Download The immundefence

The impact of Interleukin 2 on rapid T cell expansion
Arian Sadeghi
Project Report 20p MN3 Biology / molecular biology
Department of clinical immunology
Uppsala University Hospital
“Science is nothing but developed perception, interpreted intent, common sense
rounded out and minutely articulated”.
George Santayana (1863-1952)
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Index:
1.0
1.1
1.2
2.0
3.0
4.0
4.1
4.2
4.3
4.4
4.5
4.6
5.0
5.1
5.1.1
5.1.2
5.1.3
5.2
5.3
5.4
5.5
5.6
6.0
6.1
6.2
6.3
6.4
6.5
7.0
8.0
9.0
The immune system
Innate and adaptive immunity
Adaptive immune response
The major histocompatibility complexes and antigen presentation
T lymphocyte activation
Immunotherapy
Cancer vaccines
Dendritic cells
Viral vectors
Adoptive cell transfer therapy
Adoptive T cell transfer therapy for treatment of EBVand CMV
Adoptive T cell transfer for treatment of melanoma
Material and methods
Isolation and expansion of CMV specific CD8+ T cells
Separation of lymphocytes and monocytes
Differentiation and maturation of dendritic cells
Generation of CMV-directed T cells
Stimulation of mononuclear cells with irradiated autologous LCLs
Isolation and expansion of TIL microcultures from tumor tissue
Rapid Expansion Protocol
Tetramer analysis
Intercellular interferon gamma staining of T cells
Results
Generation of dendritic cells from monocytes
Generation of Cytotoxic T lymphocytes specific for CMV pp65 495-503
peptide using peptide loaded mature DC
Rapid expansion of CMV restricted T cells
Generation and expansion of EBV specific T cells
Rapid expansion of TILs
Discussion
Future perspectives
References
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Abbreviations
ACT
CTL
CMV
CpG
DC
EBV
ER
FITC
GM-CSF
GVHD
HLA
IFN
IL
imDC
MHC
PBMC
PE
PerCP
TAP
TCR
TGF
TIL
TNF
Adoptive cell transfer therapy
Cytolytic T lymphocyte
Cytomegalovirus
Cytosine-phosphate-Guanine
Dendritic cell
Epstein-Barr virus
Endoplasmic reticulum
Flourescein-isothiocyanate
Granulocyte macrophage colony stimulating factor
Graft versus host disease
Human leukocyte antigen
Interferon
Interleukin
Immature dendritic cell
Major histocompatibility complex
Peripheral blood mononuclear cell
phycoerythin
Peridinin chlorophyll protein
Transporters associated with antigen processing
T cell receptor
Tissue growth factor
Tumor infiltrating lymphocyte
Tumor necrosis factor
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Abstract
In this work isolation and rapid expansion of cytotoxic antigen specific CD8+ T cells
have been studied. The T cells used were directed against Cytomegalovirus, Epstein-Barr
virus and melanoma, since such T cells have been adoptively transferred to treat patients
in numerous clinical trails. In many of these clinical trails the T cells have been
expanded to clinical relevant numbers using an agonistic anti-CD3 antibody, IL-2 and
irradiated allogenic feeder cells before transfer. The focus has been on increasing T cell
numbers with sustained phenotype and function using modified versions of this protocol.
In particular the influence of IL-2 on T cell expansion rate, phenotype and function has
been extensively studied. IL-2 is a great catalyst in T cell evolution, growth and
proliferation. Results indicate that IL-2 aided the expansion of T cells but not during the
whole two week expansion phase. The rapid growth of the T cells proved to have
influence upon T cell phenotype whereas cell function was maintained. In conclusion,
expansion probably changed the bias towards function as to phenotype.
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1.0 The immune system
The goal of the living is to survive and to preserve life and to this end an organism must
be able to distinguish between self and non-self. Non-self in this case is the actual
physical surrounding of the organism e.g. dust, pollens, microorganisms, drugs,
chemicals, etc. Therefore, protection against such agents is an absolute necessity for
survival and an elaborated systematic defense, namely the immune system has evolved
for this purpose. The immune system is built up of defensive networks and barriers
spread all over the body which all collaborate in a well-orchestred manner to efficiently
recognize, control and dispose of foreign matters whenever such gain accesses into the
body.
1.1 Innate and adaptive
The immune system can be divided into innate and adaptive immunity. The innate
immunity exists and acts without memory of previous pathogenic encounters. It is
manifested in form of cellular and biochemical mechanisms that reacts rapidly to
infections. Such reactions are always constant and in the same manner no matter how
repetitious an infection might be. Examples of innate immunity are the skin/surface
barriers including mucous membranes and cilia apparatus. The phagocytes, natural killer
cells, cytokines and interferons are other examples of innate immunity. Adaptive
immunity in contrast is stimulated by exposure to foreign agents and the response
escalades with each successive exposure. Such increase in magnitude is due to the ability
of the adaptive immunity to keep a record of previous convergences with harmful
pathogens1. This delicate specificity and adapting ability is not only due to the power of
remembering and acting more vigorously on second encounters, but also on expanded
capacity to remember different antigens and the ability to distinguish between closely
related molecules or microbes. Adaptive immunity is thus antigen-specific and the
response elicited is solely depending on the type of antigen and the number of pervious
encounters1. The adaptive immunity is divided into two subtypes; humoral immunity and
cell-mediated immunity. Humoral immunity is based on antibody producing B
lymphocytes, which recognize a specific antigen, neutralize it or tag it for destruction by
other cells or mechanisms. Antibodies are abundant, of enormous variation and highly
specialized. Different antibodies can elicit different responses e.g. phagocytosis or release
of inflammatory mediators. The limitation of humoral immunity is that it only acts
extracellulary. The cellular immunity is mediated by T lymphocytes and deals with
viruses and bacteria that survive and proliferate inside host cells. T lymphocytes are
divided into Helper T and Cytolytic T cells. Helper T cells are activated upon antigen
recognition and in turn activate other immune cells like phagocytes and cytolytic T cells.
