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R egul at ory T-cel ls for immunothe ra py J.H. Pe te rs
Regulatory T-cells
for immunotherapy
J.H. Peters
RegulatoRy t-cells
for immunotherapy
The research presented in this thesis was performed at the Department of Laboratory Medicine - Medical Immunology, Radboud University Medical Centre, Nijmegen, The Netherlands.
Part of the work described in this thesis was supported by the Dutch Kidney Foundation (grant C05-2106).
ISBN: 978-90-6464-639-3
Printed by GVO drukkers & vormgevers B.V. | Ponsen & Looijen, Ede, The Netherlands
Cover design by J.H. Peters and GVO drukkers & vormgevers B.V. | Ponsen & Looijen, Ede, The Netherlands
RegulatoRy t-cells
for immunotherapy
Proefschrift
ter verkrijging van de graad van doctor
aan de radboud universiteit nijmegen
op gezag van de rector magnificus
prof. mr. s.c.J.J. Kortmann,
volgens besluit van het college van decanen
in het openbaar te verdedigen
op maandag 25 maart 2013
om 13.30 uur precies
door
J.H. Peters
geboren op 16 september 1982
te Wijchen
Promotores
Prof. dr. I. Joosten
Prof. dr. L.B. Hilbrands
Co-promotor
Dr. H.J.P.M. Koenen
Manuscript commissie
Prof. dr. G.J. Adema (voorzitter)
Prof. dr. M.G. Netea
7 29 59 ... 83 ... .... 107 .... 129 ... ... 149 167 175 179 General introduction
Immunotherapy with regulatory T-cells in transplantation
Human secondary lymphoid organs typically contain
polyclonally activated proliferating regulatory T-cells Co-culture of healthy human keratinocytes and T-cells promotes keratinocyte chemokine production and RORγt-positive IL-17 producing T-cell populations
Ex vivo generation of human alloantigen-specific regulatory T-cells from CD4posCD25high T-cells for immunotherapy Clinical grade Treg: GMP isolation, improvement of purity by CD127pos depletion, Treg expansion, and Treg cryopreservation
Summary and discussion
Nederlandse samenvatting Dankwoord List of publications Curriculum vitae Chapter 1: Chapter 2: Chapter 3: .... Chapter 4: .... .... Chapter 5: .... Chapter 6: .... .... Chapter 7: Chapter 8: Chapter 9: Chapter 10:
TabLe Of CONTeNTs
1
CHAPTER 1
1
THE
IMMUNE
sysTEM
The immune system is crucial to human survival. Even minor infections can prove
fatal in the absence of an immunological defense. However, in spite of having a
functional immune system, all humans will suffer from a number of infections during their lifetime. This is because building a strong immune response towards invading microorganisms takes time. Meanwhile, the microorganisms can multiply and cause disease. Our body is permanently involved in battles with many intruding
viruses, bacteria and fungi. Next to that, there are endogenous dangers that have
to be averted, such as uncontrolled cells that are potential sources of cancer. In these battles, the human body makes use of an army with many different types
of soldiers, in the form of various cell types and soluble molecules. Many cells
of peripheral tissues, such as keratinocytes in the skin, pneumocytes in the lung
and mucosal cells in the gut defend the body from invasions by microorganisms. When microorganisms pass these defenses, the immune system comes in to help. Generally, the immune system is divided into two legions: the innate and
the adaptive immune system. Innate responses provide a crude non-specific but fast first line of defense, disarming many of the intruders. NK-cells, monocytes, granulocytes and mast cells are the most important effector cell types of the innate immune system. On the other hand, the adaptive system is capable of mounting responses aimed against specific pathogens. This adaptive response can be immensely powerful but it takes days to develop fully. The adaptive system forms long lasting memory cells, providing a faster and stronger response in the case of subsequent attacks by the same pathogen. In the adaptive immune response, conventional CD4pos helper T-cells (Tconv) are important orchestrators.
