Induction of Immunological tolerance via T regulatory cells

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1 Induction of Immunological tolerance via T regulatory cells A clinical reality or just a nice experiment in mice? Manuela Battaglia

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3 Induction of Immunological tolerance via T regulatory cells A clinical reality or just a nice experiment in mice? Inductie van immunologische tolerantie via regulatoire T- cellen Een klinische realiteit of gewoon een leuke experiment bij muizen? (met een samenvatting in het Nederlands) Proefschrift ter verkrijging van de graad van doctor aan de Universiteit Utrecht op gezag van de rector magnificus, prof.dr. G.J. van der Zwaan, ingevolge het besluit van het college voor promoties in het openbaar te verdedigen op dinsdag 18 november 2014 des ochtends te uur door Manuela Battaglia geboren op 30 april 1971 te Milaan, Italië

4 Promotoren: Prof.dr. A.B.J. Prakken Prof.dr. M.G. Roncarolo The research described in this thesis was financially supported by: the Telethon Foundation and the Juvenile Diabetes Research Foundation.

5 Tolerance and patience should not be read as signs of weakness. They are signs of strengths. - Dalai Lama

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7 Contents Chapter 1: General Introduction Chapter 2: Rapamycin selectively expands CD4 + CD25 + FoxP3 + T regulatory cells 9 32 Chapter 3: Rapamycin promotes expansion of functional CD4 + CD25 + FOXP3 + T regulatory cells of both healthy subjects and type 1 diabetic patients Chapter 4: Rapamycin and Interleukin- 10 treatment induces T regulatory type 1 cells that mediate antigen- specific transplant tolerance Chapter 5: Induction of tolerance in type 1 diabetes via both CD4 + CD25 + T regulatory cells and T regulatory type 1 cells Chapter 6: Rapamycin monotherapy in patients with type 1 diabetes modifies CD4 + CD25 + FOXP3 + T regulatory cells Chapter 7: Summary and Discussion Chapter 8: Nederlandse Samenvatting Acknowledgments Curriculum Vitae List of Publications

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9 1. General Introduction This chapter consists of parts gathered from the following manuscripts: Roncarolo MG, Battaglia M. T regulatory cell immunotherapy for tolerance to self- and alloantigens in humans. Nature Reviews Immunology 2007, 7: Battaglia M. Potential T regulatory cell therapy in transplantation: how far have we come and how far can we go? Transplantation 2010; 23(8): Roncarolo MG, Gregori S, Bacchetta R, Battaglia M. Tr1 Cells and the Counter- Regulation of Immunity: Natural Mechanisms and Therapeutic Applications Current Topics in Microbiology and Immunology 2014; 380:39-6 with the addition of updated references and newly emerging concepts in the field of Treg cells. 9

10 T regulatory cells The concept of a dominant form of immunological tolerance involving a specialized population of suppressor T cells that act to terminate conventional immune responses and to prevent autoimmune pathology was proposed more than 40 years ago (1). Early studies hypothesized a suppressor cell cascade involving multiple suppressor factors, anti- idiotypic T cell networks, suppressor- inducer', and contra- suppressor' cells (2). However, the mechanisms responsible for these suppressive phenomena were never characterized at the molecular and biochemical levels primarily because of the difficulty in isolating suppressor T cells at the single cell level. Moreover, key findings of those studies could not be reproduced and the field of suppressor T cell biology was largely discredited (3). In the last few years modern technologies and new experimental approaches consented a rebirth of suppressor cells (now called T regulatory cells), which are, at present, considered as one of the central players in immune regulation. Over the years several types of T regulatory (Treg) cells have been identified. The best characterized among the CD4 + T cells, are the Treg cells expressing the forkhead box P3 (FOXP3 + Tregs) and the T regulatory type 1 (Tr1) cells. FOXP3 + Tregs can be either thymus- derived (ttregs), or induced peripherally in vivo (ptregs) or experimentally in vitro (itregs) (4). Regardless of their origin, both subsets (ttregs and p/itregs) are characterized by constitutive cell surface expression of the IL- 2Rα chain (CD25), and of FOXP3, which is widely recognized as the master transcription factor for their function (5). On the contrary, Tr1 cells are induced in the periphery independently from FOXP3. Tr1 cells are distinct from other CD4 + T cell subsets because of their unique cytokine production profile. Their differentiation and function rely on the presence of IL- 10, a potent immunosuppressive cytokines. However, a Tr1- cell specific transcription factor related to the IL- 10 pathway has not been yet identified. For clarity, I hereby will refer to Treg cells as to all FOXP3- expressing cells independently on their origin (thymic or induced) and to Tr1 cells as to the inducible IL- 10 producing FOXP3 - Treg cells. Studies in murine models and in primary immunodeficiency patients have contributed to elucidate the biological properties and the function of these two subsets of Treg cells and to understand the pathogenesis of the diseases caused by altered generation and function of Treg and Tr1 cells. FOXP3 + Treg cells CD4 + CD25 + FOXP3 + Treg cells are strictly FOXP3- dependent and are subject to a unique pattern of DNA demethylation of the FOXP3 gene promoter, which guarantees their stability. FOXP3 natural mice mutants, the scurfy mice, in which FOXP3 gene is partially deleted, its expression is abrogated, and Treg cells are absent, have demonstrated the essential role of FOXP3 as cytokine suppressor gene and of Treg cells as key suppressor of lymphoproliferation (6). Conversely, FOXP3 transgenic mice showed the power of the suppressor function transferred by FOXP3 overexpression to different cell types (7). Finally, FOXP3 knock- in and reporter mice have elucidated the lineage specifications and timing of gene expression during development (8). Although the mechanism/s of action of Treg cells is not fully elucidated it is clear that they potently suppress activation, proliferation, and/or effector functions of both CD4 + and CD8 + T cells and of possibly natural killer, natural killer T, B, and dendritic cells. Their ability to control such a range of target cells in varying phases of the immune response presumably derives from the implementation of multiple modes of suppression in a multi- step manner. Said modes 10

