Treg Therapy Research Articles

Development of GMP-Compatible Protocols for Thymus-Derived Regulatory T Cell Expansion

Introduction: Animal and early clinical studies have shown that regulatory T cell (Treg) therapy can prevent graft-versus-host disease (GVHD) following hematopoietic stem cell transplant. Despite these promising results, challenges remain in administering Treg therapy including the difficulty in isolating pure Tregs and expanding them to a therapeutic dose. We have previously found that the thymus, which is routinely removed during pediatric cardiac surgery, is a plentiful source of Tregs. These Tregs can be easily isolated, and can prevent xenoGVHD in mice. However, to use this therapy in humans, standard GMP-compliant methods for isolation and large-scale expansion of thymic Tregs must be developed. Methods: CD4+CD8-CD25+ thymic Tregs were isolated from pediatric thymuses using GMP-compatible magnetic bead-based separation. Tregs were expanded using artificial antigen-presenting cells or GMP-compatible antibody tetramer complexes that stimulate CD3 and CD28, with or without additional activation of CD2. Cells were cultured with rapamycin and IL-2, and were restimulated after 7 days without rapamycin. Results: Thymic Tregs expand in vitro when cultured with tetrameric antibody complexes. About 50-fold expansion was achieved using the anti-CD3/CD28/CD2 antibody tetramers, which represents the expansion that will be necessary to reach a clinically relevant dose. A higher expansion rate was obtained using the anti-CD3/CD28/CD2 antibody tetramer complexes than the anti-CD3/CD28. Cells cultured with these antibody tetramers express high levels of FOXP3, similar levels of Treg-characteristic markers as those expanded with artificial antigen-presenting cells, and do not produce inflammatory cytokines. Conclusion: Thymic Tregs can be isolated and expanded with GMP-compliant methods. Once the protocol is optimized, we can transfer our protocols to a GMP facility for further process development and to generate data required for a clinical trial application. We are the first group to pursue clinical application using allogeneic thymic Tregs, which provide an opportunity for an “off-the-shelf” therapy to treat GVHD. Canadian National Transplant Research Program.

MHC class II-peptide complexes displayed on activated T cells guide Treg suppression

Abstract Regulatory T cell (Treg) therapy is a promising strategy for extending allograft survival. Treg suppression requires initial T cell receptor (TCR)/peptide-MHC-II (pMHC-II) contacts, the nature of which remains elusive. As inflammation facilitates transfer of pMHC-II from antigen presenting cells (APCs) to activated T cells, we hypothesize that during transplantation associated inflammation, pMHC-II relocate on alloreactive T effector cells and serve as Treg activation signals. To test this, suppression assays were performed using murine CD4+ CD25− (Teff), CD90 depleted APCs, and CD4+ CD25+ Treg cells of different MHC haplotypes. We also used a transplantation model of skin allografts in RAG1 deficient mice injected with Teff and Treg cells from various MHC backgrounds. Data from more than 150 co-culture experiments show that Treg suppression occurs only when Treg and Teff share the same MHC-II, but independently of Treg matching with APC MHC-II. Further experiments demonstrate that pMHC-II complexes, displayed on activated Teff cells, are crucial to the induction of Treg suppression. In vivo results recapitulate the in vitro data: Treg suppression, leading to graft survival, occurs only when Teff and Treg cells are MHC-II matched. Collectively, these findings suggest that suppression of alloreactivity is directed by Treg TCR recognition of pMHC-II complexes exposed on Teff cells. The nature, origin (host vs. donor), and significance of MHC-II loaded peptides have yet to be established. Future studies to improve targeted Treg therapies for transplantation tolerance will focus on mechanisms coupling Treg function with Teff cell activation.

IL-33 Augments the Capacity of Human Dendritic Cells to Expand Suppressive, but IFN-Gamma hi Regulatory T Cells

