Purified CD4+ CD25 cells or purified
CD4+CD25+ cells were double stained
for CD25 and the indicated surface
molecules. (C) Proliferative
responses of CD4+ CD25+ cells. Cells
(5 X 104) were incubated with the
indicated agents (10 ng/ml PMA, 1
mM ionomycin, 3 ng/ml IL-2, 3.0
µg/ml soluble anti-CD3, 3.0 µg/ml Con
A, 10 µg/ml anti-CD28, or wells
coated with 10 µg/ml anti-CD3) in the
presence of AC (5 X 104). Results are
expressed as the mean of triplicate
cultures.
CD4+CD25+ cells suppress the proliferation of CD4+ cells;
need to be stimulated through their TCR
. (A) CD4+CD25 cells (5 X104) were incubated with plate bound anti-CD3 (triangles) or with 1.0 µg/ml
soluble anti-CD3 (squares) in the presence of AC (5 X 104) and the indicated number of CD4+CD25+ cells.
(B) CD4+CD25+ cells do not suppress antigen-specific CD4+ cells from TCR transgenic mice. CD4+CD25
cells (5 ? 104) were purified from DO.11.10 SCID mice on a BALB/c background and stimulated with 0.5
µM ovalbumin peptide (amino acid 323-339; triangles) or 0.5 µg/ml anti-CD3 (squares) in the presence of
AC (5 ? 104) and the indicated number of CD4+CD25+ cells. Results are expressed as the mean of triplicate
A soluble factor does not mediate
CD4+CD25+ induced suppression.
(A) CD4+CD25- cells (5
× 105) were cultured
in 24-well plates in the
presence of 3.0 µg/ml
soluble anti-CD3 and
AC (5 × 105). The
indicated number of
CD4+CD25+ cells was
added directly to the
culture (squares) or to
the transwell in the
absence (triangles) or
presence (circles) of
AC (5 × 105).
Fig. 7. CD4+CD25+
cells suppress IL-2
production.
(A) CD4+ CD25- cells (5 × 104),
AC (5 × 104), and 0.5 µg/ml antiCD3 were cultured in the
presence or absence of
CD4+CD25+ cells (2.5 × 104),
supernatants were taken at the
indicated times and IL-2 was
quantified. Each sample was
tested in triplicate. (B) RNA was
purified from CD4+ cells
stimulated in the presence or
absence of CD4+CD25+ cells
after 16 h using RNAzol B (Teltest). RNA was separated by gel
electrophoresis, transferred to
nitrocellulose and probed for IL-2
message (top) or -actin message
(bottom) by using an IL-2 PCR
fragment or a -actin PCR product
as a probe.
In vitro seems to work this way but in vivo data
demonstrate some role for TGFB and IL-10 in some
systems (in vivo vs in vitro or diverse
Treg populations)
• Ciriaco A. Piccirillo, John J. Letterio, Angela
M. Thornton, Rebecca S. McHugh, Mizuko
Mamura, Hidekazu Mizuhara, and Ethan M.
Shevach. CD4+CD25+ Regulatory T Cells
Can Mediate Suppressor Function in the
Absence of Transforming Growth Factor ß1
Production and Responsiveness
J. Exp. Med., Jul 2002; 196: 237 - 246
.
What are the requirements for Treg
development?
• TCR ENGAGEMENT?
• THYMOCYTE SELECTION
Thymic selection of CD4+CD25+ regulatory T cells
induced by an agonist self-peptide Nature Immunology 2,
301 - 306 (2001) Martha S. Jordan…. & Andrew J. Caton
• TCR transgenics (TS1) specific for the I-Ed/ HA
peptide(S1)were crossed to HA transgenic (HA28) mice
• The S1-specific T cells were not deleted
• Many of the S1-specific CD4+ T cells were CD25+ and
functioned as Treg cells.
• Specificity for a self-peptide “directs” CD4+CD25+ T cell
development.
•Selection of CD4 requires a TCR with high affinity for a self
peptide because thymocytes that bear TCRs with lower affinity do
not develop along this pathway.
Thymic selection of CD4+CD25+ regulatory T cells
induced by an agonist self-peptide Nature
Immunology 2, 301 - 306 (2001) Martha S.