11 include cell contact- dependent suppression, functional modification or killing of antigen- presenting cells (APC), and secretion of immunosuppressive cytokines (reviewed in (5)). Treg cells are important early in life, and loss of their function is life- threatening at very young age, as it is demonstrated in patients with Immune dysregulation Polyendocrinopathy Enteropathy X- linked (IPEX) syndrome, bearing FOXP3 mutations. Treg- cell expansion and function is IL- 2 dependent, as shown by both IL- 2 or CD25 knock- out (KO) mice and CD25- deficient patients, whose phenotype resembles FOXP3 deficiency (9). Tr1 cells Tr1 cells are memory T lymphocytes identified by cell surface expression of the lymphocyte- activation gene 3 (LAG- 3) and the integrin alpha2 subunit (CD49b) (10). Upon activation, Tr1 cells secrete high levels of IL- 10, and minimal amounts of IL- 4 and IL- 17 (10). Tr1 cells secrete also TGF- β, variable amounts of IL- 5, GM- CSF and IFN- γ, but low levels of IL- 2 (11). Tr1 cells proliferate poorly upon polyclonal T Cell Receptor (TCR)- mediated or antigen- specific activation in vitro (11-13). The autocrine production of IL- 10 by Tr1 cells contributes to their low proliferative capacity, since addition of neutralizing anti- IL- 10 antibody partially restores their proliferation (11; 12; 14). Tr1 cells modulate immune responses mainly through the secretion of IL- 10 and TGF- β (15). To exert their suppressive function Tr1 cells need to be activated via their TCR, but, once activated, Tr1 cells can mediate bystander suppressive activity against other antigens. IL- 10 and TGF β directly inhibits T- cell responses by suppressing IL- 2, and IFN- γ ((16) and by preventing T- cell proliferation (17; 18). IL- 10, locally released by activated Tr1 cells, acts also on APC, by down modulating co- stimulatory molecules and production of pro- inflammatory cytokines (19; 20) and on B cells by promoting isotype switching (21; 22). Overall, Tr1 cells control immune responses by modulating directly and indirectly T-, APC- and B- cell functions. Interestingly, IL- 10 KO mice have originally revealed the importance of this cytokine in the gut homeostasis and in preventing overwhelming inflammation (23). These mice spontaneously develop intestinal inflammation characterized by transmural lesions affecting the small and large intestine and uncontrolled generation of IFN- γ producing T cells. Similarly, the genetic loss of IL- 10R or IL- 10 in humans (24; 25) leads to a devastating inflammatory disease with predominant pathology in the gut where it is now well known that Tr1 cells are required to maintain tolerance to commensal antigens and bacteria. 11

12 Treg cells control tolerance in humans Several studies have shown that Treg cells play a crucial role in controlling tolerance to self- and allo- antigens in humans. This section describes the correlation between, on the one hand, the presence of Treg cells and tolerance and, on the other hand, the absence of Treg cells and immunopathology. Furthermore, the human genetic diseases in which Treg- cell dysfunction occurs is discussed. Role of Treg cells in responses to self- antigens Negative selection of developing autoreactive thymocytes is not a wholly efficient process and some T cells with high affinity for autoantigens can escape the deletion process in the thymus and migrate to the periphery. Peripheral tolerance is required to suppress those autoreactive T cells that escape thymic selection. Proliferative responses of human peripheral- blood mononuclear cells (PBMC) from healthy individuals toward the self antigens heat- shock protein 60 (26), myelin oligodendrocyte glycoprotein (MOG) (27), the type 1 diabetes- associated antigen glutamic- acid decarboxylase 65 (GAD65), and the vitiligo- associated antigen (28) are indeed significantly enhanced in vitro upon removal of Treg cells. Similarly, Tr1 cells specific for self- MHC molecules (29) and for islet- derived peptides (30) have been found in healthy individuals. These observations indicate that autoreactive T cells are present and circulate in healthy subjects and that Treg cells actively suppress their function. Also consistent with an important role for Treg cells in controlling autoreactivity is the demonstration that a wide variety of autoimmune diseases are associated with defects in Treg- cell function, raising the interesting possibility that this may be a common basis for the uncontrolled immune responses to self antigens (31). Tr1 cells have been isolated from the synovium of patients with rheumatoid arthritis, but they seem to be present in significantly lower numbers compared with control individuals with non- autoimmune- mediated joint inflammation (32). Similarly, Treg- cell defects in peripheral blood of patients with multiple sclerosis, type 1 diabetes, psoriasis, myasthenia gravis, rheumatoid and juvenile idiopathic arthritis have been described (31). Role of Treg cells in responses to alloantigens Preclinical studies clearly demonstrate that Treg cells are associated with transplantation tolerance and Treg- cell therapy efficiently controls graft rejection (see below). Nonetheless, what clinical evidence do we have that Treg cells play a fundamental role in inducing and/or maintaining long- term tolerance after solid organ transplantation in humans? In principle, it seems quite an easy task to identify Treg cells in the periphery of transplanted patients and to associate the presence or absence of Treg cells with a given clinical condition (e.g., engraftment versus rejection versus tolerance). Unfortunately, this is not the case. For instance, FOXP3 expression, which is constitutively high in Treg cells, can be up- regulated in non- Treg cells upon activation (33; 34). Thus, functional in vitro assays performed with purified Treg cells are fundamental to precisely discriminate between cells with regulatory and those with non- regulatory activity. However, cell- sorting strategies are often subjective and they might lead to isolation of cells with various degrees of purity. Without mentioning that suppressive assays are time- consuming experiments unlikely to be used routinely. There has been a growing interest on the characterization of bona fide ttreg cells by the analysis of the Treg- specific demethylated region (TSDR) within the FOXP3 locus. This region is constantly demethylated in bona fide Treg 12

