Allogeneic Treg Research Articles

Local administration of regulatory T cells promotes tissue healing

Regulatory T cells (Tregs) are crucial immune cells for tissue repair and regeneration. However, their potential as a cell-based regenerative therapy is not yet fully understood. Here, we show that local delivery of exogenous Tregs into injured mouse bone, muscle, and skin greatly enhances tissue healing. Mechanistically, exogenous Tregs rapidly adopt an injury-specific phenotype in response to the damaged tissue microenvironment, upregulating genes involved in immunomodulation and tissue healing. We demonstrate that exogenous Tregs exert their regenerative effect by directly and indirectly modulating monocytes/macrophages (Mo/MΦ) in injured tissues, promoting their switch to an anti-inflammatory and pro-healing state via factors such as interleukin (IL)-10. Validating the key role of IL-10 in exogenous Treg-mediated repair and regeneration, the pro-healing capacity of these cells is lost when Il10 is knocked out. Additionally, exogenous Tregs reduce neutrophil and cytotoxic T cell accumulation and IFN-γ production in damaged tissues, further dampening the pro-inflammatory Mo/MΦ phenotype. Highlighting the potential of this approach, we demonstrate that allogeneic and human Tregs also promote tissue healing. Together, this study establishes exogenous Tregs as a possible universal cell-based therapy for regenerative medicine and provides key mechanistic insights that could be harnessed to develop immune cell-based therapies to enhance tissue healing.

Planning for success with an allogeneic Treg cell sourcing strategy

Planning for success with an allogeneic Treg cell sourcing strategy

Murine CAR19 Tregs suppress acute graft-versus-host disease and maintain graft-versus-tumor responses.

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) efficacy is complicated by graft-versus-host disease (GVHD), a leading cause of morbidity and mortality. Regulatory T cells (Tregs) have shown efficacy in preventing GVHD. However, high Treg doses are often required, necessitating substantial ex vivo or in vivo expansion that may diminish suppressor function. To enhance in vivo suppressor function, murine Tregs were transduced to express an anti–human CD19 chimeric antigen receptor (hCAR19) and infused into lethally irradiated, hCD19-transgenic recipients for allo-HSCT. Compared with recipients receiving control transduced Tregs, those receiving hCAR19 Tregs had a marked decrease in acute GVHD lethality. Recipient hCD19 B cells and murine hCD19 TBL12-luciferase (TBL12luc) lymphoma cells were both cleared by allogeneic hCAR19 Tregs, which was indicative of graft-versus-tumor (GVT) maintenance and potentiation. Mechanistically, hCAR19 Tregs killed syngeneic hCD19+ but not hCD19– murine TBL12luc cells in vitro in a perforin-dependent, granzyme B–independent manner. Importantly, cyclophosphamide-treated, hCD19-transgenic mice given hCAR19 cytotoxic T lymphocytes without allo-HSCT experienced rapid lethality due to systemic toxicity that has been associated with proinflammatory cytokine release; in contrast, hCAR19 Treg suppressor function enabled avoidance of this severe complication. In conclusion, hCAR19 Tregs are a potentially novel and effective strategy to suppress GVHD without loss of GVT responses.