When these cells are activated, they in turn activate an array of other immunologic effector cells of the adaptive immune system, such as B-cells and CD8pos cytotoxic
T-cells, but also macrophages and other cells of the innate immune system (Figure 1). Immune responses can destroy intruders, but also healthy bystander tissue
figure 1. activation of cD4pos helper t-cells ultimately leads to activation of an array of other immune effector mechanisms.
cells. Therefore, the immune system is equipped with brake-mechanisms. These
prevent detrimental effects on tissues caused by escalating immune responses and prevent responses against harmless agents or self-antigens. There are a number of cell populations specialized in guiding and suppressing immune responses. Of these populations, regulatory T-cells (Treg) are the most well-known and well-studied.
REGUlATORy T-CEllS
IDENTIFyING REGUlATORy T-CEllS
Naturally occurring Treg reside within the CD4pos T-cell lineage. They develop
in the thymus [1], and were initially thought to form as a stable thymus-derived cell lineage with limited plasticity. These thymus-derived Treg are referred to as
naturally occurring Treg, or nTreg. However, it has recently been shown in vivo in
mouse models that thymus-derived Treg can convert into Tconv [2-4]. Vice versa, CD4pos Tconv can be converted into Treg depending on the microenvironment.
These Treg are called induced Treg, or iTreg, and they resemble nTreg in many of
their phenotypic characteristics. A number of iTreg subsets have been described. For example, iTreg can be formed after activation of Tconv in the presence of
IL‑10 (Tr1 cells) [5], TGFβ (Th3 cells) [6] or Il‑35 [7]. These iTreg regulate immune
responses by increased production of Il‑10, TGFβ or Il‑35, respectively.
To complicate matters, identification of CD4pos Treg is not straightforward. The first
Treg were initially described as CD4posCD25high T-cells [8]. However, co-expression
of CD4 and CD25 is not unique for Treg, since human Tconv can also express CD25 upon activation. likewise, the Treg key transcription factor FoxP3, which is stably expressed by Treg, is also transiently expressed by recently activated human Tconv [9;10]. Moreover, the fact that FoxP3 is expressed intracellular and not on the cell surface limits the usefulness of this marker for the isolation of viable Treg. More recently, CD127 was described as a useful marker to identify Treg, since Treg show a high CD25 and a low CD127 expression, in contrast to Tconv, which have a CD25lowCD127high phenotype [11;12]. Again, recently activated Tconv can resemble
Treg with their CD25highCD127low phenotype [13]. Despite the possible contamination
with activated Tconv, most studies use the CD4posCD25highCD127lowFoxP3pos
phenotype to define Treg.
Other markers to further characterize nTreg have entered and/or left the stage, such as CD49d [14], PD-1 [15], GITR [16;17], CTlA-4 [18], membrane bound TGFβ [19], FR4 [20], CD39 and CD73 [21;22], none of them being uniformly expressed
on all Treg or being unique for Treg. Recently, GARP, a protein expressed on the
surface of activated Treg that is involved in the regulation of the bioavailability and activation of TGFβ, has been described as a marker to distinguish activated Treg from activated Tconv [23-28]. like GARP, NP-1 was also described to be involved in the binding of latent TGFβ on the Treg cell surface [29]. NP-1 has been described as a useful tool to distinguish naturally occurring Treg from induced Treg in animal models [30;31], but expression characteristics seem to be different in human cells [32].
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different properties have been identified. The differentiation of Treg resembles that of Tconv, with CD45RAposCD25pos naïve Treg [33;34], memory Treg expressing
the lymph node homing markers CCR7 and CD62l, and memory Treg which do not express lymph node homing markers but are positive for multiple peripheral homing markers such as selectin ligands, adhesion markers and chemokine receptors other than CCR7 and CD62l [35;36]. Alternative subdivisions of Treg have been based on expression of CD27 [37-39] HlA-DR [40], or integrin αE [41].