13 cells and not in inducible Treg cells or activated FOXP3 + non- Treg cells (35). Therefore, the TSDR analysis represents the ideal tool to discriminate among ttreg and non- Treg cells and so far it is considered a reliable and environmentally- uninfluenced marker for bona fide ttreg cells. Unfortunately, this assay is based on DNA analysis and thus does not allow for isolation of viable pure cells. The identification and quantification of Tr1 cells have been even more complex due to the lack for several years of any specific surface marker that could exclusively associate a tolerogenic state with the Tr1- cell presence and abundance. Until 2013, when Tr1- cell specific markers have been described (10), IL- 10 production was the only hallmark of Tr1 cells but the very same cell has to produce very low levels of IFN- γ and IL- 2 in the absence of IL- 4 (36). Intracytoplasmic staining was the only in vitro read- out which provided such information but it requires TCR- mediated activation which has to be quite powerful (i.e., non physiological) to detect appreciable numbers of circulating Tr1 cells. T- cell cloning was the generally accepted technique that could give a rough idea of Tr1- cell frequency in vivo (37). This was however extremely time- consuming and could not be proposed as routine screening for transplanted individuals. Given the drawbacks of these approaches, it is very likely that the data generated in transplanted patients may not be comprehensive until 2013 when our group reported for the first time Tr1- cell specific surface markers (10). We are therefore facing important technical restraints that could possibly justify some contradictory results generated in the last few years and might have led to our partial view of the role played by Treg cells in promoting and maintaining transplantation tolerance in humans. By keeping this in mind, in case the reader craves for an overview of all the papers that positively correlate FOXP3 expression with graft tolerance and all the papers that negatively correlate graft outcome with FOXP3 expression, excellent investigators already did a tremendous job (38) which I shall not attempt to replicate. Considering the lack of a bona fide Treg cell marker it is my opinion that it is almost impossible to provide a unique interpretation of all the published data in an attempt to correlate FOXP3 expression with operational tolerance. Similarly, Tr1 cell detection in transplanted patients is often limited to IL- 10 yes / IL- 10 no, which leads to a inevitably limited view. Should humans be treated like guinea- pigs, one could deplete/block Treg cells in tolerant patients and see whether the graft is rejected. Since patients are definitely not guinea- pigs, we have to provide alternative convincing ways to prove that Treg cells are crucial for tolerance maintenance after solid organ transplantation. Defect of Treg cells in genetic diseases IPEX, WAS (Wiskott- Aldrich syndrome), APS- II (autoimmune polyglandular syndrome type 2), and ALPS (autoimmune lymphoproliferative syndrome) are the few autoimmune diseases caused by known genetic defects. Interestingly, patients with IPEX (39), WAS (40) and APS- II (41) have a clear defect in Treg- cell suppressive function. Patients with ALPS type Ia are characterized by expansion of CD3 + T cells, but a reduction in the CD4 + CD25 + T- cell subset and this defect may be indicative of disturbed lymphocyte immunoregulation in ALPS. Furthermore, data generated in our laboratory in a preclinical animal model of the Omenn syndrome, point to a defect in Treg cells in this genetic disease as well (42). Although the molecular mechanisms underlying these 13

14 defects in Treg cells are still under scrutiny, these data suggests that Treg- cell based therapy might represent a good immunomodulatory strategy also in these genetic diseases (43). 14