Introduction: To achieve successful translation of T regulatory cell (Treg) therapy in human organ transplantation, it will be essential to generate large quantities of Treg ex vivo. This is necessary as rodent studies suggest an increased number of Treg relative to T effectors (Teff) is required for experimental allograft tolerance. IL-33 administration controls alloimmunity and promotes graft survival by increasing endogenous Treg in rodent models. IL-33 also selectively expands mouse Treg expressing the IL-33 receptor ST2 when added to Treg/allogeneic DC cultures. We attempted to translate these rodent findings to expansion of human Treg and to determine whether IL-33 could enhance the ability of allogeneic DCs (allo-DCs) to stimulate expansion of flow-sorted Treg. Methods: Peripheral blood immunobead-isolated CD14+ cells were cultured with GM-CSF and IL-4 for 7 days to generate mono-DC. Irradiated DCs or anti-CD3/28 beads were co-cultured with flow-sorted peripheral blood CD4+CD25+CD127− Treg with or without 50 ng/ml IL-33 for 12 days in the presence of IL-2. Anti-CD3 Ab-loaded L cells were used to stimulate 2nd round Treg expansion, with or without IL-33 in the presence of IL-2 for an additional 7 days. DCs were incubated with or without IL-33 or IL-33 plus IL-2 for 18 hours for phenotypic analysis. Proliferation of autologous Teff in the presence or absence of the expanded Treg was used to test Treg suppressive function. Expanded alloreactive Treg were stimulated with anti-CD3/28 beads for 3 days to assess cytokine production. Results: Treg stimulated by DCs in the presence of IL-33 were expanded approximately 40% more in the 1st round and 50% more in the 2nd round than in the absence of IL-33. However, such differences were not observed when Treg were stimulated by anti-CD3/28 beads. Allo-DC increased expression of CD86 when co-cultured with IL-2 and IL-33, suggesting stronger stimulatory potency. Interestingly, while IL-33 cultured Treg expressed lower Helios, modestly downregulated Foxp3, and higher levels of IFNγ expression relative to control allo-DC Treg (Fig. 1), they retained a potent ability to suppress T effector proliferation (Fig. 2). Conclusion: IL-33 enhances DC-induced expansion of human Treg exhibiting strong suppressive capacity, but enhanced IFNγ production. Future studies will determine the functional relevance of increased IFNγ in Treg expanded during IL-33/Allo-DC culture. This work was supported by funding from the NIH (R01HL122489: HRT).FigureFigure

Modulation of CD27/CD70 Co-Stimulatory Pathway may Allow for the Generation of a More Potent Human Regulatory T Cell Product for Cell Therapy

Introduction: Regulatory T cell (Treg) therapy is a promising strategy to promote transplant tolerance and aid in the reduction of immunosuppression[1]. This study aims to identify new strategies to increase potency, stability and specificity of human Tregs to produce an effective clinical therapy. Ligation of CD70 to CD27, a costimulatory molecule member of the tumour necrosis factor superfamily, is known to be sufficient and indispensable for the generation of effective and long-term antigen-specific Tcell immunity[2][3][4][5]. In human Tregs, CD27 correlates with suppressive potency after in vitro expansion [6][7]. We hypothesised that CD27/CD70 pathway may have a direct role in the activity of Tregs, and modulation of CD27/CD70 may allow for the generation of Tregs with enhanced suppressive properties. Methods: TotalTregs (CD4+CD127low/-CD25+) and CD27Tregs (CD4+CD127low/-CD25+CD27+) were sorted from healthy donor PBMCs and expanded in vitro for 14 days using αCD3/αCD28 coated beads in the presence of rhIL-2. Cells were then flow sorted according to CD27 expression and expanded for a further 14 days. Treg phenotype, suppressive function and cytokine production were analysed after expansion by flow cytometry and ELISA. Results: CD27 expression identified in vitro-expanded human Tregs with regulatory cytokine profile and epigenetically stable Foxp3 expression, studied by demethylation of the TSDR region. Suppressive activity was confined to the CD27+expanded Treg population, and blocking the CD27/CD70 pathway within an in vitro suppression assay potentiated Tcell proliferation and Treg suppression. On ligation of CD27, there was a robust activation and proliferation response in conventional Tcells but high death levels in Tregs, suggesting that CD27 costimulation activates distinct intracellular pathways in these two cell populations.FIGURE 1: Blocking the CD27/CD70 pathway potentiates T cell proliferation and Treg suppression. Suppressive capacity of expected DC27+ Tregs andCD27-was analyzed in the absence (black) and in the pessure of 10ug/ml of isotype (striped) of αCD27 (red), αCD27 (blue) of αCD27 +αCD70 (green) blocking mAbs. 10x104 autologous CD3+ cells (responder T cells) labeled with VPD and 2X104 dendritic cells (DCs) for 80-96h. The graph represents the division index of responder cells; the average number of cell divisions that a cel in the original population has undergone. Mean with SEM is represented and statistical significance was assessed by a Mann-Whitney test. (*p<0.05, **p<0.01), ***p<0.001. No statistical differences between different isotype mAbs and control media were observed. 1 representative donor is displayedConclusion: Isolation of Tregs by expression of CD27 may facilitate the identification of a more potent and stable Treg product for cellular therapy. All indicates that CD27/CD70 pathway has a direct role in the activity of human Tregs, and its modulation may facilitate the generation of Tregs with enhanced suppressive properties. CD27 costimulation may activate distinct intracellular pathways in conventional and regulatory Tcells, and therefore CD27/CD70 axis could be exploited for regulating the balance between conventional and regulatory T cell responses.FIGURE 2: CD27 co-stimulation promotes proliferation of CD4+ T cells and cell death in Tregs. Total Tregs and CD4+ T cells were isolated from PBMCs from healty donors, strained with VPD and stimulated in vitro for 7 days in round-bottom plates coated with 1ug/ml of αCD3mAb and 5ug/ml soluble αCD28mAb or 5ug/ml soluble CD27L protein (sCD27L). After 7 days of culture A) cell proliferatuion was studied by VPD dilution and B) cell death was analysed by 7AAD expression using flow cytometry. Mean with SEM is represented (Mann-Whitney test *p<0.05, **p<0.01, ***p<0.001). 1 representative donor is displayed.