Activated cytolytic T cells can subsequently kill target cells upon antigens recognition
and as such eliminate the source of a possible infection.
1.2 Adaptive T cell response
Lymphocytes mature in generative lymphoid tissue where they are presented to the “selfantigens” in the absence of other antigens and subsequently self-reactive T cells are
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deleted. Maintenance of self-tolerance is a fundamental property of the immune system
and failure in establishing self-tolerance leads to autoimmune diseases. After maturation
the T lymphocytes leave the lymphoid organs and enter circulation. Once in circulation
antigen specific clones might be activated by their specific antigens. If the initial antigen
specific signal is followed by a second signal, originally generated by the innate immune
system, an antigen specific immune response is initiated. This also ensures that a T cell
response is triggered at the correct location i.e. the inflammatory effect. The T cell
response to antigen and inflammation is cellular proliferation and differentiation into
effector and memory T cells.
2.0 The major histocompatibility complexes and Antigen presentation
Cell-associated antigens must be displayed and presented for T cells
recognition/activation. This task is performed by proteins encoded by genes in the major
histocompatibility complex (MHC) loci. There are two main types of MHC molecules;
class I and class II and they present antigens from different sources. MHC class I
predominantly presents antigens originating from cytosolic proteins whereas class II
presents antigens originating from extra cellular compartments (Figure 1 and Figure 2).
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The MHC class I molecule in humans is known as HLA-ABC and is the product of one
of the most polymorphic loci in the genome. The molecule consists of an MHC coded αchain of ~45 kD and a non MHC coded β2-microglobulin chain. CD8+ T cells are the
cells that recognize these molecules and the antigen they present. All nucleated cells,
except spermatocytes, express MHC class I and can present associated peptides. All
intracellular proteins become proteolytically degraded by the proteasome through
ubiquitination tagging. The proteasome cleaves the protein into peptides and peptides of
6-30 residues are transported from the cytosol into the ER by the TAP (Transporters
associated with antigen processing) proteins. The peptides are subsequently loaded into
the peptide binding cleft of the MHC class I molecules, which are produced inside the
ER. Peptide/MHC class I complex is next transported through the Golgi by exocytic
vesicles to the cell surface where they interact with CD8+ T cells.
The MHC class II is known as HLA-DR/DQ in humans and consists of highly
polymorphic α and β chains ~30-34 kD. These molecules exist only on professional
antigen presenting cells like dendritic cells, phagocytes and B lymphocytes and are
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recognized by CD4+ T cells. MHC class II presents antigens originating from the extra
cellular environment. Initially, professional antigen presenting cells endocytose extra
cellular proteins into endosomal vesicles. These proteins are subsequently degraded into
peptides by lysosomal proteases. MHC class II molecules are produced inside the ER and
transported through the cytosol by exocytic vesicles. Such vesicles merge with the
endosomal/lysosomal antigenic peptide containing vesicles and peptides (15-24 residues)
are loaded into the peptide binding cleft of the MHC class II molecules, which are
subsequently transported to the cell surface (figure 2). When a professional APC
phagocytose surrounding antigens a process known as cross-presentation might occur. In
this process extra cellular antigens are presented by MHC class I molecules2. Crosspresentation is only preformed by dendritic cells. Likewise, DCs are able to present
endogenous antigens on MHC class II molecules3.
3.0 T lymphocyte activation
T cells use membrane proteins for antigen recognition, signal transduction and adhesion
as depicted by figure 3. Different antigens are distinguished by the heterodimeric T cells
receptor (TCR) consisting of the α and β chains4. Proteins responsible for signal
transduction come in great variation depending on the signal being transmitted. Common
for the T cells are the CD3 and ξ proteins that are non-covalently associated with the
TCR and when activated by TCR antigen recognition lead to general T cell activation.
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The CD4 and CD8 molecules are distinguishing factors between T cell subtypes. The
CD4 is a 55-kD monomer that recognizes peptide parts of the MHC class II molecule.
The CD8 molecule is a αβ or αα dimer, and recognize the MHC class I molecule5. Other
accessory molecules necessary for T cell function are adhesion molecules that facilitate
the migration and docking of the T cell with antigen presenting cells. Examples of
adhesion molecules are: integrins and selectins6.The CD28 molecule on T cells provides
the second signal needed for full T cell activation. This signal occurs when the CD28
molecule is associated with its ligand the B7-1/B7-2 (CD80 and CD86) molecules on
professional APC.
The initial response from T cells upon antigen recognition is clonal expansion and
differentiation into effector cells. This is facilitated by secretion of cytokines (IL-12
among others) in an autocrine fashion and through direct costimulation by professional
APCs in the microenvoirment. After clonal expansion and differentiation the T cells
migrate to peripheral tissue where they either become effector cells or memory cells.
Effector CD4+ T cells promotes the function of CD8+ T cells by releasing
immunostimulatory cytokines like IL-2. In addition, effector CD4+ T cells, activate
macrophages and antibody producing B cells. Effector CD8+ T cells directly kill antigen
displaying target cells in a MHC class I-antigen derived peptide-TCR specific manner.
Activated CTLs secrete cytotoxic granule proteins that trigger apoptosis in the target
cells. Expression of Fas ligand is another mechanism by which the CTLs can destroy
antigen displaying target cells. Binding of the Fas ligand to its target Fas protein,
expressed on most cells, results in apoptosis of the target cell. A fraction of the antigen
stimulated T cells develop into memory T cells, which live longer than the effector cells
and do not multiply. Acceleration and refinement of a secondary immune response on
subsequent infection is among the tasks of these cells.
