Treg Therapy Research Articles

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.

Protein Kinase C-θ and vimentin modulate multiple facets of Regulatory T-cell function.

Abstract Regulatory T-cells (Tregs) prevent autoimmune and alloimmune reactions. Augmenting Treg function may enhance Treg therapies for these diseases. Treg-specific inhibition of protein kinase C-theta (PKC-θ) enhances Treg function. However, it is unclear whether PKC-θ inhibition can boost Treg function in systemic inflammatory conditions. In a mouse model of acute GVHD, we found that Tregs treated with the PKC-θ inhibitor AEB071 reduced GVHD mortality and severity significantly better than DMSO treated Tregs. Compared to DMSO, AEB071 treated Tregs significantly reduced conventional T-cells (Tcon) proliferation on D4 after transplant. Multi-photon microscopy showed that AEB071 treated Tregs significantly increased Tcon velocity and displacement compared to DMSO. Mechanistically, AEB071 augments expression of Neuropilin-1 (Nrp1) and Lymphocyte activation gene 3 (Lag3). Antibody blockade of Nrp1 and Lag3 in transwell suppression assays reduced the effect of AEB071 on Treg function. PKC-θ inhibition also reduces phosphorylation of mTORC2 targets FoxO3a and Akt phospho-site S473, but not mTORC1 targets S6, 4E-BP1 or Akt phospho-site T308. Compared to DMSO, AEB071 treatment significantly increased fatty acid uptake and oxygen consumption rate (OCR). Phosphoproteomic analysis identified a significant alteration in the interaction between PKC-θ and the intermediate filament vimentin after AEB071 treatment, which was confirmed by confocal. Vimentin siRNA treatment also significantly reduces PKC-θ/vimentin interaction, increases Treg function, Nrp1 expression and OCR. In summary, PKC-θ and vimentin modulate multiple aspects of Treg function, and altering these molecules may enhance the efficacy of Treg therapeutics.

Factor VIII-specific CAR regulatory T cells modulate murine anti-factor VIII immune responses

Abstract The immune response to factor VIII protein (FVIII; F8 in constructs) limits the effectiveness of treatments for hemophilia A (HemA) patients. Previously we demonstrated that regulatory T cells (Tregs) play a pivotal role in modulating anti-FVIII immune responses. For application of adoptive Treg therapy, we successfully expanded highly suppressive murine polyclonal Tregs antigen specifically in vitro. However, the FVIII-specific Tregs in the polyclonal population are still in very small numbers. Thus, 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 scFv linked to the CAR signaling domains and fused with a 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 FVIII-specific suppressive assay showed that transduced cells had significantly higher suppressive activity than untransduced cells towards murine effector T cells. In addition, 1×106 transduced cells and untransduced cells were adoptively transferred into HemA mice and the treated mice were subsequently 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 in HemA mice. We anticipate that compared with nonspecific or polyclonally expanded Tregs, FVIII-specific CAR Tregs will exert superior suppressive activity towards anti-FVIII immune responses without triggering systemic immune suppression.

Human intrahepatic regulatory T cells are functional, require IL-2 from effector cells for survival, and are susceptible to Fas ligand-mediated apoptosis.

Regulatory T cells (Treg) suppress T effector cell proliferation and maintain immune homeostasis. Autoimmune liver diseases persist despite high frequencies of Treg in the liver, suggesting that the local hepatic microenvironment might affect Treg stability, survival, and function. We hypothesized that interactions between Treg and endothelial cells during recruitment and then with epithelial cells within the liver affect Treg stability, survival, and function. To model this, we explored the function of Treg after migration through human hepatic sinusoidal‐endothelium (postendothelial migrated Treg [PEM Treg]) and the effect of subsequent interactions with cholangiocytes and local proinflammatory cytokines on survival and stability of Treg. Our findings suggest that the intrahepatic microenvironment is highly enriched with proinflammatory cytokines but deficient in the Treg survival cytokine interleukin (IL)‐2. Migration through endothelium into a model mimicking the inflamed liver microenvironment did not affect Treg stability; however, functional capacity was reduced. Furthermore, the addition of exogenous IL‐2 enhanced PEM Treg phosphorylated STAT5 signaling compared with PEMCD8. CD4 and CD8 T cells are the main source of IL‐2 in the inflamed liver. Liver‐infiltrating Treg reside close to bile ducts and coculture with cholangiocytes or their supernatants induced preferential apoptosis of Treg compared with CD8 effector cells. Treg from diseased livers expressed high levels of CD95, and their apoptosis was inhibited by IL‐2 or blockade of CD95. Conclusion: Recruitment through endothelium does not impair Treg stability, but a proinflammatory microenvironment deficient in IL‐2 leads to impaired function and increased susceptibility of Treg to epithelial cell‐induced Fas‐mediated apoptosis. These results provide a mechanism to explain Treg dysfunction in inflamed tissues and suggest that IL‐2 supplementation, particularly if used in conjunction with Treg therapy, could restore immune homeostasis in inflammatory and autoimmune liver disease. (Hepatology 2016;64:138–150)