15 Therapy with Treg cells in mice Compelling data generated in preclinical animal models indicate that Treg cells can be used as therapeutic agents for treating immune- mediated diseases. This section summarizes the results generated with adoptive transfer of Treg cells in experimental models of autoimmune diseases and allogeneic transplantation. Adoptive transfer of Treg cells in animal models of autoimmune diseases Several preclinical animal studies have established that the adoptive transfer of Treg cells can prevent various autoimmune diseases. As the development of autoimmunity in humans is rarely a foreseeable phenomenon, to be of use as therapeutic agents Treg cells must inhibit ongoing T- cell responses and reverse established pathology. However, among the numerous published studies only three showed that Treg- cell transfer is efficacious in reverting active disease (44-46). Interestingly, of these three studies, only one showed that reversal of disease can be mediated by polyclonal wild- type Treg cells (44), whereas the other two studies used transgenic Treg cells expressing a TCR specific for the pathogenic antigen (45; 46). Importantly, the positive results obtained with polyclonal wild- type Treg cells (44) were generated in lymphopaenic hosts in whom activation or expansion of Treg- cell subsets can be influenced by homeostatic proliferation. Overall these data indicate that the suppression of an ongoing autoimmune disease by adoptive transfer of Treg cells might only be feasible when the Treg cells are specific for the pathogenic antigen. Alternatively, transfer of polyclonal Treg cells may cure ongoing disease only in lymphopaenic hosts, in which their massive expansion of Treg cells may lead to the generation of a sufficient number of antigen- specific Treg cells. If this is the case also in humans, it may represent a significant limitation for the clinical application of Treg cells in autoimmune diseases, as, to date, human self- antigen- specific Treg cells have not been successfully expanded ex vivo. By contrast, Tr1 cells can be generated in an antigen- specific manner ex vivo and/or directly in vivo and therefore may represent an ideal therapeutic alternative. However, additional preclinical studies need to be carried out to show that antigen- specific Tr1 cells can revert ongoing autoimmunity. Adoptive transfer of Treg cells in animal models of transplantation Transfer of freshly isolated Treg cells together with the bone- marrow allograft has been shown to ameliorate graft versus host disease (GVHD) and facilitate engraftment in mouse models of bone marrow transplantation (BMT) (47-49). GVHD was also the first model in which it was shown that the adoptive transfer of donor Treg cells that were polyclonally expanded ex vivo is as efficient as the transfer of freshly isolated Treg cells in curing transplanted mice (50-52). However, contrary to what was observed in the experimental models of autoimmunity, the transfer of Treg cells enriched for alloantigen specificity showed only moderately improved efficacy compared with the transfer of a polyclonal Treg- cell population (53). These different results might be ascribed to the higher frequency of alloantigen- specific T cells compared with cells specific for self antigens within the Treg- cell subset. However, this hypothesis contrasts with the concept that Treg cells are enriched for self- antigen specificity because they develop in the thymus. An alternative explanation for the different outcome in the experimental models of autoimmunity versus BMT might be the presence of a lymphopaenic environment that supports the expansion of transferred effector T cells and Treg cells in BMT recipients and that it may be different to that in 15

16 normal mice. Thanks to these promising results generated in the animal models and to the lack of antigen- specific requirements for the transferred Treg cells, BMT is the setting for the first human clinical trial with Treg cells generated ex vivo, which were carried out to test the ability of Treg cells to suppress GVHD (see below). Tr1 cells generated ex vivo upon stimulation with alloantigens in the presence of IL- 10 and TGFβ have been shown to be potent regulators after allogeneic BMT. Infusion of unmanipulated cultured T cells induced lethal GVHD in all recipients, whereas only 25% of mice receiving Tr1 cells died (54). A clinical trial with ex vivo generated alloantigen- specific Tr1 cells in BMT was performed by our group at the San Raffaele Scientific Institute (see below). In contrast to what has been reported in GVHD, to our knowledge there are no reports showing that the transfer of freshly isolated Treg cells can prevent the rejection of allogeneic solid organ transplantation. Several studies indicate that Treg cells generated in vivo in transplanted animals by various approaches (such as by treatment with vitamin D3 or mycophenolate mofetil (55)) do transfer tolerance in secondary transplant recipients. The lack of data proving efficacy after transfer of freshly isolated Treg cells might be due to unreported unsuccessful experiments and/or to the requirement of antigen- specific Treg cells for protection. Alloantigen- specific Treg cells may be absent in freshly isolated cells but may be generated upon in vivo expansion in the presence of specific alloantigens. Positive results generated with transfer of antigen- specific transgenic Treg cells are consistent with this view (56). At present, the lack of data clearly showing that transfer of Treg cells do protect from allograft rejection in several preclinical animal models represents a major hurdle to the use of Treg- cell- based immunotherapy in solid organ transplantation, although some attempts are already ongoing (see below). 16

17 Therapy with Treg cells in humans As mentioned above, the first animal model used to test the in vivo regulatory activity of Treg cells was the allogeneic stem cell transfer in mice. It was shown that donor- derived Treg cells do not induce GVHD. By contrast, Treg cells prevent GVHD when co- transplanted with effector T cells (50-52). Experimental GvHD represents the ideal model for the examination of Treg- cell mediated suppression in vivo since: (i) the time of disease onset is known and thus Treg- cell administration can be performed either prophylactically or therapeutically; (ii) the lymphopenia in conditioned recipients supports the activation and in vivo expansion of the transferred Treg cells; and (iii) it is clinically relevant. As a matter of fact, the first in- man clinical trials with Treg cells have been performed in stem cell transplanted patients with the aim of preventing/curing GVHD. However, new attempts in autoimmune diseases have been also reported. The results of these trials are briefly described below. Clinical trials with CD4 + CD25 + Treg cells The first study was reported by P. Trzonkowski and colleagues who performed a clinical trial with ex vivo expanded CD4 + CD25 + CD127 Treg cells for the treatment of acute or chronic GVHD. The therapy gave significant alleviation of the symptoms and reduction of pharmacological immunosuppression in the case of chronic GVHD. However, in the case of grade IV acute GVHD, it improved the clinical condition only temporarily (57). The group led by M. Martelli reported that transfer of freshly isolated CD4 + CD25 + T cells (consisting of CD25 high 25.6%±11.2 and FOXP3 + cells 64%±1 mean±sd) three days prior to transplantation of haploidentical CD34 + hematopoietic stem cells (HSC) favors immune reconstitution. No GVHD was observed in 17 out of 20 valuable patients. Two patients developed grade I cutaneous self- limited untreated GVHD and 1 developed grade III GvHD (this patient had received the lowest Treg cell doses). This study demonstrates that, in the setting of haploidentical stem cell transplantation, the infusion of freshly purified Treg cells prior to transplant provides long- term protection from GVHD and robust immune reconstitution (58). A more recent clinical trial performed by the same group also demonstrated that the immunosuppressive potential of Tregs cells can be used to inhibit GVHD without loss of the benefits of graft- versus- leukemia (GVL) activity (59). The group of J. Wagner performed adoptive transfer of umbilical cord blood (UCB)- derived Treg cells to recipients of non- myeloablative unrelated UCB transplantation (UCBT). CD25 + cells were obtained from a 3 rd UCB unit and expanded in the presence of anti- CD3/CD28 mab coated beads and IL- 2 for an average of 18 days. Expanded Treg- cell dose escalation levels (from 1 to 30 x 10 5 /kg) were transferred on day +1, and 30 x 10 5 /kg on days +1 and +15 after UCBT. After infusion an increased proportion of peripheral blood comprising CD4 + FOXP3 + CD127 - cells was observed. Donor Treg cells were clearly detected in all patients receiving Treg cells that were HLA disparate. The co- infusion of ex vivo expanded and activated UCB- derived Treg cells to recipients of non- myeloablative UCBT: 1) was safe at the tested dose levels, 2) led to a detectable increase in donor - derived circulating Treg cells, and 3) resulted in an increased proportion of mixed chimerism (60). However, a more in depth analysis of the data collected in this same trial led to the unexpected conclusions that there was a significantly higher cumulative density of opportunistic infections in patients treated with Treg cells as compared to historical patient groups not receiving cell therapy (61). There was potentially a higher viral infection risk within 30 17