Thymus-Derived Treg Infusion to Prevent Graft Rejection in Heart-Transplanted Children

Introduction: Immune graft rejection is the main obstacle to successful transplants. Immunosuppressive drugs fail to prevent chronic rejection and also impair the immune system, resulting in reduced lifetime survival rates. Transfer of regulatory T cells (Treg) has acquired growing interest in attempts to achieve indefinite graft survival [1]. The limited Treg numbers that can be purified from peripheral blood, along with low survival and limited suppressive capacity of expanded Treg obtained from adults, constitute important drawbacks for the effectiveness of this strategy. Methods: We established a collaboration amongst several teams working in the field to overcome current barriers and make Treg therapy a reality equipped to achieve indefinite graft survival. The major innovation is the employment of thymus, the site of Treg generation, as a new source of Treg [2]. Our goal is to perform a first-in-human trial treating heart-transplanted children with autologous thymus-derived Treg (thy-Treg) to prevent graft rejection. Results: We purified Treg from thymuses discarded routinely during cardiac surgeries in children with congenital diseases, obtaining massive quantities of thy-Treg with >98% purity. The final thy-Treg product shows high suppressive capacity, decreasing the proliferation of CD4+ and CD8+ T cells in co-cultures by more than 70%. Next, we propose to conduct a clinical trial transferring thy-Treg to heart-transplanted children. In the first instance, thymuses removed during transplant surgery will be processed in GMP-conditions, and autologous thy-Treg will be infused back to the patient at day +10, when immunosuppressant doses are routinely reduced. Due to the high quality and quantity of these thy-Treg and the favorable features of the pediatric immune system, we are confident that this strategy will boost Treg-mediated tolerance and reduce the incidence of graft rejection. Conclusion: Thymic tissue constitutes an alternative source of Treg that could be employed as cellular immunotherapy to prevent rejection. The massive quantity of Treg obtained from thymus, their high suppressive capacity, stability and survival, could overcome the drawbacks of employing Treg from peripheral blood. We propose that thy-Treg will constitute a therapeutic arsenal with infinite possibilities for the treatment of immune-related diseases, including graft rejection or autoimmune disorders.FIGURE 1: Purification and enrichment of thymus-derived Treg. Thymus tissue from one 5-months old chridren. Left: frequency of Treg cells in the total of thymocytes before pirification. Right: Highty Treg obtained after purification. Right: pure thynus-derived Treg obtained after pirification and 10-day culture.This work was supported by grants from Instituto de Salud Carlos III (ISCIII) co-financed by FEDER funds (PI15/00011; ICI14/00282).FIGURE 2: Suppressive capacity of thymus-derived Treg. Allogenic PBMCs were stained with CFSE and then co-cultured alone or with thymus derived Treg. (ratioPBMC:Treg 21). Proliferation ofCD4+ and CD8 T cells was analysed after 3 bays culture.

Low-dose interleukin-2 promotes STAT-5 phosphorylation, Treg survival and CTLA-4-dependent function in autoimmune liver diseases.

SummaryCD4+CD25highCD127lowforkhead box protein 3 (FoxP3+) regulatory T cells (Treg) are essential for the maintenance of peripheral tolerance. Impaired Treg function and an imbalance between effector and Tregs contribute to the pathogenesis of autoimmune diseases. We reported recently that the hepatic microenvironment is deficient in interleukin (IL)−2, a cytokine essential for Treg survival and function. Consequently, few liver‐infiltrating Treg demonstrate signal transducer and activator of transcription‐5 (STAT‐5) phosphorylation. To establish the potential of IL‐2 to enhance Treg therapy, we investigated the effects of very low dose Proleukin (VLDP) on the phosphorylation of STAT‐5 and the subsequent survival and function of Treg and T effector cells from the blood and livers of patients with autoimmune liver diseases. VLDP, at less than 5 IU/ml, resulted in selective phosphorylation of STAT‐5 in Treg but not effector T cells or natural killer cells and associated with increased expression of cytotoxic T lymphocyte antigen‐4 (CTLA‐4), FoxP3 and CD25 and the anti‐apoptotic protein Bcl‐2 in Treg with the greatest enhancement of regulatory phenotype in the effector memory Treg population. VLDP also maintained expression of the liver‐homing chemokine receptor CXCR3. VLDP enhanced Treg function in a CTLA‐4‐dependent manner. These findings open new avenues for future VLDP cytokine therapy alone or in combination with clinical grade Treg in autoimmune liver diseases, as VLDP could not only enhance regulatory phenotype and functional property but also the survival of intrahepatic Treg.