Jordan…. & Andrew J. Caton
Figure 1. CD4+CD25+ S1–specific T cells are generated intrathymically. (a) CD25+ thymocyte
development in TS1HA28 mice. Flow cytometry was used to determine 6.5 expression on CD4+
LN cells and CD4+ SP thymocytes from TS1 and TS1HA28 mice. The 6.5Hi cells (indicated by
horizontal lines) were then analyzed for CD25 expression. The percentages of 6.5Hi CD4+CD25and CD25+ cells in TS1 versus TS1HA28 mice are shown. Data are representative of at least six
experiments. (b) CD25 expression during ontogeny. Thymi and spleens from 2-day-old, 7-dayold and adultTS1HA28 mice were collected and cells stained with antibodies to CD4, CD8,
CD25 and 6.5. The percentages of CD4+ SP, 6.5Hi, CD25+ and CD25- thymocytes and
splenocytes from five mice per time-point were determined and absolute numbers calculated. ..
TReg cells are a normal product of thymic selection.
Figure 1. TR cells may arise from relatively high-avidity interactions with selfpeptide–MHC complexes, just below the threshold for negative selection (green
area). This narrow avidity selection window ensures that Tr cells will constitute
only a small fraction of the mature T cell pool and have a greater sensitivity to
self-peptide–MHC than potentially pathogenic autoreactive T cells.
Expression of class II in cortical
thymic epithelial cells is sufficient
for Treg development
• While some CD25+, CD4+ T cells can be
found in class II knockouts, they do not
suppress in vitro
• Furthermore K14, but not class II -/-,
CD25+CD4+ suppress IBD in an adoptive
transfer model.
• More proof that TCR engagement is
required for survival/selection?
Tregs Develop in T Laufers K14
Mice (J Exp Med 194;p 427,
2001
Thymocytes
from K14
mice have
Treg
population
K14 Tregs are anergic and suppressive,
Expression of class II in cortical thymic epithelial
cells is sufficient for Treg development
J Exp Med 198,1665, 2003
Autoimmune uvitis model
Small amounts (barely
detectable) of auto antigen
expressed in the thymus required
for Treg generation.
What are the requirements for Treg
development?
• TCR engagement in the thymus (high affinity but not so
high as negative selection)
– Higher percentage of thymocytes with high affinity TCR in context
of auto antigen develop into regs
– T cells of appropriate affinity are “instructed” to become Tregs
• Selective sparing of pre-committed cells from negative
selection or promotion of Treg lineage development?
– higher percentage, but not higher absolute number
– Preferential eliminaton of non-regulatory T cells rather than
increased production of Tregs
– TCR engagement serves as survival or expansion signal of Tregs
pre-committed to that lineage.
Foxp3 transcription factor
required for Treg lineage
specification
Is a good marker except
intracellular location limits its
utility in purifying and
identifying Tregs in nonpermeabilized/live cells
see discussion paper for solution
Nature Immunology 4, 330 - 336 (2003) Foxp3 programs the
development and function of CD4+CD25+ regulatory T cells
Jason Fontenot…Alexander Rudensky
Figure 1. Foxp3 is
specifically expressed in
CD4+CD25+ regulatory T
cells. (a) Real-time quantitative
PCR for Foxp3 from purified T
cell subsets. (b) Western blot
analysis of Foxp3 in purified T
cell subsets using rabbit antiFoxp3 IgG.
Death at 4 weeks
Figure 2. Generation
and analysis of Foxp3
conditional mutant
micea (d) Western blot
analysis of Foxp3 in purified
CD4+ T cells and (e) spleens
and lymph nodes, at 21 d of age
from littermate male Foxp3+
and Foxp3-. Black bar, 1 cm. (f)
FACS analysis of CD4+ T cells
from 28-day-old Foxp3- and
littermate Foxp3+ male mice.
Plots are gated on CD4+ cells
and are representative of >5
mice analyzed.
Foxp3 knocked
out everywhere
Foxp3 knockouts
have big spleens
and ln containing
activated T cells
by day 28,
same phenotype
at scurfy
mutant/foxp3
mutant
Figure 3. Foxp3 is required
for development of
CD4+CD25+ regulatory T
cells and does not affect
CD4+CD25- T cell
proliferation.
(a) FACS analysis of lymph node
cells and thymocytes from male
Foxp3- and littermate Foxp3+
mice. Lymph node cell plots are
gated on TCR+ cells. Thymocyte
plots are gated on CD4+CD24lo
cells.
Demonstrates what a bad
marker CD25 is since cant
distinquish Tregs from activated
Figure 4. Neonatal
transfer of CD4+CD25+
regulatory T cells rescues
the lymphoproliferative
syndrome in Foxp3deficient mice.