18 days of UCB- derived Treg- cell infusion due to suppression of the immune response. However, there was no adverse effect on the longer term outcomes, including later risk of opportunistic infections. Although observation bias is a possibility, this data suggesting higher density of viral infections argues for close observation in studies of the adoptive transfer of CD4 + CD25 + Treg cells to reduce GVHD in allogeneic hematopoietic cell transplantation and in solid organ transplantation (61). The only clinical trial thus far published with CD4 + CD25 + Treg cells in settings other than BMT/GVHD is the one performed by the group of P. Trzonkowski and colleagues in type 1 diabetes (62). Ten children with type 1 diabetes within 2 months from diagnosis were infused with autologous CD3 + CD4 + CD25 high CD127 - sorted Treg cells expanded in vitro polyclonally for a maximum of 14 days (average 10 days). The study was feasible and safe and suggested plasma C- peptide level preservations in treated patients. This data was recently confirmed in a longer follow up study (63). All the trials thus far reported with CD4 + CD25 + Treg cells demonstrated to be feasible and overall safe although particular attention should be given to the recently reported opportunistic infections after cell therapy (61). Clinical trials with Tr1 cells Our group was the first to perform a proof of concept clinical study to establish the safety and efficacy of a cellular therapy with alloantigen specific donor- derived IL- 10 anergized cells containing host- specific Tr1 cells in patients transplanted with CD34 + HSC from haploidentical donors (the ALT TEN trial) (64). PBMC were collected from both the donor prior to stem cell mobilization and the host prior to conditioning. Subsequently, a mega- dose of T- cell depleted CD34 + hematopoietic stem cells was infused in the myeloablated host. Once there were signs of neutrophyl engraftment, the donor- derived host- specific IL- 10 anergized cells were infused in the host in the absence of immunosuppression for GVHD prophylaxis, with the ultimate goal of providing immune reconstitution without severe GVHD. Sixteen patients received CD34 + selected stem cells and 12 patients were treated with IL- 10 anergized cell therapy at day +30 post- transplant, at the dose of 10 5 CD3 + cells/kg with the exception of one patient who received 3x10 5 CD3 + cells/kg. Five patients died from infections because of lack of immune- reconstitution and two patients dropped out because of graft rejection. Five patients achieved immune reconstitution followed by progressive normalization of the TCR repertoire, memory/näıve phenotype, and T- cell functions in vitro and in vivo. Acute GVHD grade III was observed in the patient who received 3x10 5 CD3 + cells/kg; GVHD grade II responsive to short- term treatment was observed in four patients who received 10 5 CD3 + cells/kg and were successfully immune- reconstituted. These four patients are alive, in complete disease remission and immune suppression- free at 7.2 years (median follow- up) after haplo- HSC transplantation. Persistent host- specific hypo- responsiveness of donor T cells in vitro and enrichment of cells with Tr1- cell specific biomarkers in vivo was observed. Gene expression profiles of immune- reconstituted patients showed a common signature of tolerance. Overall, the ALT TEN trial proves that cellular therapy with cells comprising donor- derived host- specific Tr1 cell- precursors is safe and feasible. In addition, based on follow up of the long- term surviving patients, this trial suggests that IL- 10 DLI infusion can sustain immune reconstitution with no severe GVHD and no disease relapse (64) 18