Suppression of allograft rejection by CD8+CD122+PD-1+ Tregs is dictated by their Fas ligand-initiated killing of effector T cells versus Fas-mediated own apoptosis

Mounting evidence has shown that naturally occurring CD8+CD122+ T cells are regulatory T cells (Tregs) that suppress both autoimmunity and alloimmunity. We have previously shown that CD8+CD122+PD-1+ Tregs not only suppress allograft rejection, but also are more potent in suppression than conventional CD4+CD25+ Tregs. However, the mechanisms underlying their suppression of alloimmunity are not well understood. In an adoptive T-cell transfer model of mice lacking lymphocytes, we found that suppression of skin allograft rejection by CD8+CD122+PD-1+ Tregs was mostly dependent on their expression of Fas ligand as either lacking Fas ligand or blocking it with antibodies largely abolished their suppression of allograft rejection mediated by transferred T cells. Their suppression was also mostly reversed when effector T cells lacked Fas receptor. Indeed, these FasL+ Tregs induced T cell apoptosis in vitro in a Fas/FasL-dependent manner. However, their suppression of T cell proliferation in vitro was dependent on IL-10, but not FasL expression. Furthermore, adoptive transfer of CD8+CD122+PD-1+ Tregs significantly extended allograft survival even in wild-type mice if Tregs lacked Fas receptor or if recipients received recombinant IL-15, as these two measures synergistically expanded adoptively-transferred Tregs in recipients. Thus, this study may have important implications for Treg therapies in clinical transplantation.

Treg therapy in transplantation: a general overview.

Solid organ transplantation remains the treatment of choice for end-stage organ failure. Whilst the short-term outcomes post-transplant have improved in the last decades, chronic rejection and immunosuppressant side effects remain an ongoing concern. Hematopoietic stem cell transplantation is a well-established procedure for the treatment of patients with haematological disorders. However, donor T cells are continually primed and activated to react against the host causing graft-versus-host disease (GvHD) that leads to tissue damages and death. Regulatory T cells (Tregs) play an essential role in maintaining tolerance to self-antigens, preventing excessive immune responses and abrogating autoimmunity. Due to their suppressive properties, Tregs have been extensively studied for their use as a cellular therapy aiming to treat GvHD and limit immune responses responsible for graft rejection. Several clinical trials have been conducted or are currently ongoing to investigate safety and feasibility of Treg-based therapy. This review summarizes the general understanding of Treg biology and presents the methods used to isolate and expand Tregs. Furthermore, we describe data from the first clinical trials using Tregs, explaining the limitations and future application of these cells.

Expression of a Chimeric Antigen Receptor Specific for Donor HLA Class I Enhances the Potency of Human Regulatory T Cells in Preventing Human Skin Transplant Rejection.

Regulatory T cell (Treg) therapy using recipient-derived Tregs expanded exvivo is currently being investigated clinically by us and others as a means of reducing allograft rejection following organ transplantation. Data from animal models has demonstrated that adoptive transfer of allospecific Tregs offers greater protection from graft rejection compared to polyclonal Tregs. Chimeric antigen receptors (CAR) are clinically translatable synthetic fusion proteins that can redirect the specificity of T cells toward designated antigens. We used CAR technology to redirect human polyclonal Tregs toward donor-MHC class I molecules, which are ubiquitously expressed in allografts. Two novel HLA-A2-specific CARs were engineered: one comprising a CD28-CD3ζ signaling domain (CAR) and one lacking an intracellular signaling domain (ΔCAR). CAR Tregs were specifically activated and significantly more suppressive than polyclonal or ΔCAR Tregs in the presence of HLA-A2, without eliciting cytotoxic activity. Furthermore, CAR and ΔCAR Tregs preferentially transmigrated across HLA-A2-expressing endothelial cell monolayers. In a human skin xenograft transplant model, adoptive transfer of CAR Tregs alleviated the alloimmune-mediated skin injury caused by transferring allogeneic peripheral blood mononuclear cells more effectively than polyclonal Tregs. Our results demonstrated that the use of CAR technology is a clinically applicable refinement of Treg therapy for organ transplantation.