231.1: MHC-deficient CD8+ Tregs as a Universally Compatible Cell Therapy

Introduction: Adoptive Treg therapy has shown to be effective in a variety of immune diseases in preclinical studies (Bézie et al., Front Immunol.2017, Blood Adv.2019) and is currently moving to phase I and II to demonstrate its effectiveness. However, autologous Tregs are expensive and slow to produce, which is why we are considering using ready-to-use allogeneic Tregs for future cell therapy. Method: CD8+CD4-CD45RClow/-CD56- T cells were sorted by FACS Aria II, stimulated with anti-CD3 and CD28 mAbs for 2 days, nucleofected with CRIPR/Cas9 to Knock Out MHC molecules, and cultured for 19 additional days in medium supplemented with rapamycin, IL-2 and IL-15, and stimulated with anti-CD3 and CD28 mAbs every week. Suppression capacity was assessed in vitro on allogeneic CD4+T cells proliferation in response to third party APCs and in vivo on 1.5 Gy-irradiated NSG mice co-injected with allogeneic human PBMCs. Allogenicity of KO Tregs was assessed on allogeneic CFSE-labeled effector T cells. Results: First, to explore the feasibility of off-the-shelf CD8+Treg-cell based therapy, we investigated the capacity of CD8+Tregs to control allogeneic immune responses. Interestingly, CD8+Treg could inhibit third-party APC-induced allogeneic effector T cell proliferation in vitro as efficiently as syngeneic cells. In addition, CD8+Tregs still control the development of GVHD induced by PBMCs from of foreign donor in vivo in humanized NSG mice. However, as CD8+Tregs express high levels of MHC-I and -II after ex vivo expansion, we evaluated their immunogenicity against allogeneic T cells. As expected, CD8+Tregs activated and initiated the proliferation of allogeneic CD8+ and CD4+ effector T cells. Thus, we developed a method to invalidate HLA molecules using CRISPR/Cas9 technology. By targeting B2M and CIITA genes encoding the proteins required for the expression of MHC-I and -II respectively, we succeeded in generating MHC-I and MHC-II deficient CD8+Tregs with preserved suppressive function, and which, unlike MHC+CD8+Tregs, did not activate effector T cell proliferation in vitro. Conclusion: We have shown the therapeutic potential of MHC-deficient CD8+Tregs for off-the-shelf cell therapy. Experiments are underway to explore the impact of MHC-I deficiency of CD8+Tregs on their susceptibility to NK cell lysis.

Combined treatment of graft versus host disease using donor regulatory T cells and ruxolitinib

Donor derived regulatory T lymphocytes and the JAK1/2 kinase inhibitor ruxolitinib are currently being evaluated as therapeutic options in the treatment of chronic graft versus host disease (cGvHD). In this work, we aimed to determine if the combined use of both agents can exert a synergistic effect in the treatment of GvHD. For this purpose, we studied the effect of this combination both in vitro and in a GvHD mouse model. Our results show that ruxolitinib favors the ratio of thymic regulatory T cells to conventional T cells in culture, without affecting the suppressive capacity of these Treg. The combination of ruxolitinib with Treg showed a higher efficacy as compared to each single treatment alone in our GvHD mouse model in terms of GvHD incidence, severity and survival without hampering graft versus leukemia effect. This beneficial effect correlated with the detection in the bone marrow of recipient mice of the infused donor allogeneic Treg after the adoptive transfer.

Mesenchymal stem cells transfer mitochondria to allogeneic Tregs in an HLA-dependent manner improving their immunosuppressive activity

Cell-based immunotherapies can provide safe and effective treatments for various disorders including autoimmunity, cancer, and excessive proinflammatory events in sepsis or viral infections. However, to achieve this goal there is a need for deeper understanding of mechanisms of the intercellular interactions. Regulatory T cells (Tregs) are a lymphocyte subset that maintain peripheral tolerance, whilst mesenchymal stem cells (MSCs) are multipotent nonhematopoietic progenitor cells. Despite coming from different origins, Tregs and MSCs share immunoregulatory properties that have been tested in clinical trials. Here we demonstrate how direct and indirect contact with allogenic MSCs improves Tregs’ potential for accumulation of immunosuppressive adenosine and suppression of conventional T cell proliferation, making them more potent therapeutic tools. Our results also demonstrate that direct communication between Tregs and MSCs is based on transfer of active mitochondria and fragments of plasma membrane from MSCs to Tregs, an event that is HLA-dependent and associates with HLA-C and HLA-DRB1 eplet mismatch load between Treg and MSC donors.