REGUlATORy T-CEll FUNCTION
The exact mechanisms used by Treg to regulate immune responses remain
incompletely understood. In general, Treg can modulate Tconv actions directly through cell-cell contact, by the production of cytokines, by cytokine scavenging, and/or indirectly by modulation of antigen presenting cells (APCs), of which dendritic cells (DCs) are the most important [42] (Figure 2). Here, the most important Treg mechanisms of action are listed.
subsets of Treg produce immunosuppressive cytokines such as IL‑10 [5], TGFβ [6],
IL‑35 [7], and regulatory proteins such as galectin-1 [43]. These cytokines inhibit
Tconv proliferation, suppress APC activity and/or lead to conversion of Tconv into iTreg. These cytokines are likely to play a role in the immunoregulatory function of Treg in the draining lymphoid tissue as well as at the peripheral site of inflammation. Treg can regulate immune responses through interactions with other cell types, such as Tconv, DCs, B-cells and NK-cells. Multiple interactions between Treg and DCs have been described. Binding of Treg CD152 (also known as CTlA-4) to its ligands CD80 and CD86 on DCs can reduce expression of these costimulatory molecules on the DCs. It also leads to IDO production by DCs, resulting in a local deprivation of the essential amino acid tryptophan. Both mechanisms attenuate activation and proliferation of CD4pos and CD8pos effector T-cells [44;45]. In lymph nodes, Treg
have been found to limit the contact between Tconv and DCs in vivo and ex vivo in
mice [46;47], further reducing Tconv activation and expansion in lymphoid organs. Other Treg/DC interactions take place in the periphery. In a mouse model of cutaneous immune responses, Treg rigorously eliminated DCs, thereby preventing Tconv activation and limiting the responses [48]. More subtle interactions between
Treg and DCs at peripheral sites have also been described. Upon contact with Treg,
DCs decrease chemokine production [49] and show reduced endocytosis capacity [50], both effects leading to a decreased immune response. Next to that, in vitro
activation of DCs in the presence of Treg yields semi-mature DCs with a normal lymph node homing phenotype but with an impaired capacity to activate Tconv [51]. Treg interactions with Tconv lead to a reduction of Tconv proliferation in secondary lymphoid organs in mice [52;53]. In another mouse model, Treg did not reduce Tconv proliferation, but rather limited the egress of Tconv from the lymph nodes by inducing downregulation of S1P1, thereby reducing the number of Tconv
available for an immune response in the periphery [54]. At the site of inflammation, Treg limit Tconv proliferation [52] and local Treg delay the influx of Tconv [55]. Treg have also been found in follicles in mouse and human lymph nodes [56-59], and these Treg suppressed B-cell responses and follicular T-cell proliferation, indicating another function for a subset of Treg within the secondary lymphoid organs. Additionally, Treg have been shown to suppress NK-cell maturation in mouse lymph nodes [60].
OTHER REGULATORy CELL sUBsETs
Besides the CD4pos Treg described above, there are a number of other T-cell
populations with the capacity to down-regulate immune responses. CD8posCD28neg
T-cells exert their regulatory capacity via cell-contact dependent inhibition of T-cell activation by APCs and via cell-contact independent inhibition through Il-10 [61;62]. CD4negCD8neg T-cells can diminish immune responses by killing antigen-specific
T-cells, and by suppressing and killing APCs [63]. Subsets with immunomodulatory functions have been described within natural killer T-cells (NKT-cells). These cells function through the production of anti-inflammatory cytokines such as Il-4, and by inhibiting the production of pro-inflammatory cytokines such as IFNγ and TNFα by other cell types [64;65]. γδ T-cells have been shown to exert local regulatory activity in the gut mucosa, although their mechanism of action is unclear [66].