19 Finally, an open- label, multicenter, single- injection, escalating- dose, phase I/IIa clinical study with Ovalbumin (OVA)- specific Tr1 cells clones was performed in 20 patients with refractory Chron s disease (CD) in whom objective indicators of inflammation were measured (the CATS1 trial) (65). OVA- specific Tr1 cells were generated with a manufacturing process that included a first step of culturing patient PBMC with native OVA, IL- 2, and IL- 4 to enrich and expand OVA- specific T cells, followed by cloning and expansion on a layer of feeder cells (i.e., Schneider cells) transfected to express a membrane- bound anti- CD3 antibody, as well as human CD80, CD58, IL- 2, and IL- 4. OVA- Tr1 cell clones were selected based on an OVA- specific IL- 10 production. A single dose of escalating numbers of autologous OVA- specific Tr1 cells was administered to 20 patients with moderate to severe refractory CD. To ensure activation of OVA- specific Tr1 cells migrating to the gut, patients ingested an OVA- enriched diet. The adoptive transfer of Tr1 cells was safe, with 11 adverse events (AEs) possibly related to Tr1- cell infusion. Seven patients experienced severe AEs (SAEs) and of these, 2 patients reported 3 suspected unexpected serious adverse reactions (SUSARs). All patients with SUSARs fully recovered. This trial resulted in a 40% response rate in the intention- to- treat population. CD flares were documented in 7 patients. Six out of the 8 CD patients in the lowest dose group (10 6 Treg cells) had a measurable response, with 3 patients being in remission at week 5 and 2 patients at week 8 (65). Overall, the lowest dose group had the highest percentage of patients in remission, based on the evaluation of the Crohn's Disease Activity Index. From a mechanistic standpoint, OVA- specific proliferative responses diminished in responders, especially in the lowest Treg- cell dose group. The clinical effect was time limited, reaching the maximum 5 weeks after treatment and declining thereafter. This observation suggests that the infusion of multiple doses of Tr1 cells may be required for long- term control of the disease. This study demonstrates that administration of antigen- specific Tr1 cell clones to patients with refractory CD was well tolerated and had some efficacy. The OVA- specific immune response correlated with clinical response, supporting Ag specific immune- regulatory mechanisms of OVA- Tr1 cells (65).. Overall, Tr1- cell based therapy has been shown thus far to be feasible and safe. The ONE Study An interesting ongoing project is The ONE Study, an integrated European Union- funded project that aims at developing and testing different subsets of regulatory cell products in kidney transplanted recipients allowing a direct comparison of the safety, clinical practicality and therapeutic efficacy of different cell types ( Results from this trial, in which we participate with donor specific Tr1 cells generated ex vivo, will define which regulatory cell subset has the highest possibility to be functional in vivo at inducing donor specific tolerance after kidney transplantation. 19

20 Possible mechanisms of action of Treg cell- based therapy To control complex immune- mediated diseases, such as acute GVHD after HSCT and autoimmune diabetes, the immune system needs to be modulated at different levels. Here I describe the known immunological events that occur in the two abovementioned pathological settings in an attempt to delineate, based on our present knowledge, the level at which Treg cells could intervene. Treg cells and the control of acute GVHD The pathophysiology of acute GVHD after HSCT can be considered as a three step process in which both the innate and adaptive immune systems interact (Figure 1). As draining lymph nodes are the sites of donor T- cell priming by host antigen- presenting cells (APCs), it seems likely that protection from GVHD by adoptively transferred Treg cells might also occur at these sites. Therefore, an important prerequisite for Treg cells to function is their ability to migrate to the correct site where they can encounter alloantigens presented by host APCs and be activated (48; 66). Early robust expansion of Treg cells in draining lymph nodes followed by their migration and localization into peripheral tissues was reported in mice (67), further supporting this hypothesis. After activation, Treg cells exert their suppressive function by inhibiting effector T- cell proliferation, cytokine production and migration. Treg cells also down- modulate DC function by preventing their maturation in a cell- contact- dependent manner (68). Treg cells can also influence monocyte and macrophage function by inhibiting lipopolysaccharide- induced monocyte survival through the FAS/FASL apoptotic pathway (69). Alternatively, Treg cells restrain monocytes by reducing their activation state, leading to reduced pro- inflammatory cytokine production and impaired APC function (70). Interestingly, Treg cells also inhibit neutrophil activity and promote their apoptosis and death (71). Tr1 cells, by producing IL- 10, induce anergy in effector T cells and affect APC function by down- regulating the expression of MHC and co- stimulatory molecules. In addition, IL- 10 promotes the secretion of IL- 1 receptor antagonist and soluble tumor- necrosis factor receptor rather than pro- inflammatory cytokines by APCs (15). Of particular interest is the observation that both interferon- γ (IFNγ) and nitric oxide (NO), which are produced at high levels during GVHD, have paradoxical functions (72). The pathogenic role of IFNγ as a T helper 1 (Th1)- cell- associated cytokine that is involved in the development of immune- mediated diseases has been well documented (73). Similarly, NO is a cytotoxic molecule that may contribute to immune- mediated diseases. However, in the presence of Treg cells these two molecules have immunoregulatory activity (72). In the presence of Treg cells, IFNγ stimulates APCs to produce NO and heme oxygenase 1 (HO1) through increased expression of the enzymes inducible nitric- oxide synthase (inos) and indoleamine 2,3- dioxygenase (IDO), which degrades the essential amino acid tryptophan and greatly affects T- cell proliferation and survival. NO then can diffuse into neighbouring T cells, influencing their function and triggering apoptosis, whereas HO1 prevents T- cell proliferation and modulates their activation through the degradation of the pro- oxidant heme into carbon monoxide, iron and biliverdin (72). 20