4.0 Immunotherapy
Any attempt to mobilize or manipulate a patient’s immune system in order to cure or treat
a disorder is referred to as immunotherapy. This approach is appropriate to help patients
suffering from autoimmune diseases, chronic inflammations and infectious diseases.
Immunotherapy generally divided in active and passive immunotherapy7. Examples of
active immunotherapy are different therapeutic vaccines, such as peptides and proteinvaccines to mobilize patients own immune system de novo. Examples of passive
immunotherapy are administration of monoclonal antibodies, cytokines or previously
activated immune cells.
4.1 Cancer vaccines
The most frequently used approaches to stimulate the immune system to elicit an immune
response against cancer are vaccines consisting of proteins or peptides administered
together with an adjuvant. Adjuvants are compounds that provoke an inflammation where
monocytes, neutrophils, T cells and other immune cells are recruited. Adjuvants can
consist of bacterial cell components, immunostimulatory DNA i.e. cytosine/guanosinerich motifs (CpG)8,9 or cytokines such as Interleukin 12 or granulocyte macrophage
colony stimulating factor (GM-CSF)10. Dendritic cells, macrophages or other
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phagocytosing cells are activated by such adjuvants, capture the antigen, process and
present it on their MHC molecules to which T cells and other effector cells respond.
Some of the most extensive and successful peptide vaccinations on cancer patients are in
melanoma and prostate cancer11,12. Results from these studies have revealed antigen
specific immune responses, instances of complete or partial regression and prolonged
survival13,14. Tumor cell-based vaccines can also be used15. In this case tumor cells
extracted from biopsies or established cancer cell lines have been used as source of
antigen16,17.Tumor cells have been irradiated and injected into patients with the hope to
activate a cancer directed immune response. The cell-based vaccines have also been
administered in combination with various adjuvants, like BCG 17. Additional strategies
involve tumor cells transduced with vectors expressing different inflammation inducing
genes18.
4.2 Dendritic cells
Dendritic cells (DCs) have many attributes that makes them suitable for human
immunotherapy. Tumor cells or virus-infected cells express tumor associated antigens or
pathogen specific peptides originating from these antigens, displayed in the cell surface
by MHC molecules. However, most tumor cells or virus-infected cell can not initiate a
primary T cell response due to the lack of co-stimulatory molecules. DCs have a distinct
and highly regulated mechanism to capture and process antigens, migrate to sites of high
lymphatic activity and optimally present antigenic peptides to lymphocytes. For this
purpose DCs express a large array of T cell stimulation molecules such as CD40, CD54,
CD80, CD86 in addition to MHC class I and II. DCs are also capable of antigen cross
presentation and secretion of immunostimulatory cytokines. These attributes makes DCs
very lucrative in active immunotherapy and they have been used in many clinical trails,
primarily on cancer patients19. The antigen presenting and T cell activation abilities of
matured DCs is far superior to that of immature DCs 20. DCs can be modified with Tumor
antigens by many means21. DCs pulsed with viral or tumor antigenic peptides can trigger
tumor or viral specific CD8+ T cell responses. Peptides pulsed onto DCs replace native
peptides already bound to MHC22.DCs can also be incubated with protein antigens.
Protein antigens are applicable independently of HLA restrictions and prior knowledge of
peptide immunogenicity is not required. DCs can also be pulsed with tumor cell lysate.
Lysates have the advantage of containing all relevant antigens and therefore no prior
identification of tumor antigens are needed.23
Viral and plasmid vectors encoding tumor antigens have been used for in vivo and ex vivo
immunization. This method takes advantage of the unique antigen presenting ability of
dendritic cells24.Genetically delivered antigens utilities the patients own antigen
processing machinery and relevant peptides are presented to the T cells. One advantage
of this method is that no prior knowledge about immunogenic peptide epitopes are
required. DNA and RNA vectors have been used for gene expression in DCs, with DNA
being most frequently applied due to stability, manipulation capability and possibility to
be produced in large quantities25. RNA transfection of DCs is currently being studied
since it has proven advantageous in several aspects. The mRNA content of tumor cells
can be isolated and amplified using PCR techniques before transfection into DCs26. Other
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advantages of RNA transfection is the benefit of expressing several tumor-derived genes
within the DCs at the same time. This leads to the translation of several tumor antigens
within the same DC. The short half-life of RNA and heterogeneous levels of intact
protein expression achieved by RNA/DNA may also impose limitations.
The vaccination strategies mentioned have in many cases been successful in increasing
the number of circulating antigen reactive lymphocytes. Unfortunately, the results have
been highly inconsistent and only sporadic clinical responses have been reported. In
melanoma for example, peptides used for vaccination did successfully generate tumorreactive CTLs, but vaccination alone did only in very few cases result in tumor
regression30. Reasons for the lack of tumor rejection by immunized patients are not well
characterized. Mechanisms that could limit the immune response and compromise the
effects of reactive CD8+ T cells are the lack of T helper cells and the suppressive status of
CD4+ CD25+ regulatory T cells27. Furthermore, the CD8+ CTLs could be in insufficient
amounts or be deficient in receptor avidity/signaling and other T cell functions.
Production of immunosuppressive chemokines by the tumor cells or failure of the T cells
to home to tumor areas could be other factors. In addition, tumor cells can acquire
different escape mutations like loss of tumor antigen expression, loss or down-regulation
of HLA-expression or acquire resistance to CTL lysis28.
4.4 Adoptive cell transfer therapy
Adoptive cell transfer (ACT) therapy has proven to be one of the most fruitful approaches
to treat cancer and infectious diseases in both murine models and clinical trials29,30. The
foundation of ACT therapy is based on the fact that tumor or virus antigen restricted T
cells can be isolated, characterized and expanded ex vivo. The method is based on
selection of T lymphocytes with high avidity for tumor-associated antigens or viral
antigens, massive ex vivo cell expansion in the absence of regulatory T cells or other
suppressor mechanisms and subsequent infusion. The presence of high affinity antigenspecific CD8+ and CD4+ T cells is a prerequisite for successful ACT therapy and
efficiency of the treatment have so far been directly correlated with the number of
transferred tumor/virus antigen specific T cells.