OP028 Gut specific regulatory T cells – a new frontier for Crohn's disease therapy

Background: We have recently shown that Tregs isolated from the peripheral blood of patients with Crohn's Disease (CD) play a critical role in controlling both phenotype and expansion of auto-reactive T cells (1). This is a critical step to developing cell based therapy for Crohn's disease, where where primary and secondary non response rates to biologics remain unacceptably high. The immune system used retinoic acid (ATRA) to induce a4b7 and thus prime Tregs to home to the gut. We sought use ATRA in-vitro in order to engineer gut specific Tregs for our Phase 1 trial in Crohn's Disease. We then validated our findings in-vitro and in-vivo. Methods: Tregs were isolated from peripheral blood of CD patients. ATRA supplementation was tested in standard culture conditions. The expression of a4b7 was assessed by flow cytometry. Suppression assays were performed using autologous effector T cells (Teff). An ibidi flow chamber system coated with recombinant human MAdCAM-1 was used for in-vitro trafficking experiments. SCID mice xenografted with foetal intestinal small bowel were used for in-vivo experiments. Parametric and non-parametric data were calculated as the mean ± s.d. and median (IQR). For comparison of parametric and non-parametric data, t-test, or ANOVA were used. Results:Ex-vivo expansion of Tregs in the presence of ATRA significantly induced the expression of a4b7 compared to Rapamycin alone (5.57%±3.12 vs 82.8%±9.5, p=0.0057) Cells treated with Rapa+ATRA maintained their superior suppressive ability compared to Rapamycin treated Tregs (95.8%±3.5% vs 91.15%±10.1% p=ns; at Treg:Teff 1:1 ratio). RAPA+ATRA Tregs did not produce IFNy or IL17 under pro-inflammatory cytokine challenge. When flowed through a MAdCAm-1 coated chamber, significantly higher numbers of Rapa+ATRA treated cells were observed to roll (Rapa 0.83±0.40 vs Rapa+ATRA 10.17±2.54 p=0.005), crawl (Rapa 0 vs Rapa+ATRA 4±0.89 p=0.001) and firmly adhere (Rapa 0.33±0.21 vs Rapa+ATRA 36.8±1.78, p<0.001) than those treated with Rapa alone. When Tregs were transferred into mice, a higher proportion of Tregs were found in xenografts of animals treated with Rapa+ATRA Tregs compared Rapa Tregs (12.10 (7.54–22.83) vs 4.97 (1.72–7.63), p=0.0056). Importantly there was a higher proportion of Tregs in inflamed xenografts of animals treated with Rapa+ATRA Tregs compared to those treated with Rapa Tregs (18.35 (12.95–28.63) vs 6.78 (2.65–9.61), p=0.0095). Conclusions: The addition of ATRA to Treg culture in-vitro confers a gut homing phenotype. This is functionally relevant in-vitro and in-vivo. The treatment maintains the highly suppressive and phenotypically stable phenotype of these cells. These gut specific Tregs will be implemented in our first in man trial Treg therapy for Crohn's disease.

Generation of human islet-specific regulatory T cells by TCR gene transfer

Based on the success in animal models of type 1 diabetes (T1D), clinical trials of adoptive regulatory T cell (Treg) therapy are underway using ex vivo expanded polyclonal Tregs. However, pre-clinical data also demonstrate that islet-specific Tregs are more potent than polyclonal Tregs at reversing T1D. Translation of this approach into man will require methods to generate large populations of islet-specific Tregs which, to date, has proved to be a major hurdle. Here we demonstrate the feasibility of lentiviral-mediated T cell receptor (TCR) gene transfer to confer antigen specificity on polyclonal human Tregs. Targeting has been achieved using TCRs isolated from human islet-specific and viral-specific CD4+ T cell clones. Engineered T cells demonstrated expression of ectopically-delivered TCRs, resulting in endowment of cognate antigen-specific responses. This enabled antigen-specific suppression at increased potency compared to polyclonal Tregs. However, cells transduced with islet-specific TCRs were less responsive to cognate antigen than viral-specific TCRs, and in some cases, required additional methods to isolate functional antigen-specific Tregs. This study demonstrates the potential of TCR gene transfer to develop islet-specific Treg therapies for effective treatment of T1D, but also highlights that additional optimisation may be required to achieve its full potential.