T-cells are not the only cell type with immunomodulatory subsets. In fact,
subsets with immune inhibitory actions have been described for most leukocyte populations. CD19posCD24highCD38high regulatory B-cells constitute a small subset
of the total B-cell pool and they regulate immune responses by producing Il-10 [67;68]. Regulatory macrophages, formed upon contact with Treg or B-1 cells (a sub-class of B-cells), are characterized by specific morphological features and surface phenotype, and by the ability to suppress T-cell proliferation in vitro
through the production of Il-10 [69]. DCs are known for their strong capacity to activate T-cells and promote immune responses, but even within this leukocyte population regulatory subsets have been defined, called tolerogenic DCs [70]. Immature DCs can induce T-cell tolerance [71], and certain types of plasmacytoid DC can induce the generation of antigen-specific Treg [72]. Myeloid-derived suppressor cells (MDSCs) form a heterogeneous population of progenitor cells with suppressive immune functions. Several subsets of MDSCs have been described and most exert their function in tissues during inflammation [73]. Activated MDSCs suppress T-cell, B-cell and NK-cell proliferation and cytokine production in vitro
through various soluble mediators, and promote Treg induction in an IFNγ and Il-10 dependent manner. Mesenchymal stromal cells, the only non-leukocyte cell population mentioned in this paragraph, migrate to sites of inflammation and exert local immunomodulatory and regenerative properties, for example through induction of Treg [74].
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Immune regulation is a complex process that involves multiple mechanisms and the co-operation of many cell populations. It is very likely that all regulatory
cell subsets described above co-operate in guiding immune responses, each
functioning at different locations and at different stages of immune responses.
REGUlATORy T-CEll BASED IMMUNOTHERAPy
Solid organ and stem cell transplantation procedures are very valuable treatment modalities for patients suffering from a number of life-threatening medical conditions. However, due to the recognition of foreign antigens, these procedures
evoke undesired immunological responses against the transplant or the host. In
autoimmune diseases unwanted immune responses against self-antigens also lead to significant morbidity and mortality. Treg immunotherapy is a potential treatment
modality to dampen such harmful immune responses. Below, immune responses in
solid organ and stem cell transplantation as well as in autoimmunity are described, followed by a brief summary of the current status of Treg therapy in these settings.
sOLID ORGAN TRANsPLANTATION
While solid organ transplantation can solve many life-threatening medical conditions due to end-stage failure of kidneys, liver, lung, heart or other organs,
it also introduces new problems. One of the major problems is caused by our own
immune system. Immediately after transplant surgery, activation of the innate immune system by the injured tissue can cause significant damage. In addition, the adaptive immune system can recognize the graft as foreign and dangerous, which will induce effector mechanisms that cause deterioration of graft function and ultimately lead to loss of the transplant. In the past decades, advances in immunosuppressive drug therapy have greatly lowered the risk of early rejection [75], and the 1-year survival of renal allografts is about 90%. Despite these advances, the slowly progressive process of chronic immune rejection remains problematic and is the main cause of late allograft loss [76].
like in many adaptive immune responses, CD4pos T-cells play a central role in
rejection of allografts. When these cells are activated, they in turn activate an array of other immunologic effectors, such as macrophages, B-cells and CD8pos
cytotoxic T-cells (see Figure 1). Once activated, these effectors result in delayed type hypersensitivity reactions against donor antigens, alloantibody production and direct lysis of graft cells, respectively. All these processes cause damage to the graft.
Donor antigens are largely divided into two main categories: major histocompatibility complex (MHC) antigens and minor histocompatibility antigens. In humans, MHC antigens are called human leukocyte antigens (HlA). HlA is important for physiological T-cell activation, as it is involved in antigen presentation. HlA class II is constitutively expressed by APCs such as DCs. These cells scout their surroundings for antigens, take them up through phagocytosis and process them into peptides. These foreign peptides are then bound to HlA class II molecules and presented to CD4pos T-cells. CD4pos T-cells with a T-cell receptor specific for the
all nucleated cells and is used to
present peptides derived from
intracellular pathogens to CD8pos
T-cells, and CD8pos T-cells with
a T-cell receptor recognizing the peptide/HlA complex are activated.