4.5 Adoptive T cell transfer therapy for treatment of EBV, CMV
Cytomegalovirus (CMV) and Epstein-Barr (EBV) virus are members of the herpes virus
group. Between 50-80% of adults in Europe are infected with CMV and more than 90%
are infected with EBV31. After an initial CMV or EBV infection the virus remains latent
within myeloid cells and causes as such lifelong infections. These infections are kept
under control by virus specific T cells which constantly remove virus producing cells.
In patients that are immunocompromised because of diseases, immunosuppressive
therapy after transplantation or chemotherapeutical treatments, the viruses can become
threatening. Immunosuppression often involves a reduction of immune cells in order to
inhibit graft versus host disease or host versus graft disease. However, it also removes
CMV- and EBV-specific T cells and a wide-spread virus infection might be initiated.
Immunosuppressed stem cell transplanted patients can regain immunity towards CMV
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and EBV through recovery of virus-specific T cells in vivo. However this process can
take up to several years, especially since these patients are continuously treated with
immunosuppressive drugs. For some individuals particularly elderly ones, full immunity
will never be reached32-34.
Transplant patients are immunosuppressed at various degrees depending on the
transplant. Stem cell transplantation for example requires high level of
immunosuppression. These patients receive a profound suppression of the immune
system for preventing GVHD. Therefore, stem cell transplanted patients more susceptible
to primary virus infection and have a higher risk of latent virus re-activation then solid
organ transplanted patients. CMV and EBV are in these cases most commonly reactivated and the biggest causes of viral related complications after transplantation35.
Although rarely occurring, these infections cause two types of complications. The direct
effect caused by the virus is manifested as tissue invasive disease and the indirect effects
are manifested as acute rejection, cardiac complications, diabetes and lymphoma. The
outbreak of the viral disease is categorized into early and late onset with approximately
70% of infections occurring within 5-12 weeks after transplantation36. During recent
years the prophylactic administration of anti-viral drugs such as Ganciclovir has
significantly reduced the incidence of early onset. However, this has increased the
incident of late onset. Furthermore, prolonged treatment with antiviral drugs in attempt to
prevent late acquired infection is undesirable due to side effects such as nephrotoxicity
and myelosuppression which in turn lead to severe bacterial and fungal infections37.
Therefore, new and alterative methods are needed.
Increased knowledge of cell-mediated immunity and the mechanisms by which antigen
directed T cells can be selected and cultured ex vivo has increased the interest in adoptive
transfer of virus antigen specific T cells. Mainly, virus-directed T cells have been used to
treat patients suffering for CMV and EBV related post-transplant complications38,39. A
rigid selection of T cell clones with specificity for CMV or EBV antigens is imperative in
order to avoid or reduce the risk of graft-versus host disease (GVHD) 40. In vitro
stimulation of EBV specific lymphocytes is possible by using EBV-transformed
autologous lymphoblastoid B cell lines (LCLs) as antigen presenting cells39. According to
studies conducted by Rooney et al 2002, EBV associated lymphoproliferative disorders
(LPD) after transplantation is a direct result of T cell dysfunction41. Rooney et al also
proved that prophylaxic infusion of EBV specific T cells were effective in preventing
EBV-LPD in patients receiving T cell compared to the historical controls. Cellular
immunity persisted up to 80 months and significantly reduced high virus load in 12% of
patients42.
CMV-specific lymphocytes have been generated through stimulation with DCs pulsed
with immunodominant peptides from the CMV coat protein pp6543. Ex vivo T cell
stimulation enriches for CMV-specific T cells and reduces the frequency of T cells able
to cause GVHD. In studies conducted by Riddell and Walter et al 44and later by Einsele
et al 45,46 it appear that survival of the administered CD8+ T cells depends on the
endogenous reconstruction of CMV specific CD4+ T cells and vice versa. Studies by
Peggs et al have shown that out of 13 patients, with CMV antigen detected in the blood
by PCR, none developed CMV disease after receiving CMV-specific T cells30. In this
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system the CMV HLA-matched immunodominant peptides pulsed DCs were used to
stimulate naïve CD8+ T cells. After 12 days of specific stimulation the cells were rapidly
expanded before infusion. Other autologous systems to generate CMV-specific T cells
involve recombinant viral vectors, CMV lysate and recombinant proteins47.
4.6 Adoptive cell transfer for treatment of melanoma
Adoptive T cell transfer therapy is one of the most promising therapeutical methods to
treat metastatic melanoma resistant to standard treatment. With this method, tumor
antigen-specific, high avidity effector T cells are selected and expanded. Melanoma
associated antigens include MART-1, gp100 (Glycoprotein 100) and Tyrosinase. Most
melanoma patients have natural immunity against several of these antigens, i.e. they have
circulating antigen-reactive T cells. The most reactive melanoma antigen-specific
lymphocytes are isolated from tumor tissue and are called tumor infiltrating lymphocytes
(TILs)48. Nearly all melanoma tumors and metastases are infiltrated by TILs. TILs
specific for tumor antigens (and other lymphocytes) are easily educed from excisional
lesions by addition of IL-2. TILs with tumor antigen-specific reactivity can subsequently
be analyzed through several in vitro assays, interferon gamma release among others.
Tumor antigen-reactive TILs are thereafter rapidly expanded using an agonistic anti-CD3
antibody, IL2 and irradiated allogeneic peripheral blood mononuclear cells (PBMCs)
before being transfused back to the patient, as shown in figure 4.