T cell receptor repertoires after adoptive transfer of expanded allogeneic regulatory T cells

Regulatory T cell (Treg ) therapy has been exploited in autoimmune disease, solid organ transplantation and in efforts to prevent or treat graft-versus-host disease (GVHD). However, our knowledge on the in-vivo persistence of transfused Treg is limited. Whether Treg transfusion leads to notable changes in the overall Treg repertoire or whether longevity of Treg in the periphery is restricted to certain clones is unknown. Here we use T cell receptor alpha chain sequencing (TCR-α-NGS) to monitor changes in the repertoire of Treg upon polyclonal expansion and after subsequent adoptive transfer. We applied TCR-α-NGS to samples from two patients with chronic GVHD who received comparable doses of stem cell donor derived expanded Treg . We found that in-vitro polyclonal expansion led to notable repertoire changes in vitro and that Treg cell therapy altered the peripheral Treg repertoire considerably towards that of the infused cell product, to different degrees, in each patient. Clonal changes in the peripheral blood were transient and correlated well with the clinical parameters. We suggest that T cell clonotype analyses using TCR sequencing should be considered as a means to monitor longevity and fate of adoptively transferred T cells.

Adoptive Cell Therapy with Tregs to Improve Transplant Outcomes: The Promise and the Stumbling Blocks.

The contribution of regulatory T cells (Treg) to the induction and maintenance of tolerance is well-recognized in rodents and may contribute to long-term human organ allograft survival. The therapeutic efficacy of adoptively-transferred Treg in promoting tolerance to organ allografts is well-recognized in mouse models. Early phase 1/2 clinical studies of Treg therapy have been conducted in patients with type-1 (autoimmune) diabetes and refractory Crohn's disease, and for inhibition of graft-versus-host disease following bone marrow transplantation with proven safety. The feasibility of adoptive Treg therapy in the clinic is subject to various parameters, including optimal cell source, isolation procedure, expansion, target dose, time of infusion, as well as generation of a GMP-cell product. Several phase 1/2 Treg dose-escalation studies are underway in organ transplantation. Recent evidence suggests that additional factors are critical to ensure Treg safety and efficacy in allograft recipients, including Treg characterization, stability, longevity, trafficking, concomitant immunosuppression, and donor antigen specificity. Accordingly, Treg therapy in the context of organ transplantation may prove more challenging in comparison to other prospective clinical settings of Treg immunotherapy, such as type-1 diabetes.

Short-term intratracheal use of PEG-modified IL-2 and glucocorticoid persistently alleviates asthma in a mouse model.

Regulatory T (Treg) cells play an important role in allergic airway diseases, and upregulation of Treg cells is a potential therapeutic strategy for asthma. In this study, we show that short-term intratracheal use of IL-2 combined with glucocorticoid alleviates antigen-induced airway inflammation and reduces airway hyperresponsiveness by expanding antigen-nonspecific Treg cells, with a decrease in T helper 2 (Th2) cells and Th2-associated cytokines. We also designed a long-acting polyethylene glycol (PEG)-modified IL-2 and demonstrated that the optimal dosage form is IL-2(PEG) plus budesonide, which can upregulate Treg cells and ameliorate asthma at a lower dose. The therapeutic effect was faster than treatment with dexamethasone and was effective at a low dose suitable for humans that could last for at least 6 weeks. This study unveils a new therapeutic regimen and suggests that such endogenous Treg therapy could be a useful tool to persistently alleviate asthma.

CD4+CD25hiFoxp3+ Cells Exacerbate Bleomycin-Induced Pulmonary Fibrosis

Idiopathic pulmonary fibrosis is a fatal lung disease with a median survival of 2 to 5 years. A decade of studies has downplayed inflammation contributing to its pathogenesis. However, these studies preceded the discovery of regulatory T cells (Tregs) and all of their functions. On the basis of human studies demonstrating Tregs can decrease graft-versus-host disease and vasculitides, there is consideration of their use to treat idiopathic pulmonary fibrosis. We hypothesized that Treg therapy would attenuate the fibroplasia involved in a preclinical murine model of pulmonary fibrosis. IL-2 complex was used invivo to expand CD4(+)CD25(hi)Foxp3(+) cells in the lung during intratracheal bleomycin challenge; however, this unexpectedly led to an increase in lung fibrosis. More important, this increase in fibrosis was a lymphocyte-dependent process. We corroborated these results using a CD4(+)CD25(hi)Foxp3(+) cellular-based therapy. Mechanistically, we demonstrated that CD4(+)CD25(hi)Foxp3(+) cells undergo alterations during bleomycin challenge and the IL-2 complex had no effect on profibrotic (eg, transforming growth factor-β) or type 17 immune response cytokines; however, there was a marked down-regulation of the type 1 and augmentation of the type 2 immune response cytokines from the lungs. Collectively, our animal studies show that a specific lung injury can induce Treg alterations, which can augment pulmonary fibrosis.