(a) Lymph node cellularity
and total donor cells
recovered from 21-day-old
mutant and wild-type male
mice. (b) Spleen and lymph
nodes from 21-day-old male
recipient mice. (c) Flow
cytometric analysis of
activation markers expressed
by host CD4+ T cells from
recipient male mice.
Histograms are gated on
CD4+Ly5.1+ cells. (d) Flow
cytometric analysis of hostand donor-derived T cell
populations. Plots are gated
on TCR+ cells. All data are
representative of at least
four mice per experimental
condition.
Figure 5 Foxp3 programs the
development and function of
CD4+CD25+ regulatory T cells
a) MSCV MigR1 retroviral constructs and
representative CD4+CD25- T cell population
before infection: MigR1[Foxp3] (top), and
MigR1[empty] (bottom). (b) Retroviral
transduction efficiency of transferred cell
populations. (c) Suppression of wasting
disease and colitis by Foxp3-transduced cells.
Percent weight change of RAG1-/- mice
injected with retrovirally transduced cell
populations.
IPEX IN HUMANS CAUSED
BY FOXP3 MUTATION
•Immune dysregulation, Polyendrocrinopothy, Enteropothy, X-linked
•Early onset autoimmunity with
•Lymphoproliferation
•Diabetes, thyroditis, enteropathy,
Food allergies preventing normal food intake
• autoimmune hemolytic anemia and thrombocytopenia,
•severe infections
•Onset 3-4 weeks
•Usually, fatal in boys during early infancy or early childhood
Twenty-first century Foxp3; Nature Immunology
Anne O'Garra & Paulo Vieira
Figure 1. Equilibrium in the control of effector T cells.TReg cells expressing Foxp3 are produced in
the thymus, but CD4+ cells in the periphery may also acquire regulatory function. Neonatal thymectomy
or loss-of-function mutations in Foxp3 abrogate regulatory cells and cause wasting disease,
autoimmunity and lymphoproliferation. The human disease IPEX is also caused by mutations in Foxp3.
TReg cells inhibit activation and CD4+ cell effector function, thereby preventing these disorders.
Triggering of dendritic cells (DCs) by bacterial products overcomes this suppression. The equilibrium
between TReg cells and DCs is therefore crucial for the proper regulation of the immune response.
4, 304 - 306 (2003)
Requirements for Treg activity
Antigen specific activation necessary for suppressive
CD28 very important (IL-2 needed)
IL-2 important
IL-2 knockouts lethal lymphoproliferation
Treg wanabees are there but need IL-2
added back to function as regs
IL-2 alpha and beta receptor chain knockouts
• Tregs are stimulated in
an antigen specific
manner, but once
activated they inhibit
both CD4 and CD8
responses in an antigen
non-specific manner.
•May act indirectly (at
least in part) through
APCs (I.e decrease B7 on
APC)
•In in vitro models, a
soluble factor does not
mediate CD4+CD25+
suppression (cell:cell
contact required); in vivo
IL-10 is important
•CD4+CD25+ cells
suppress IL-2 production
–IL-2 mRNA and
secreted IL-2 protein
Figure 1. Linkage of central and peripheral tolerance by thymic production of natural CD25+CD4+ Treg
cells that contribute to peripheral self-tolerance.
A reduction in CD25+CD4+ Treg cells or attenuation of their suppressive activity may elicit
autoimmunity, tumor immunity, microbial immunity and allergy. In contrast, an increase in the number
of Treg cells or augmentation of their suppressive activity may establish transplantation tolerance and
maintain feto-maternal tolerance. Treg cells may control effector T cells (Teff cells) either directly or
indirectly through APCs. †, death.
Powrie Model, NI2:816 (2001)
In our quantitative model, the maintenance of self-tolerance is
dependent on the ratio of TR cells to potentially pathogenic
autoreactive T (TPATH) cells that respond to a given
peripheral antigen. In a given lymph node, this ratio will be dynamic
and fluctuate depending on the infectious status of the local tissue. In
the steady-state, immature DCs may traffic through peripheral tissues;
here they can efficiently phagocytose proteins and apoptotic debris
arising from normal cell turnover in the tissue without becoming
activated(Fig. 3a). Even in the absence of inflammation, a few of these
immature DCs will migrate to the draining lymph nodes where they
will present a panel of self-peptides to both TR cells and TPATH
cells108-110. However, autoimmunity will not occur, perhaps because
the autoreactive T cells are insufficiently activated by immature DCs
or, alternatively, because the immature DC preferentially stimulate TR
cells. Consistent with the latter hypothesis, TR cells can respond to
much lower concentrations of cognate peptide ligands than
conventional naïve CD4+ T cells25 and immature DCs express
relatively low amounts of MHC class II and costimulatory molecules.