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In clinical studies, patients up for adoptive T cell therapy receive non-myeloablative but
lymphodepleting chemotherapy prior to transfusion46. This causes a transient elimination
of circulating lymphocytes. Highly selected and expanded TIL cultures where then
administered together with IL-2. In the studies by Dudley et al 18 out of 35 (51%)
patients showed an objective tumor regression at all tumor sites. The metastatic deposits
showing regression were found in lungs, brain, liver, cutaneous and subcutaneous tissues
and lymph nodes. Four of the patients developed vitiligo (skin depigmentation) while five
experienced autoimmune destruction of normal melanocytes49. Examples of additional
tumors that could generate TILs suitable for adoptive cell transfer therapy is renal cell
carcinomas50,51.
AIM:
The main aim of this study is optimization of the rapid expansion protocol of antigen
specific T cells. Since IL-2 is the catalyst of T cell growth, adjustments of IL-2
concentrations can be beneficial considering the maintenance of phenotype and function
of T cells. Previous research protocols have used rather high IL-2 concentration. If same
results can be obtained with lower IL-2 concentrations it is salutary for the T cells and
economically beneficial.
5.0 Material and Methods
Informed and signed consent was obtained from all blood and tissue donors.
Ethical committee approval numbers are for melanoma 2005:383 and for CMV UPS01085 and UPS99-250.
5.1 Isolation and expansion of CMV specific CD8+ T cells
5.1.1 Separation of lymphocytes and monocytes.
PBMCs were obtained from healthy CMV+ adult donors by centrifugation of buffycoat
blood over Ficoll-Paque gradients (Amersham biosciences). Monocytes were separated
from lymphocytes through plastic adhesion, 90 minutes at 37°C where the monocytes
fractions adhere to the bottom of a T-75 culture flask (Corning, NY, USA). Lymphocytes
were collected as free floating cells in the medium. PBMCs were cultured in RPMI1640
(Invitrogen, Carlsbad, CA, USA), supplemented with 1% pooled human AB serum
(Uppsala University Hospital), 1% PEST (Invitrogen) , 1% HEPES (Invitrogen) , 0.5%
1mM L-Glutamine (Invitrogen) and 0.2% 20 µM 2-mercaptoethanol (Invitrogen).
5.1.2 Differentiation and Maturation of Dendritic cells.
Monocytes were differentiated to immature dendritic cells by using 50ng/ml Granulocyte
macrophage colony stimulating factor (GM-CSF) (Leucomax, Schering-Plough,
Novartis, Kenilworth, NJ, USA) and 25ng/ml interleukin 4 (R&D Systems, Minneapolis,
MN, USA) for six days. The media and cytokines were replaced every other day.
Immature DCs were then matured by adding 40ng/ml TNFα (R&D Systems) for 48
hours. Mature dendritic cells were analysed by flow cytometry using antibodies against:
HLA–ABC, HLA–DR, CD14, CD83, CD54, CD80, CD 86 and CD40 (BD biosciences).
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As a negative control, DCs were stained with isotype relevant negative control
antibodies.
5.1.3 Generation of CMV directed T cells.
The HLA-A0201 immunodominant CMV pp65495-503 peptide NLVPMVATV (amino
acids 495 – 503 of pp65) was synthesized at the department of Medical Biochemistry and
Microbiology, Uppsala University. Peptide purity was higher than 95% as assessed by
HPLC. Mature DCs were pulsed for 4 hours at 37°C with the pp65495-503 peptide (10
g/ml), washed, mixed with autologous lymphocytes at a 30:1 lymophocyte:DC ratio and
resuspended at 1.5x106 T cells per ml. To promote CTL activation and expansion IL-7
(20 ng/ml) (R&D Systems) and IL-12 (0.1 ng/ml) (R&D Systems) was added. After
seven days of co-culture half of the media was replaced and new IL-7 (20 ng/ml) was
added. After an additional 5 days the T cells were ready for use.
5.2 Stimulation of mononuclear cells with irradiated autologous LCLs.
EBV-specific T cells were generated from donor’s peripheral blood mononuclear cells by
co-culture with autologous EBV-transformed B cell lines (Lymphoblastoid cell lines –
LCLs). In short, LCLs are generated by adding 200 ul of concentrated B95-8 EBV
supernatant to 5x106 PBMCs. LCLs are subsequently cultured to appropriate cell
numbers for T cell stimulation in culture media containing RPMI1640, 10% fetal bovine
serum (Invitrogen) and 1% PEST. LCLs were kindly provided by Dr A Loskog. LCLs
were irradiated (40Gy), washed and resuspended at 5x104 cells/ml. Responder cells
(autologous PBMCs) were added to LCLs at a 4:1 ratio (2.5x105 stimulator cells per well
of a 24 well plate). Cells were co-cultured for 4 days before changing the medium.
Thereafter the cells were re-stimulated and expanded by weekly stimulations with IL-2
(100 IU/ml) and LCL (responder to stimulator ratio 4:1) before harvest52.
5.3 Isolation and expansion of TIL microcultures from tumor tissue.
Tumor-tissue was extracted by surgery and cut into 12-36 small pieces measuring 2-4
mm in diameter. Single pieces were placed in individual wells of a 12-well tissue culture
plate in 2 ml of complete medium together with 6000 U/ml of recombinant human IL-2
(Chiron Corp. Emeryville, CA). Complete medium was RPMI1640, 1% HEPES and 1%
PEST, 2mmol/L L-Glutamine 5.5x10-5 mol/L β-mercaptoethanol supplemented with 10%
human serum. Plates were placed in humidified incubator at 37ºC with 5% CO2 and
cultured until lymphocyte proliferation became visible. Normally, after 1-2 weeks a
carpet of lymphocytes would cover the plate surrounding the tumor fragment. TIL
cultures were continuously stimulated with IL-2 in fresh media until at least 1x107 cells
were obtained, at which point the TIL cultures were phenotyped and tested for antigen
reactivity. Frozen TILs generated from patient tumor-tissue were kind gifts of Dr Björn
Carlsson.