In the transplantation setting,
there are two well-described
pathways for activation of recipient CD4pos T-cells: direct
and indirect allorecognition (Figure 3). In the direct route, recipient T-cells are activated
directly by donor APCs. The foreign HLA on these APCs
results in the activation of a high number of alloreactive CD4pos
T-cells and CD8pos T-cells. several
studies indicate that the direct
route is the most important pathway in the pathophysiology of acute rejection [77-80]. On the other hand, it seems not to be a significant contributor to chronic rejection, possibly because the donor APCs disappear over time [81-83]. In indirect alloantigen recognition, recipient APCs present alloproteins (particularly peptides derived from allo-HlA) to CD4pos T-cells, thereby activating them. The indirect
activation route resembles the normal route of eliciting a response against other antigens. Thus, non-self HlA-molecules are highly immunogenic proteins in both the direct and the indirect pathway of allorecognition. An explanation for this high immunogenicity is that the thymus selects T-cells with a low affinity for self-HlA to prevent autoimmune reactions. These same T-cells can have a high affinity for foreign HlA molecules that differ slightly from self-HlA. To keep immunogenicity
of donor organs to a minimum, donors are selected for maximum HLA-matching
with the recipient. To complicate matters, many other foreign donor-proteins (such as minor histocompatibility antigens) are capable of activating recipient T-cells in the indirect alloreactivity pathway. These proteins are capable of causing chronic rejection in the absence of HlA-mismatches [84]. Even though the number
of precursor T-cells in the indirect route is much lower than in the direct route
(at least 10-fold lower in mice [77] and even 100-fold lower in humans [78]), the indirect route is of great importance because it does not fade over time, and it is associated with acute [83;85;86] as well as chronic rejection [81;87-90] in both
animal models and human transplant recipients.
solid organ transplant recipients currently receive lifelong treatment with
immunosuppressive drugs to prevent unwanted immune responses to alloantigens. These drugs are certainly not ideal, as they do not offer complete protection from rejection, and the required long-term immunosuppression has major adverse effects, such as an increased incidence of infections and malignancies [91;92]. Infections in transplant recipients can trigger alloimmune responses by molecular
figure 3. cD4pos t-cells can be activated by donor antigens through two pathways: the direct pathway (left) and the indirect pathway (right).
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mimicry, activation of APCs carrying alloantigen, and by local increase in the concentrations of cytokines and chemokines [93;94].
There are a number of strategies aiming at minimizing generalized immunosuppression while still preventing graft rejection, so-called donor-specific immunosuppression. Generally, these include therapies that lower relative frequencies or functional activity of alloreactive Tconv, and those that increase the frequency or activity of alloreactive regulatory cell subsets. The use of
immunosuppressive drugs that permit or promote Treg generation and function in vitro, such as rapamycin [95] might have clinical benefit. Another option is to induce lymphopenia at the time of transplantation by administration of antibodies that
deplete leukocyte populations such as anti-thymocyte globulin (depleting mainly T-cells, but also B-cells) or Alemtuzumab (anti-CD52, depleting T-cells, B-cells, monocytes, and granulocytes) to transplant recipients. Treg are activated by a lymphopenic environment [96]. Also, different subsets of leukocytes are repopulated at different rates and interestingly, regulatory immune cells can outnumber effector cells in the early stages of repopulation [97;98], which can help to create a tolerogenic environment for the transplant. Anti-thymocyte globulin has been shown to promote the generation of Treg [98], while Alemtuzumab can foster the generation of Treg [97] as well as CD8pos regulatory cells [99] and regulatory B-cells [100].