Regulatory T cell therapy: An option to induce operational tolerance in liver transplantation

Regulatory T cells (Treg) may play an important role in operational (clinical) tolerance (OT), a stable graft function without immunosuppression in an otherwise immunocompetent host, that is spontaneously observed in some patients many years after transplantation. Several ongoing clinical trials are currently testing the effects of donor-specific or non-specific Treg infusion with the goal to induce this state of OT a few months after liver transplantation (LT). The preliminary results of two of these trials have been recently published, and raise a number of comments and issues: (1) These two papers demonstrate that a 100 to 1000-fold ex-vivo expansion of Treg is possible in humans after 2 weeks of culture. The optimal human Treg dose is however not clearly established, and might be higher than the dose that would be expected from translating murine data. (2) A lot of concerns are remaining regarding the Treg purity before expansion, the Treg stability during in vitro culture and the in vivo fate of infused cells. A strict monitoring of Treg should thus be done at each step. (3) Since Treg may play a detrimental role in some conditions, such as viral diseases and cancer, potential LT recipients with such diseases should probably be excluded from the initial trials of Treg infusion. (4) The follow-up of tolerant liver recipients should include repeated liver biopsies and detection of autoantibodies and humoral response, in addition to conventional liver graft assessment, in order to prevent the development of immune complications related to immunosuppression withdrawal. (5) The final issue raised by Treg therapy in LT is the choice of the immunosuppressive regimen used before tapering or withdrawal, appropriate to preserve OT establishment.

PWE-027 Feeding Tregs for Therapeutic In Vitro Expansion – Retinoic Acid not SCFA Provides The Best Diet

Introduction We have shown that Tregs expanded with rapamycin from FACS-sorted Crohn’s Disease (CD) peripheral blood (PB) CD4+CD25hiCD127loCD45RA+ precursors, yield an epigenetically stable FOXP3+ cell population that is resistant to pro-inflammatory cytokine expression.1 We will utilise this for a first in man clinical trial of Treg therapy for Crohn’s disease. We wished to optimise the expression of gut homing molecules on these cells as Tregs are required at the site of action for optimal suppressive ability. Methods Tregs were isolated from peripheral blood of CD patients and healthy controls. Retinoic acid (RA) and Rapamycin supplementation was tested in standard culture conditions as well as the addition of short chain fatty acids (SCFA). The effect of SCFA was assessed on ex-vivo expanded Tregs from CD4+CD25hiCD127loCD45RA+ precursors and induced Tregs (iTreg) from naive T cell precursors. The expression of gut homing molecules integrin b7 and GPR15 was assessed by flow cytometry. Suppressive ability was tested in vitro using autologous effector T cells (Teff). Parametric and non-parametric data were calculated as the mean±s.d. and median (interquartile range, IQR) respectively. For comparison of parametric and non-parametric data, t- test, one- or two-way ANOVA. Results In comparison to Rapamycin treated cells, the addition of RA significantly increased the expression of integrin beta 7 (38.7%±11.78% Rapamycin alone vs 72.26%±8.98% Rapamycin + RA p = 0.04, N = 7). RA treatment also significantly increased GPR15 expression. Cells treated with RA maintained their superior suppressive ability compared to Rapamycin treated Tregs (95.8%±3.5% vs 91.15%±10.1% p=ns; at Treg:Teff 1:1 ratio). SCFA treatment led to diminished cell viability and did not induce the expression of colonic homing molecule GPR15 in CD4+CD25hiCD127loCD45RA+ Tregs (74.4%±13.2% Rapamycin and RA treated culture vs 43.4%±17% with the addition of SCFA). Conclusion Contrary to existing evidence in mouse, SCFA did not increase the proportion of CD4+FOXP3+ Tregs in human iTreg cultures. In conclusion, the addition of Retinoic acid not SCFA provides the optimal conditions for ex-vivo Treg expansion for cell based therapy of Crohn’s disease. Reference 1 Canavan JB, Scotta C, Vossenkamper A, Goldberg R, Elder MJ, Shoval I, et al. Developing in vitro expanded CD45RA+ regulatory T cells as an adoptive cell therapy for Crohn’s disease. Gut. 2015. Disclosure of Interest None Declared

181 - Difference in Nutrient Requirements for Optimal CD4+ and CD8+ T Cells Expansion Under Chemically-Defined Condition

Antigen specific suppressive immunotherapy such as Tregs therapy and highly selective targeted immunotherapy such as chimeric antigen receptor and T-cell receptor therapy have shown great benefits and therapeutic value to the patient population. These immunotherapy applications require ex vivo expansion of T cells to high cell densities without loss of T cell phenotypes and functionality for successful adoptive transfer. Moreover, a serum-free, chemically-defined basal medium is ideal to reduce cost, limit variability and lower the risk of foreign contaminants.

Development of citrullinated-vimentin-specific CAR for targeting Tregs to treat autoimmune rheumatoid arthritis.