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5.4 Rapid Expansion Protocol.
1x105 viable antigen specific CTLs were cultured with 4x107 irradiated (50Gy)
allogeneic PBMCs. A PBMC pool was isolated from eight healthy blood donors using
Ficoll separation. Rapid expansion cultures were kept in complete RPMI1640 media
(described above) supplemented with the agonistic anti-CD3 antibody OKT3TM (1mg/ml)
(Ortho Biotech Products, Bridgewater, NJ, USA) in upright standing T-25 cm2 culture
flasks. IL-2 was added every third day, beginning on day 2, over a 2 week period. Five
different doses of IL-2 were tested for influence on T cell expansion rate and quality
(0U/ml, 6U/ml, 60U/ml, 600U/ml and 6000U/ml). The media was replaced on day five
and then every third day. On day eight an aliquot of cells was removed counted,
phenotyped and tested for anti-peptide activity by FACS. The T cells were harvested on
day 14 and again counted and assayed for phenotype and function by FACS.
5.5 Tetramer analysis.
The HLA-A0201/pp65495-503 tetramer binding status of T cells was determined with a
phycoerythin (PE)-labelled tetramer (Beckman Coulter Immunomics Operations, San
Diego, CA, USA) along with allophycocyanine (APC) labelled anti-CD3 antibody
(Becton Dickinson, San Diego, CA, USA) and a peridinin chlorophyll protein (PerCP)
labelled anti-CD8 antibody (Becton Dickinson). Cells were incubated with
antibodies/tetramer for 30 minutes at 4ºC, washed twice and subsequently fixed with 1%
paraformaldehyde in PBS. The cells were analysed on a FACSCalibur (Becton
Dickinson), and at least 30,000 events were collected.
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5.6 Intercellular interferon gamma staining of T cells.
Stimulator cells for CMV specific T cells, were DCs pulsed with the pp65495-503 CMV
peptide or an irrelevant peptide originating from the Vesicular monoamine transporter 1
VMAT-1 (LLDNMLFTV). DCs were pulsed for 2 hours at 37ºC. Stimulator cells for the
melanoma antigen directed TILs, were HLA semi-matched melanoma cell lines.
Stimulator cells for EBV specific T cells were autologous LCLs. T lymphocytes were
mixed with stimulators at a 1:1 ratio. The cells were incubated for two hours at 37ºC
before Brefeldin A (Sigma) (8µg/ml) was added to block the secretion of IFNγ. The
incubation was subsequently carried out for an additional five hours. Next, the cells were
permeabilized (i.e. made permeable for flourochrome labelled antibody staining (BDPerm; Becton Dickinson) and labelled with APC-labelled anti-CD3, PE-labelled antiCD8 and FITC-labelled anti-IFNγ (Becton Dickinson) for 30 minutes at 4ºC. After the
staining the cells were washed twice and fixed with 1% paraformaldehyde in PBS .The
cells were analysed on a FACSCalibur, and at least 30,000 events were collected.
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6.0 RESULTS
6.1 Generation of Dendritic cells from monocytes
Immature DCs were generated by culturing monocytes with IL-4, GM-CSF for 6 days.
The immature DCs were thereafter matured with TNF-α for 48 hours. As depicted in
figure 6 the mature DCs express high levels of the T cell activation and interaction
molecules like HLA-ABC, HLA-DR, CD83, CD86 and CD54. No expression of the
CD14 could be found on the mature DCs.
6.2 Generation of Cytotoxic T lymphocytes specific for CMV/pp65495-503 peptide using
peptide loaded mature DC.
Matured DCs from CMV+, HLA-A*0201+ blood donors were pulsed with a synthetic
peptide originating from the CMV coat protein pp65. The CMV pp65 peptide (amino
acids 495-503, NLVPMVATV) is immunodominant in HLA-A*0201+ individuals e.g. is
naturally presented on HLA-A*0201 molecules in CMV infected cells. This
immunodominans is in many HLA-0201+ CMV+ individuals manifested by circulating
pp65495-503 specific CD8+ T cells. Such circulating CD8+ T cells can be seen in figure 7A.
In this case 0.7% of the CD8+ T cell population bind the HLA-A*0201pp65495-503
- 17 -
tetramer. A tetramer is an immunological FACS-staining reagent that consists of four
HLA molecules which are all loaded with the same HLA-binding peptide. The tetramer is
able to bind TCR that are specific for the tetramer’s HLA and the HLA tetramer loaded
peptide (figure 6). After extra cellular peptide loading the DCs were used to stimulate
autologous lymphocytes. Following 12 days of co-culture a notable increase in tetramer
binding CD8+ lymphocyte could be observed. As shown in figure 7A, tetramer binding
had increased from 0.7% to 10.5%.
Interferons are usually secreted by activated B and T cells. T cells secrete interferon
gamma in response to antigen stimulation, normally viral antigen. IFNγ regulates the
immune response by inducing the production of other interferons and by stimulating antiviral/tumor-directed immune cells. Therefore, IFNsecretion can be used as a sign of
antigen specific T cell activation. The activity of the CMV pp65495-503 specific CD8+ T
cells was measured by intracellular IFN FACS staining after stimulation with pp65495-503
peptide pulsed dendritic cells. IFNproduction was measured directly after the initial 12
days of DC-T cell co-culture and as such prior to the rapid expansion protocol. As shown
in figure 7B the percentage of the CD8+ T cells that produced interferon gamma in
response to antigen stimulation was 1.2%. This indicates that only about 10% of the
pp65495-503 specific T cells are able to produce IFN upon peptide stimulation. Dendritic
cells loaded with an irrelevant peptide did not induce IFNproduction nor was it
observed from T cells alone.