Adoptive Cord Blood T Regulatory Cell Therapy Leads to Resolution of Inflammation and Decreased Proteinuria in Lupus Nephritis

Background. Systemic lupus erythematosus (SLE) is associated with widespread inflammation with multi-organ involvement where renal failure is the most dreaded and fatal complication. Adoptive therapy with cord blood (CB) derived T regulatory cells has been shown to improve outcomes in disease driven by inflammation including graft versus host disease (GvHD), inflammatory bone marrow disorder, COVID-19 induced acute respiratory distress syndrome. We hypothesize that CB Treg therapy can treat lupus nephritis. Methods. The suppressive abilities of CB Tregs expressing CD4+CD25highCD127lowFoxP3+ were assayed by human cytokines assay kits (IL-10, IFN-γ, IP-10, TNF-α, IL-6, and IL-17A) in the cell culture supernatants. For examining the efficacy of CB Tregs in vivo, SLE xenograft model was created with female Rag2-/-γc-/- mice transplanted with 3x106 human SLE-peripheral blood mononuclear cells (PBMCs) by intravenous injection on day 0. The mice were allowed to develop disease and on day 30 post-transplant, they were divided into 2 groups: i) control and ii) treatment. 1x107 ex vivo-expanded, cryopreserved, allogeneic, non-HLA matchedCB Tregs were injected into SLE xenografts intravenously once per week for 4 weeks through the tail vein. Serial blood draws were performed for the phenotypic analysis, cytokine assay and anti-double stranded (ds)DNA IgG antibody analysis. Serial examination of the urine samples was performed for creatinine and albumin quantification. Histopathologic examination of the harvested organs was performed at the time of planned euthanasia at 13 weeks. Results. Co-culture of CB Tregs with the pathogenic SLE-PBMCs decreased the secretion of inflammatory cytokines including IFN-γ, IP-10, TNF-α, IL-6, and IL-17A (Figure A) with a reciprocal increase in the secretion of the anti-inflammatory IL-10 cytokine (Figure B). Adoptive therapy with CB Treg cells led to a significant decrease in circulating CD8+ effector T cells and an increase in CD4+ helper T cells (Figure C). CB Treg recipients showed preserved weight gain (Figure D), lower GvHD score (Figure E) and improved overall survival (Figure F). A significant decrease in proteinuria at 9 weeks post-transplant (Figure G) correlated with a decrease in anti-dsDNA IgG Ab levels (Figure H) and soluble CD40 ligand levels (Figure I). Histopathological results from two index cases from each arm (Figure J) demonstrated that CB Treg recipients show reduced T-cells (CD3+) (Figure K) and B-cells (CD20+) (Figure L) in the kidneys, as well as a decrease in the lymphoid infiltration into glomeruli and renal parenchyma as compared to the control arm (Figure M). Conclusion. We are the first to demonstrate the benefit of allogeneic CB Treg cell therapy for treatment of lupus nephritis. We propose to examine such a strategy in the clinical setting. Figure Disclosures Nishimoto: Janssen Pharmaceutical K.K.:: Research Funding; Bayer Yakuhin, Ltd:: Research Funding. Sadeghi:Cellenkos Inc.: Current Employment. Parmar:Cellenkos Inc.: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding.

Adoptive transfer of regulatory T cells stimulated by Allogeneic Hepatic Stellate Cells mitigates liver injury in mice with concanavalin A-induced autoimmune hepatitis