Abstract Rheumatoid arthritis (RA) is one of the most common chronic autoimmune diseases with a frequency of 0.5–1.0% in the adult population of developed countries. RA is characterized by an aberrant inflammation of the synovial membrane that causes irreversible joint and bone damages. It has been previously described that regulatory T cells (Tregs) were defective in RA patients and that whereas Treg depletion increased the severity of the disease, Treg transfer efficiently reversed this effect in the collagen-induced arthritis (CIA) model, an animal model of RA. These studies strongly suggest that Treg therapy may be able to treat RA patients by inducing a long-term restoration of appropriate tolerance. Moreover, adding Ag-specificity to the expanded Tregs may promote their migration to the site of the abnormal inflammation and enhance their function. The aim of our study has been to engineer and expand chimeric antigen receptor (CAR)-expressing Tregs specifically targeting an antigen present in the joint of RA patients to induce a localized and effective immunosuppressive response. We developed a CAR directed against citrullinated vimentin (CV), a posttranslational modified intermediate filament protein known to be exclusively located in the extracellular matrix of the synovial tissue in 50% of the RA patients. We are currently working on transducing the CV-specific CAR into Tregs to then study the functional activity of these cells in vitro. We are also developing an in vivo protocol to assess the therapeutic efficacy of the CAR Treg transfer in CIA model. In conclusion, our project aiming to combine Treg therapy and CAR technology may lead to the development on a promising therapeutic tool to cure RA and potentially other autoimmune disorders.

434. Adoptive Treg Cell Therapy Using Factor VIII-Specific CAR Regulatory T Cells Regulates Anti-Factor VIII Immune Responses in Hemophilia A Mice

The immune response to factor VIII (FVIII; F8 in constructs) protein limits the effectiveness of treatments for hemophilia A (HemA) patients. Our previous studies demonstrated that regulatory T cells (Tregs) play a pivotal role in modulation of anti-FVIII immune responses. In particular, adoptive transfer of Tregs isolated from FVIII-primed HemA/Foxp3 mice attenuated anti-FVIII immune responses induced by gene transfer of FVIII plasmid in HemA mice. For developing adoptive Treg therapy, we successfully expanded FVIII-sensitized polyclonal Tregs using an FVIII-specific expansion protocol in vitro and showed that these cells had increased FVIII-specific suppressive activity compared with nonspecific Tregs. However, FVIII-specific Tregs in the polyclonal population are still in very small numbers. In this study, we explored the strategy to generate FVIII-specific Tregs using the chimeric antigen receptor (CAR) approach. Lentiviral vector (LV) incorporating a high-binding anti-FVIII antibody-derived variable region (scFv) linked to signaling and costimulatory moieties of immune receptors (third generation CAR) and fused with a murine Foxp3 cDNA (F8CAR-Foxp3-LV) was prepared and used to transduce murine CD4+T cells. Flow cytometry analysis confirmed extracellular scFv and intracellular Foxp3 expression in transduced cells (F8CAR-Tregs). In vitro suppressive assay showed that transduced CD4+T cells had significantly higher FVIII-specific suppressive activity than untransduced cells towards FVIII-specific CD4+ effector T cells (Teffs). In addition, 1×106 transduced cells and untransduced cells were adoptively transferred into HemA mice. One day after cell transfer, the treated mice were challenged with FVIII plasmid injected hydrodynamically. The anti-FVIII antibody titers are evaluated overtime. It is expected that F8CAR-Foxp3-LV transduced cells will prevent or decrease the production of anti-FVIII antibodies. We have also prepared a LV incorporating only the F8CAR region (F8CAR-LV). CD4+CD25− and CD4+CD25+ cells isolated from HemA mice were transduced with F8CAR-LV to generate FVIII-specific Teffs and Tregs, respectively. In vitro FVIII-specific suppressive assays using CD4+ Teffs from FVIII-primed HemA mice or F8CAR-LV transduced Teffs as responder cells are performed to compare the suppressive function of F8CAR-LV transduced CD4+CD25+ cells and F8CAR-Foxp3-LV transduced CD4+ cells. CD4+CD25+ cells isolated from FVIII-primed HemA mice and untransduced CD4+CD25+ and CD4+ cells from HemA mice are used as control cells. These experiments compare the extent of suppression towards specific F8CAR Teffs and polyclonal FVIII-specific Teffs as well as the potency of suppressive function between two differently engineered F8CAR Tregs. Finally, adoptive transfer experiments into HemA mice using the transduced and control cell populations are performed to evaluate their in vivo function to protect the HemA mice from anti-FVIII antibody production. We anticipate that compared with nonspecific and polyclonally expanded Tregs, FVIII-specific CAR Tregs will exert superior suppressive activity towards anti-FVIII immune responses without triggering systemic immune suppression.