- 18 -
6.3 The rapid expansion of CMV restricted T cells
1x105 T cells of which 10% were specific and CMV pp65495-503 restricted were next put
through the 2 week rapid expansion phase. Five doses of IL-2 were tested for its effect on
T cell quality and quantity. Table 1A and 1B illustrates the numerical cell expansion after
8 and 14 days of expansion. For each dose of IL-2 a triplet of T cell cultures were set up
and the average of cells counted in each triplet is represented in the tables.
- 19 -
After 8 days of expansion T cells originating from donor 1 show a steady decrease of
viable cells with decreasing IL-2 (Table 1A). At the same time, T cells originating from
donor 2 show an increase from 6.3x106 to 10x106, when the IL-2 dose was lowered from
6000U to 600U. Cell numbers then dropped with decreasing IL-2 (60U/ml, 6U/ml and
0U/ml). However, cell numbers were still higher (8.3x106) when given 600U/ml of IL-2
than 6000U/ml (6.3x106). After 14 days in rapid expansion T cell numbers seems to
increases with a decrease in IL-2 for both donors (Table 2B). This is most striking for
donor 2. The peak in cell numbers was reached when the IL-2 dose was set to 60U/ml.
Next, T cell tetramer binding and function was analyzed. As denoted in figure 8A and B,
the amount of tetramer binding CD8+ T cells is constant for the administered doses of
6000, 600 and 60 units of IL-2. The tetramer binding CD8+ T cells range from 1.5% to
3% for 6000 U/ml, from 1% to 2.2% for 600 U/ml and from 1.6% to 2.3% for 60 U/ml.
Tetramer binding decreases to 0.5 to 1% when given 6 or 0 units IL-2/ml. By comparing
figure 7 with figure 8 it is obvious that the amount of tetramer binders have dropped
dramatically from 10.5 % on day 0 to around 2% on day 14 i.e. after the rapid expansion
phase. Next, the expanded T cells were stimulated with CMV pp65495-503 peptide pulsed
dendritic cells and interferon gamma release was analyzed for donor 1. For 6000 U/ml
2.4%, for 600 U/ml 2.6%, for 60 U/ml 2.7%, for 6 U/ml 3.1% and for 0 U/ml less than
0.5% of the CD8+ T cells released interferon gamma (Figure 8B). A steady increase is
noticed for the decreasing concentrations of IL-2 starting from 2.4% at 6000 U/ml up to
3.1% at 6 U/ml. The function of the T cells given 0 U/ml of IL-2 can be compared to the
spontaneous amount of interferon gamma released or the amount released upon
stimulating with an irrelevant peptide originating from VMAT-1.
- 20 -
- 21 -
- 22 -
6.4 Generation and expansion of EBV-specific T cells
T cells were continuously stimulated with EBV transformed autologous B cells (LCLs).
When analyzed for interferon gamma release upon LCL stimulation a 7-fold increase was
observed when comparing stimulated and un-stimulated T cells (2.5% before and 17%
after stimulation) (Figure 8). IFN was in this case produced by both CD8+ and CD8- (i.e.
CD4+) T cells. Unfortunately, the cells were lost and could not be tested for phenotypical
and functional changes during the rapid expansion protocol.
6.5 Rapid expansion of TILs
Tumor infiltrating lymphocytes (TILs) were cultured from a metastasized melanoma
excisional biopsy using IL-2. The TILs were prior to expansion shown to be directed
against the melanoma antigen MART-1/Melan-A and Tyrosinase by both tetramer
staining and IFN production (data not shown). The TILs were subsequently rapidly
expanded using 4 different concentrations of IL-2, (6000, 600, 60, 6 U/ml). The increase
in cell numbers after 14 days of expansion is depicted in table 2.
6000 U
7.0 x 106
600 U
6.9 x 106
60 U
5.2 x 106
6U
1.8 x 106
Table 2A. The cell-count after 14 days of expansion with 4 different doses of IL-2.
- 23 -
A steady decrease in cell number is to be observed with decreasing IL-2. For 6000 U/ml
and 600 U/ml, no difference is noticed (7x106 cells for 6000 U/ml and 6.9x106 cells for
600 U/ml) as both concentrations reach a 70-fold cell number increase. For 60 U/ml the
count drops to 5.2x106 cells and for 6 U/ml to 1.8x106 cells. An interferon gamma release
assay was performed prior to and after rapid expansion. The patient was HLA-A*0201+
and share as such the HLA-A*0201 molecule with the melanoma cell line SK-23 (HLAA0101/0201, B-0702/0801, C-0701/0702). Prior to the rapid expansion phase 0.02 % of
the CD8+ T cells released interferon gamma upon SK-23 stimulation (Figure 10). After
14 days of rapid expansion, this increased to 2 % for cells treated with 6000 U/ml IL-2, to
0.5 % for cells treated with 600 U/ml IL-2, to 0.15% for 60 U/ml and to less then 0.1 %
for 6 U/ml treated cells, as shown in figure 10.
- 24 -
7.0 Discussion
There is no doubt that IL-2 is an important factor and the keystone to a successful
expansion of isolated, antigen-restricted T cells. Therefore the optimization of the IL-2
concentrations should be taken into consideration and evaluated. In prior studies, most of
them conducted by Riddell and Greenberg, the recommended concentration for optimal
growth and successful expansion were set to 6000 U of IL-2 per ml53. The main aim of
this study was to determine the exact role of IL-2 on the growth rate and quality of
expanded CTLs. This is highly appropriate since excessive administration could have
various negative effects on growing cells together with the massive costs linked to
extensive use of IL-2. IL-2 concentrations were chosen based on a logarithmic scale with
the highest set to the recommended 6000 U/ml. The T cells analyzed were directed
against CMV, EBV and melanoma antigen because such T cells have been extensively
used in clinical trails53-56. Also, such T cells are generated using different protocols and
come from different sources which might influence the ability of the cells to proliferate
upon CD3 stimulation. In this aspect CMV and EBV restricted lymphocytes are
stimulated ex vivo from peripheral blood where as melanoma antigen-directed T cells are
cultured from tumor tissue.