21 Figure 1. The pathophysiology of acute graft- versus- host disease (GVHD) can be envisaged as a three step process. a The first step occurs before donor- cell infusion. Prior to haematopoietic stem- cell transplantation (HSCT), the conditioning regimen (that is, irradiation and/or chemotherapy) leads to damage and activation of host tissues, especially the intestinal mucosa. This allows the translocation of microbial products, such as lipopolysaccharide, from the intestinal lumen to the circulation, which stimulates the secretion of pro- inflammatory cytokines, such as interleukin- 1 (IL- 1) and tumor- necrosis factor (TNF), from host tissues (particularly from macrophages). Activated macrophages produce chemokines that activate neutrophils, which further increases inflammation. The release of these pro- inflammatory cytokines increases the expression of MHC and adhesion molecules on host cells, enhancing their antigen- presenting capacity. b The second step is characterized by activation of donor T cells, which occurs mainly in the lymph nodes that drain GVHD target organs, such as the gut, skin and liver. Donor T- cell activation results mainly in interferon- γ (IFN- γ) production, which further increases the expression of MHC and adhesion molecules, chemokines and CD95 on antigen- presenting cells (APC). This results in further increases in antigen presentation and recruitment and the expansion of host- specific cytotoxic CD8 + and CD4 + T cells, and natural killer (NK) cells. c In the final step, effector cells then migrate to the target organs, where they mediate tissue injury that leads to multi- organ failure mediated mainly by the CD95 CD95 ligand and the perforin granzyme pathways. Treg cells can intervene at different stages to control GVHD. It is likely that Treg cells, like effector T cells, are first activated in the draining lymph nodes. Both FOXP3 + CD4 + CD25 + Treg and T regulatory type 1 (Tr1) cells, although through different mechanisms, block activation and expansion of effector T cells and/or modulate the functions of APC, monocytes, macrophages and neutrophils. Consequently, activation and proliferation of alloreactive donor effector T cells is suppressed, resulting in diminished export from the lymph nodes to the target organs. In addition, T R1 cells, through IL- 10 and transforming growth factor- β (TGFβ) production, efficiently reduce the inflammatory state. IFNγ produced in the presence of inducible regulatory T cells leads to the expression of inducible nitric- oxide synthase (inos) and indoleamine 2,3- dioxygenase (IDO) with consequent production of NO and haem oxygenase 1 (HO1), which modulate effector T- cell functions. idc, immature DC; mdc, mature DC; TCR, T- cell receptor. 21

22 Treg cells and the control of autoimmune type 1 diabetes Type 1 diabetes is a tissue- specific autoimmune disease in which inflammation plays a central role (Figure 2). The mechanisms through which Treg cells can function in vivo to block the development of diabetes are under intense investigation. The model that we propose is based on data generated mainly in mouse models, and its clinical significance is yet to be proven. Treg cells present in the pancreatic draining lymph nodes regulate the priming of autoreactive T cells by limiting their expansion and differentiation. Interestingly, Treg cells may function by interrupting the development of effector T cells through limiting the access of autoreactive T cells to DC (74). Thanks to the control of this crucial step in lymph- node priming, Treg cells limit the chance of a T cell becoming an effector cell. Treg cells inhibit CXC- chemokine receptor 3 (CXCR3) expression by T helper cells with a consequent lack of infiltration of these cells into the islets (75). Furthermore, Treg cells might restrain autoimmune aggression directly in the islets by controlling the inflammatory reaction (insulitis) (76). T11 cells produce IL- 10 and TGFβ, which both have an important immunoregulatory role in type 1 diabetes (77). Transient expression of TGFβ in the islets during the priming phase of diabetes inhibits disease onset and stimulates expansion or generation of intra- islet FOXP3- expressing Treg cells (78). IL- 10 modulates APC function, reduces inflammation, and decreases T- cell activation. Furthermore, IL- 10 produced by Tr1 cells down- regulates the expression of intercellular adhesion molecule 1 (ICAM1) on effector T cells, which prevents their migration to the target organ (79). Finally, the paradoxical effects of IFNγ and NO that are thought to be important for the immunomodulatory effects of Treg cells during GVHD might also have a role in autoimmune diabetes (80). 22

23 Figure 2. As for graft- versus- host disease, the pathophysiology of type 1 diabetes can be envisaged as a three- step process. a First, under still undefined pathogenic conditions, modified islet beta- cell antigens are released and presented by MHC class I molecules. These previously 'cryptic' antigens are presented by tissue- resident antigen- presenting cells (APCs) and recognized by CD8+ T cells that cause damage to MHC- class- I- expressing cells either through the release of cytotoxic cytokines (such as IFNγ) or through the perforin granzyme pathway. b The released islet beta- cell components are taken up by immature dendritic cells (idcs) in the pancreatic islets and transported to the draining pancreatic lymph nodes, where the antigens are processed and presented to CD4+ T cells. Lymph- node priming is thought to be the second crucial step leading to expansion of low frequency circulating autoreactive T cells. After clonal expansion, CD4+ effector T cells express adhesion molecules, such as intercellular adhesion molecule 1 (ICAM1) and lymphocyte function- associated antigen 1 (LFA1), and chemokine receptors, such as CC- chemokine receptor 4 (CCR4), CCR5 and CXC- chemokine receptor 3. This allows the effector cells to home to the pancreatic islets, tracing antigen gradients and chemokines induced by the early CD8+ T- cell- mediated inflammatory response. c Once in the pancreas, the activated CD4+ T cells recruit and activate inflammatory cells, causing insulitis. The effector phase of islet beta- cell destruction is mediated by cytokines (mainly interleukin- 1 (IL- 1) and tumor- necrosis factor (TNF)) through the induction of pro- apoptotic signaling selectively in islet beta- cells and/or by inducing the expression of CD95 by islet beta- cells, which allows direct killing by CD95 ligand (CD95L)- expressing effector T cells. There is also evidence that the production of free radicals is involved in the pathogenic events leading to islet beta- cell destruction (not shown). Treg cells can intervene at different stages to control type 1 diabetes. As in graft- versus- host disease, it is likely that Treg cells are first activated in the pancreatic lymph nodes. Activated FOXP3+CD4+CD25+ Treg cells and Tr1 cells, through distinct regulatory mechanisms, block the activation and expansion of effector T cells either directly or indirectly through APCs. Expression of adhesion molecules and chemokine receptors by effector T cells is also suppressed by regulatory T cells, with consequent reduced effector T- cell migration to the target organ. The aggressiveness of insulitis is also directly inhibited in the pancreas by regulatory T cells. TR1 cells, through IL- 10 and transforming growth factor- beta (TGFβ) production, can inhibit the onset of disease and reduce inflammation. 23