A logical approach to directly increase Treg numbers is cellular therapy using
Treg. In a variety of experimental models of solid organ transplantation, including transplantation of skin [101;101-106], heart [101-103;107] and artery [108;109], adoptively transferred Treg were able to induce tolerance or to prolong graft survival. In these models, Treg immunotherapy has proven to be a very effective strategy for controlling both acute [104-108] as well as chronic rejection [107-109]. Alloantigen-specific Treg are more efficient than polyclonal Treg and have lower potential of inducing general immune suppression. In most of the animal studies mentioned above, Treg with direct alloantigen-specificity were used. In two studies, it was shown that a combination of Treg with direct and indirect alloantigen-specificities was superior to only Treg with direct antigen-specificity [102;107].
Clinical trials on Treg immunotherapy with polyclonal Treg have been initiated. A large multicentre Phase I/II clinical trial in kidney transplant recipients is currently ongoing [110], and clinical trials have also been started in pediatric kidney transplantation recipients [111] and in liver transplantation recipients [112]. Before clinical trials using alloantigen-specific Treg can be envisioned, a number of technical difficulties have to be overcome.
sTEM CELL TRANsPLANTATION
Stem cell transplantation (SCT) is the optimal treatment modality for a number of hematological malignancies, such as multiple myeloma or leukemia, and other (hematological) diseases [113]. Although SCT has the potential to cure patients with
life-threatening diseases, it is associated with a high burden of treatment-related
morbidity and mortality. The treatment includes high dose chemotherapy and/ or radiotherapy to minimize the number of malignant cells in the host. Inevitably,
this results in temporary failure of hematopoiesis. Engrafted donor hematopoietic stem cells eventually reconstitute the hematopoietic system. The leukocytes that develop from the transplanted stem cells have the important task of eradicating recipient tumor cells that survived the conditioning regimen, which is called the graft-versus-leukemia response (Gvl) [114]. However, this new immune system can also recognize normal, healthy cells of the patient as non-self and can launch an attack. This is called graft-versus-host-disease (GvHD) [115], a complication that is fatal in approximately 15% of transplant recipients [116]. In contrast to the situation in solid organ transplantation where alloresponses are restricted to the allograft, in the case of SCT the donor immune cells can in potential attack every single healthy cell of the host. The most commonly affected organs are the gut, the liver and the skin. Similar to alloresponses in solid organ transplantation, the most important alloantigens starting these reactions are the HlA-molecules. The direct route of alloantigen-reactivity is important in graft-versus-host pathology (see Figure 3) [117]. In contrast to the situation in solid organ recipients, the direct route of alloantigen presentation does not fade over time in SCT recipients. Transplantation centers pursue a high degree of HlA-matching to prevent harmful reactions, as HlA-mismatching is associated with more severe GvHD [113;118]. To this end, the patient’s family is screened for a well-matched donor. If no suitable related donor can be found, international stem cell donor databases are searched for a matched unrelated donor. However, for patients with uncommon HlA-types, such as patients from ethnic minority groups, these searches often fail to provide a suitable donor. For these patients, HlA-locus mismatched or haploidentical stem cells are increasingly used for transplantation [119].
Nowadays, patients receive immunosuppressive drug treatment to reduce GvHD after stem cell transplantation. However, also for these patients immunosuppressive drug treatments are not ideal. Immunosuppressive drugs decrease Gvl activity, thereby increasing the risk of relapse, and increase the incidence of infections and other malignancies. Immunosuppressive regimens after stem cell transplantation
therefore need to carefully navigate between the risks of GvHD and of tumor
relapse and other adverse effects.