In the case of CMV, the adherent fraction of PBMCs were allowed to mature to dendritic
cells which were subsequently used as professional APCs for T cell stimulation. The
monocytes-derived mature DC showed all relevant cell surface markers. Peptide pulsed
DCs were able to promote a 15-fold increase in tetramer binding CD8+ T cells during the
twelve days of co-culture. Most of the tetramer-binding T cells were subsequently lost
during the rapid expansion procedure, independently of IL-2 concentration. These results
could be explained by the fact that the cells with the proper phenotype, i.e. the specific
TCR, are exhausted and unable to multiply. IL-2 might have rapidly matured these cells
into late stage effector cell and thus rendered them unable to expand and multiply. Other
early effector T cells may instead have been expanded and therefore a drop in tetramer
binder frequency is noticed. The number of cells entering rapid expansion was kept
constant and after the 14 day expansion we readily observed a 200 to 250-fold increase in
cells. When it comes to the functionality of the CMV-restricted T cells a slight increase is
noticeable after the completion of the rapid expansion phase. Before expansion and after
twelve days of stimulation with CMV pp65495-503 peptide loaded DCs the amount of IFNγ secreting CD8+ T cells was 1.2%. After the two week rapid expansion phase this figure
had increased for the cells given 6000 – 60 U/ml of IL-2. The fraction of interferon
releasing cells in the cultures given 6 – 0 U/ml of IL-2 had declined.
Taken together one can observe that high concentration of IL-2 is more beneficial during
the first week of expansion than during the later week. During the second week of
expansion a low IL-2 concentration, 100-fold less, is as good as if not better than higher
concentrations. The tetramer binding T cells were initially enriched by stimulation with
mature DCs and it is possible that such stimulation would promote development of
effector CD8+ T cells which are unable to proliferate57. This could also explain the
decrease in the percentage of tetramer binding cells and the increase in IFN-γ secreting
- 25 -
cells when comparing the cells before and after expansion. However, this does not
account for the lack of IFN production by the CD8+ T cells before expansion.
The EBV-experiment is still ongoing and the only results presented are of the stimulation
phase of the protocol. Stimulation of T cells with LCLs results in a remarkable increase
in interferon gamma releasing T lymphocytes. In this case all viral proteins are
potentially presented as antigens on MHC class I and class II on the LCLs. The advantage
of antigen presentation by both MHC classes is that both CD4+ and CD8+ T cell can be
stimulated simultaneously. This is clearly visualized by the post stimulation results where
both CD8+ (the upper right quadrant) and what is most likely CD4+ (the lower right
quadrant) T cells produce IFN. A rapid expansion of EBV-restricted T cells remains to
be conducted.
The TILs were isolated from an excisional melanoma biopsy and were only stimulated by
IL-2 during the initial culturing stage. Before rapid expansion the TILs were identified as
melanoma antigen-directed using both tetramer and interferon gamma staining. After
rapid expansion the cell number increased by 70-fold for the doses 6000 and 600 U/ml of
IL-2. The interferon gamma assay reveals an increase of IFN-γ secreting cells. When
6000 U/ml of IL-2 was administered a 100 fold increase of IFN-γ secreting portion of the
T cells were observed. While a 25 fold increase was observed when 60 U/ml was
administered. This experiment is also still ongoing and the tetramer binding abilities of
the TILs and the effects of rapid expansion thereupon is being studied.
8.0 Future perspective
There is undoubtedly much room for improvements of methods and protocol considering
the expansion of CTLs to clinical relevant numbers. To begin with one could narrow
down the logarithmic interval between the IL-2 concentrations. The range in my opinion
would be more informative if set in between 6000 to 600 units per ml. An attempt to
change the doses of IL-2 after half the expansion time would be an appropriate
consideration. According to the standard protocol the addition of IL-2 and the change of
media are set to fixed days. Instead, one could try to change media supplemented with
IL-2 when necessary by observing media color changes. Such procedures could be more
beneficial for the cells as the culture would be thriving in constantly fresh media. The
presented protocol also needs to be repeated several times to generate useful statistics
which is invaluable for protocol optimization. When it comes to EBV-restricted CTLs,
several expansions need to be done for satisfactory statistics. More function assays with
both LCLs and naïve B cell, prior and after expansion is an absolute necessity in order to
thoroughly revise the role and the effects of different IL-2 concentrations on both CD8+
and CD4+ effector cell expansion.
Considerable work remains to be done and is ongoing on the TILs. To begin with proper
tetramer binding analysis together with function analysis is to be conducted before and
after expansion. It is also of great importance to expand the “correct” T cells as the
existence of CD4+ regulatory T cells and CD8+ suppressor T cell can inhibit effector cell
function. More advanced labeling methods should be used to stain different subtypes of
- 26 -
lymphocytes. This opens up for the possibility to distinguish between naïve CD8+ T cells,
early effector CD8+ T cells, intermediate effector CD8+ T cells and late effector CD8+ T
cells57. These cells are considered to have different strengths and abilities when it comes
to antitumor responses, ex vivo expansion and most importantly in vivo expansion.
Finding, isolating and expanding the most optimal population of effector cells is the
future of cancer immunotherapy.
- 27 -
Acknowledgments
Björn Carlsson, Thank you for your invaluable sharing of your knowledge, patients and
guidance.
Magnus Essand, Thomas Tötterman, Angelica Loskog and all the other kind people
and co-workers at the GIG-group and clinical immunology.
- 28 -
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