• Model for the control of pathogenic immune responses by
TR cells.
Model for the control of pathogenic immune responses by TR cells.
(a) In the steady-state, low numbers of immature DC traffic to the draining
lymph node (LN) from uninflamed tissues and present self-peptides (yellow) to
both TR and TPATH cells. The relatively high ratio of TR:TPATH cells, together
with the intrinsically higher affinity of the TR cells, leads to low-level activation
of the TR cells (red arrow) and inhibition of TPATH cells (green arrow). iDC,
immature DC. (b) During an immune response high numbers of activated mature
DCs traffic to the LN where they present peptides derived from both self (yellow)
and foreign antigens (red). This strong stimulus (red arrows) leads to activation
not only of T cells specific for the infectious agent (TE cells), but also to full
activation of the TR cells. This results in their proliferation and a transient loss of
suppressive function, which, in turn, allows activation and expansion of TPATH
cells. mDC, mature DC. (c) After the infectious agent is cleared, in the absence of
foreign peptides, TE cells either die or become memory T cells (TM). The
regulatory phase is characterized by presentation of self-peptides, again leading to
competition between TR and TPATH cells. As both have proliferated, the ratio of
TR:TPATH cells is maintained so that regulatory activity dominates; this leads to
the inhibition of TPATH cells and down-regulation of APCs. To prevent a chronic
pathogenic inflammatory response, TR cells may also migrate to
the inflammatory site
Nature Immunology 6, 353 - 360 (2005)
Published online: 22 March 2005; | doi:10.1038/ni1181
Natural regulatory T cells in infectious disease
Yasmine Belkaid & Barry T RouseFigure 2. Manipulation of natural Treg cells as a therapeutic approach during
infection.
The interactions between a host and a pathogen range from uncontrolled pathogen growth to sterile elimination.
Blockade or enhancement of natural Treg cell function may represent a therapeutic approach at each 'extreme' of the
host-pathogen interaction. Excess control of effector immune responses by natural Treg cells can lead to
uncontrolled growth of the pathogen and eventual death of the host. In such cases, prevention of natural Treg cell
function may restore the capacity of the host to efficiently control infection. At the other 'extreme' of the hostpathogen interaction, effector immune responses can efficiently eliminate pathogens. This situation can lead to
'unleashed' effector immune responses and immunopathology. In the most extreme scenario, the host can die from
uncontrolled immune responses. For controlling immnopathology, enhancement of natural Treg cells function may
represent a therapeutic approach.
Nature Immunology 6, 353 - 360 (2005)
Published online: 22 March 2005; | doi:10.1038/ni1181
Natural regulatory T cells in infectious disease
Yasmine Belkaid & Barry T Rouse
Table 1 Microbial infections for which a regulatory function for natural Treg cells has been
suggested
Microbe
Species
Antigen specificity
Reference
Helicobacter hepaticus Mouse
ND
8,10
Human
Helicobacter pylori
Mouse
ND
12,49
Human
Listeria monocytogenes Mouse
ND
38
Pneumocistis carinii
Mouse
ND
13
Leishmania major
Mouse
Yes
16-18,23,37
Schistosoma masoni
Mouse
Yes
19,20
Candida albicans
Mouse
ND
14,80
Herpes simplex virus
Mouse
ND
22,28
Friend virus Mouse
ND
30,75
Human immunodeficiency virus
Human
Yes
32-34,59,60
Hepatitis C virus
Human
Yes
21,35,36
Cytomegalovirus
Human
ND
32
Murine AIDS
Mouse
ND
31
Feline immunodeficiency virus
Cat
ND
56,81
ND, not done.
Table 2 Functions of Treg cells during infection
Reduced Tregs
Advantages for:
Host
Pathogen clearance
Equilibrium betweenTreg cells/effector cells
Excess Tregs
Maintenance of protective immunity
Control of excessive immune responses
(immunopathology)
Microbe
Persistence and/or transmission
Transmission
Disadvantages for:
Host
Tissue damage
Maintenance of reservoir
Prevention of effector responses
Disease reactivation
Microbe
Microbe clearance
Host destruction