Treg immunotherapy is a highly attractive candidate for cellular therapy after
sCT, because these cells have the capacity to reduce GvHD while allowing GvL responses in vivo in animal models [120-122]. Even in aggressive SCT models with transplantation across complete MHC barriers, lethal GvHD can be prevented by Treg administration, although high ratios of Treg were needed (1:1 Treg:Tconv) [123-125]. Treg immunotherapy in SCT models is also very attractive because of the deeply lymphopenic setting, which activates Treg and gives them an advantage over effector cells, as described above for lymphocyte depletion after solid organ transplantation. When Treg were given a head start by administration one or two days prior to Tconv, Treg: Tconv ratios down to 1:10 were shown to be sufficient [52].
The first small-scale clinical trials in humans have been conducted. In a trial with twenty-three patients who received SCT from umbilical cord blood and polyclonally expanded Treg from third party donors [126]. The patients received additional immunosuppressive drugs. No adverse effects from the infused Treg were observed, and incidence of severe acute GvHD was significantly reduced as
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compared with historical controls. In another trial, twenty-eight patients received polyclonal Treg (not expanded) four days before haploidentical SCT from the same donor [127]. The patients did not receive immunosuppressive drugs. The patients showed faster immune reconstitution, a lower rate of GvHD, and a lower rate of
disease relapse as compared to historical controls.
While in the studies mentioned above Treg immunotherapy is used for prevention of GvHD, in one study Treg were administered to two patients with active GvHD [128]. One patient with chronic GvHD was successfully treated and immunosuppressive drugs could be withdrawn. However, the other patient suffered from acute GvHD
that progressed despite Treg immunotherapy.
Together, these results are very promising, and other trials in sCT recipients are
now ongoing [129;130].
AUTOIMMUNITy
There are many autoimmune diseases, with varying clinical presentations and limited therapeutic options. As over 3% of people are affected by autoimmune diseases, autoimmunity causes high morbidity and mortality world-wide [131].
Among the most prevalent autoimmune diseases are type I diabetes, rheumatoid
arthritis, inflammatory bowel disease, psoriasis, systemic lupus erythematosus and multiple sclerosis. The severity of autoimmunity varies widely between patients, from mild intermittent symptoms to life-threatening disease. Immune dysregulation plays a major role in the pathogenesis of autoimmune diseases, for the initiation as well as the progression of these diseases. Although the exact pathophysiology of autoimmunity is different for all autoimmune diseases, Treg seem to play a role in many of them. For example, impaired Treg function has been shown in patients suffering from type 1 diabetes [132;133], rheumatoid arthritis [134], multiple sclerosis [135;136] and psoriasis [137;138]. Currently, patients are treated with a myriad of immunosuppressive drugs and/or biologicals, with varying
success.
Of special interest are the treatments that promote Treg function. Similar to the solid organ transplantation setting, antibody treatments creating lymphopenia
are of interest in the treatment of autoimmune diseases, as lymphopenia causes
Treg activation and gives Treg an advantage over effector cells. These effects might
help to rebalance the immune system in autoimmunity. Indeed, anti-thymocyte globulin showed promising results in early-onset type 1 diabetes [139] and administration
of antibodies against the T-cell receptor CD3 induces Treg that suppress type 1 diabetes in animals [140] and improved clinical symptoms in early-onset diabetic patients [141]. The immunosuppressive drug rapamycin is of interest for the
treatment of autoimmunity as it permits or promotes Treg cell generation and function in an animal model of type 1 diabates [142], and this drug is currently tested in human patients with type 1 diabetes.
One way to directly push the immune balance towards self-tolerance in
autoimmunity is to adoptively transfer Treg. Treg immunotherapy has been shown to block development of disease in animal models of multiple sclerosis [143;144], type 1 diabetes [145-147], systemic lupus erythematodes [148]. Treg transfer
slowed progression of ongoing disease in an animal models of rheumatoid arthritis [149], and even reversed ongoing disease in an animal models of inflammatory bowel disease [150], type 1 diabetes [146] and systemic lupus erythematodes [148]. In some of these studies, antigen-specific Treg were used, which were generally more efficient as compared to polyclonal Treg [144-147].