Antigen-specificity using chimeric antigen receptors: the future of regulatory T-cell therapy?

Adoptive regulatory T-cell (Treg) therapy using autologous Tregs expandedex vivois a promising therapeutic approach which is currently being investigated clinically as a means of treating various autoimmune diseases and transplant rejection. Despite this, early results have highlighted the need for potent Tregs to yield a substantial clinical advantage. One way to achieve this is to create antigen-specific Tregs which have been shown in pre-clinical animal models to have an increased potency at suppressing undesired immune responses, compared to polyclonal Tregs. This mini review outlines where Treg therapy currently stands and discusses the approaches which may be taken to generate antigen-specific Tregs, including the potential use of chimeric antigen receptors (CARs), for future clinical trials.

Adoptive Regulatory T-cell Therapy Attenuates Subarachnoid Hemor-rhage-induced Cerebral Inflammation by Suppressing TLR4/NF-B Signaling Pathway.

Inflammation is one major cause of poor outcomes of subarachnoid hemorrhage (SAH). The recent evidence suggested that adoptive regulatory T-cell (Treg) therapy conferred potential neuroprotection by suppressing cerebral inflammation against cerebral ischemia. Therefore, we proposed that Treg transfer might protect the brain against SAH by decreasing cerebral inflammation. In this study, we injected the autologous blood into cisterna magna twice to make the SAH model and administrated Tregs by vein to SAH rats. Intriguingly, adoptive transfer of Tregs significantly ameliorated SAH-induced brain edema and increased cerebral blood flow. Moreover, Treg-afforded cerebral protection was accompanied by suppressing SAH-induced cerebral inflammation. Concurrently, administration of Tregs attenuated the activation of the toll-like receptor 4 and nuclear factor-kappa B (TLR4/NF-κB) signaling pathway, which should be involved in the suppression of SAH-induced cerebral inflammation. Altogether, our study suggested that Treg adoptive transfer could attenuate SAH-induced cerebral inflammation by suppressing the activation of the TLR4/NF-κB signaling pathway, and thus provided new insights into the potent Treg cells-based therapy specifically targeting on post-SAH inflammatory dysregulation.

Regulatory T cells inhibit CD34+ cell differentiation into NK cells by blocking their proliferation.

Graft versus Host Disease (GvHD) remains one of the main complications after hematopoietic stem cell transplantation (HSCT). Due to their ability to suppress effector cells, regulatory T cells (Tregs) have been proposed as a cellular therapy to prevent GvHD, however they also inhibit the functions of natural killer (NK) cells, key effectors of the Graft versus Leukemia effect. In this study, we have explored whether a Tregs therapy will also impact on NK cell differentiation. Using an in vitro model of hematopoietic stem cell (HSC) differentiation into NK cells, we found that activated Tregs led to a 90% reduction in NK cell numbers when added at the time of commitment to the NK cell lineage. This effect was contact dependent and was reversible upon Tregs depletion. The few NK cells that developed in these cultures were mature and exhibited normal functions. Furthermore, adoptive transfer of activated Tregs in rag-/- γc-/- mice abrogated HSC differentiation into NK cells thus confirming our in vitro findings. Collectively, these results demonstrate for the first time that activated Tregs can inhibit NK cell differentiation from HSC under specific conditions.