24 Strengths and pitfalls of Treg- cell based therapy The use of Treg cells as immunotherapy has several advantages compared to standard therapies but it is currently limited by technical, regulatory and financial issues. Strengths and pitfalls of such therapy are here briefly described. Strengths The administration of immunosuppressive drugs is the most common approach for treating immune- mediated diseases. However, the long- term administration of non- antigen- specific agents that cannot distinguish between beneficial and destructive immune responses is a major drawback. Furthermore, immunosuppressive treatment must be life- long, as, if withdrawn, there is high risk of disease relapse due to lack of tolerance induction. By contrast, Treg cells are physiological components of the immune system and their adoptive transfer should re- establish the immunological homeostasis broken under pathological circumstances. Immunotherapy with Tr1 cells can, for example, be envisaged as an in vivo transfer of a biological source of IL- 10, which, if administered exogenously as a recombinant protein, would likely not have the same therapeutic effects. Importantly, Treg- cell based therapy is an individualized medicine, customized to the patient and can therefore be created to satisfy specific patient s needs with limited side effects. This is, however, a double- edged sword, as T- cell products cannot be manufactured and distributed easily and freely as for standard medicinal products and can be prohibitively expensive in its current experimental phase. Pitfalls The obstacles that limit Treg- cell based immunotherapy at present are mainly technical and relate to cell manipulation. The Treg cells must be collected from the peripheral blood, for example by leukapheresis, and immediately processed or cryopreserved when necessary. Circulating Treg cells can be directly isolated from the circulating pool of CD4 + T cells but because of their limited number they need to be further expanded in vitro. Tr1 cells are generated from CD4 + T cells, which need first to be isolated and primed in vitro. Cell isolation and manipulation are therefore prerequisite for Treg- cell based immunotherapy. Several other points need to be considered for the development of Treg cells as a medicinal product and many different quality controls are needed (Figure 3). Cell isolation, manipulation, expansion and re- infusion into the patients are all procedures that need to be performed under GMP conditions. As a result, this therapeutic approach is extremely expensive in its experimental phase and also requires certified GMP facilities. It is therefore unavoidable that only few institutions can provide all the necessary infrastructure to make regulatory T- cell therapy a reality. However, should this immunotherapy meet its therapeutic targets, we might envisage that not only academic institutions but also pharmaceutical companies will be interested in adopting this therapeutic approach. Safety of the infused ex vivo manipulated product is clearly a high priority. The risk of uncontrolled cell proliferation, pan immunosuppression and consequent tumor development should be carefully monitored. Furthermore, superior efficacy of Treg- cell based immunotherapy over conventional therapy should be clearly demonstrated. 24

25 Finally, complying with international regulatory requirements for Treg- cell based therapy is an expensive, time- consuming process, and approval is never certain. In addition, such requirements are often different in each country making this approach extremely complex for a quick and efficient translation into the clinic. Figure 3. The steps necessary for the development of Treg cells as a medicinal product are shown. From cell collection to cell processing, expansion and differentiation, and final infusion into the patient, clinical good manufacturing practice (GMP) procedures need to be performed. Various quality controls are required in each manufacturing step and the final product can be released and infused into the patient only after approval by a qualified person. 25

26 Aim and outline of the thesis The possibility that Treg cells might have an application for the treatment of T- cell- mediated diseases has recently gained increasing momentum. The advantages of the adoptive transfer of Treg cells over conventional treatment are numerous. Some of these benefits include: the potential for antigen specificity with the lack of general immunosuppression, the possibility of inducing a physiological long- lasting regulation in vivo, and the fact that Treg- cell based immunotherapy could be a custom- made product, designed ad hoc for each patient, with very limited or absent side effects. Treg- cell based therapy can be performed in two distinct ways: (i) via infusion of ex vivo generated/expanded Treg cells or (ii) via the direct in vivo induction/expansion of antigen specific Treg cells. Both methods have advantages and disadvantages. Some of the key issues related to the infusion of ex vivo generated/expanded Treg cells that need to be addressed - and that were subjects of this thesis - are: - the definition of an efficient and safe protocol for the ex vivo expansion of Treg cells; - the demonstration that that the expanded Treg cells can promote tolerance in vivo in preclinical animal models; - the demonstration that the protocol is equally efficient and safe by using human cells obtained from both healthy donors and patients. Some of the important issues that need to be addressed in case of direct in vivo Treg- cell induction/expansion - and that were subjects of this thesis- are: - the definition of a short- term therapy that allows control of effector responses while permitting at the same time induction /expansion of functional Treg cells; - the demonstration that the Treg- cell permissive therapy is efficient in more than one disease animal models; - the clinical relevance of the findings eventually reported in animals. Taken together, in this thesis, we attempted to address a series of important and still unresolved questions in the field of Treg- cell immunotherapy. We first used mouse models and then attempted to confirm the data, when possible, using human samples. With such an approach we aimed at making Treg- cell based therapy a reality closer to the clinic. 26