The first clinical trial on adoptive Treg transfer in human autoimmune patients has been reported in inflammatory bowel disease [151]. ex vivo generated autologous
ovalbumin-specific Treg clones were administered to twenty patients with moderate to severe refractory inflammatory bowel disease (Crohn’s disease), and patients ingested an ovalbumin-enriched diet to ensure activation of gut-homing Treg. The adoptive transfer of Treg was safe, with eleven patients experiencing adverse events possibly related to Treg infusion (gastrointestinal disorders, most commonly temporary flare of inflammatory bowel disease), including two patients with serious adverse events (arterial thrombosis, septic thrombophlebitis, serious flare of inflammatory bowel disease). All patients recovered from the adverse events. Interestingly, 40% of the patients had favorable clinical responses after Treg infusion, including six patients with complete remission. However, the clinical effect was limited in time, with a maximum at five weeks after treatment and progressive decline thereafter, suggesting that multiple Treg infusions might be necessary to reach long-lasting effects. Another trial on Treg immunotherapy has recently been started in type 1 diabetes patients [152].
AIM AND OUTLINE Of THIs THEsIs
Treg play a critical role in various immunological processes, and immunotherapy based on Treg is a potential treatment for several immunological diseases, including unwanted transplantation related immune processes and autoimmune diseases. The ideal therapeutic intervention after transplantation of solid organs or stem cells should induce allospecific tolerance, defined as the absence of an immune response against alloantigen without immunosuppressive drug treatment, while immune responses against other antigens, such as antigens on tumor cells
and pathogenic microorganisms, remain intact. similarly, in autoimmunity ideally the autoimmune responses should be dampened without immunosuppressive
drug treatment, while maintaining normal immunity against other antigens. Even though the first clinical trials on Treg immunotherapy are promising, many
important issues regarding Treg biology in healthy or diseased humans, as well
as Treg behavior in the immunotherapeutic setting have to be addressed before Treg therapy can be safely used in standard clinical practice. At what location can different Treg subsets exert their function? Which Treg subsets are most effective? How about the isolation of these Treg? How many Treg are needed? How can the numbers of Treg needed be obtained? What about the timing of Treg infusions? Will adoptively transferred Treg maintain a stable Treg phenotype in the patient, or is there a risk of conversion into Tconv? In the present thesis, we address a number
of these fundamental aspects regarding Treg biology and Treg immunotherapy. We
explored the role of Treg in the human immune system, and its potential use for immunotherapy, especially in transplantation.
1
and on Treg based immunotherapy in transplantation and autoimmunity.
In chapter 2, an overview of Treg immunotherapy in transplantation is provided.
In chapter 3, we analyzed human lymphoid tissues involved in immune responses in order to elucidate where and how Treg may exert their suppressive functions. We describe the presence of activated Treg in secondary lymphoid organs, and we present clues for homing behavior of Treg towards peripheral tissues.
for Treg immunotherapy, it is important to understand the role of Treg in local
tissue immunity. We chose the skin as an organ that harbors many lymphocytes and serves as a potential port of entry for external organisms. We started out by taking a closer look at the crosstalk between epithelial cells, the keratinocytes,
and T-cells. In chapter 4, we describe a novel in vitro co-culture system with
keratinocytes, CD4pos and CD8pos T-cells and Treg. We show that the keratinocytes
and effector T-cells influence each other, and co-operate to form an effective local
immune response. Treg were found to regulate these responses only at the level of
proliferation.
In the following chapters, we focus on some practical aspects of Treg
immunotherapy. for successful immunotherapy, high numbers of Treg are needed.
In chapter 5, we describe a highly efficient protocol to expand human Treg in an alloantigen-specific manner.
Subsequently, we report on the current options of isolating Treg according to
clinical grade guidelines in chapter 6.
In chapter 7, the data presented in this thesis are summarized and discussed.
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reprogramming of regulatory T cells. Immunity. 2012. 36: 262-275.
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