Regulatory T cell (Treg)-based therapy can effectively control autoimmune hepatitis (AIH). Hepatic stellate cells (HSC) can selectively stimulate allogeneic Treg proliferation following liver transplantation. This study tested the therapeutic effect and potential mechanisms underlying the action of HSC-stimulated Tregs on AIH in a mouse model of Concanavalin A (ConA)-induced AIH. HSC were isolated from BALB/c mice and characterized. Splenic CD4+CD25+ Tregs were isolated from C57BL/6 mice by immunomagnetic beads. The cells were co-cultured with primary (HSC-0), the second generation of HSC (HSC-2) for 72 h. The proliferation of Tregs was determined by flow cytometry. Similarly, the Tregs were co-cultured with HSC in transwell plates to determine the potential cell-cell contact dependent. The CD4+CD25− effector T cells (CFSE-Teffs) were co-cultured with Teff or Tregs in the presence or absence of HSC to determine the suppressive capacity of Tregs. The effects of Tregs or HSC-stimulated Tregs on AIH severity and the frequency of splenic Tregs and Th17 cells were examined in mice. Co-culture with HSC-2 significantly promoted Treg proliferation in a dose- and cell-cell contact-dependent manner, and allogeneic HSC enhanced the suppressive activity of Tregs to inhibit the proliferation of Teff in vitro. Adoptive transfer of Tregs, particularly of HSC-stimulated Tregs, significantly reduced liver injury, inflammation and Ishak modified histology activity index in AIH mice, which were associated with improving the balance of Treg and Th17 cell responses. Our data indicated that mature HSC stimulated allogeneic Treg proliferation in a dose and cell-cell contact-dependent manner, and HSC enhanced the suppressive activity of Tregs to inhibit the proliferation of Teff. Adoptive transfer of HSC-stimulated Tregs significantly reduced liver injury in AIH mice by modulating the balance of Treg and Th17 cell responses.

Immunosuppressive therapy influences the accelerated age-dependent T-helper cell differentiation in systemic lupus erythematosus remission patients

BackgroundCD4+ T cells are of great importance in the pathogenesis of systemic lupus erythematosus (SLE), as an imbalance between CD4+ regulatory T cells (Tregs) and CD4+ responder T cells (Tresps) causes flares of active disease in SLE patients. In this study, we aimed to find the role of aberrant Treg/Tresp cell differentiation for maintaining Treg/Tresp cell balance and Treg functionality.MethodsTo determine differences in the differentiation of Tregs/Tresps we calculated the percentages of CD45RA+CD31+ recent thymic emigrant (RTE) Tregs/Tresps and CD45RA+CD31− mature naive (MN) Tregs/Tresps, as well as CD45RA−CD31+ and CD45RA−CD31− memory Tregs/Tresps (CD31+ and CD31− memory Tregs/Tresps) within the total Treg/Tresp pool of 78 SLE remission patients compared with 94 healthy controls of different ages. The proliferation capacity of each Treg/Tresp subset was determined by staining the cells with anti-Ki67 monoclonal antibodies. Differences in the autologous or allogeneic Treg function between SLE remission patients and healthy controls were determined using suppression assays.ResultsWith age, we found an increased differentiation of RTE Tregs via CD31+ memory Tregs and of RTE Tresps via MN Tresps into CD31− memory Tregs/Tresp in healthy volunteers. This opposite differentiation of RTE Tregs and Tresps was associated with an age-dependent increase in the suppressive activity of both naive and memory Tregs. SLE patients showed similar age-dependent Treg cell differentiation. However, in these patients RTE Tresps differentiated increasingly via CD31+ memory Tresps, whereby CD31− memory Tresps arose that were much more difficult to inhibit for Tregs than those that emerged through differentiation via MN Tresps. Consequently, the increase in the suppressive activity of Tregs with age could not be maintained in SLE patients. Testing the Tregs of healthy volunteers and SLE patients with autologous and nonautologous Tresps revealed that the significantly decreased Treg function in SLE patients was not exclusively attributed to an age-dependent diminished sensitivity of the Tresps for Treg suppression. The immunosuppressive therapy reduced the accelerated age-dependent Tresp cell proliferation to normal levels, but simultaneously inhibited Treg cell proliferation below normal levels.ConclusionsOur data reveal that the currently used immunosuppressive therapy has a favorable effect on the differentiation and proliferation of Tresps but has a rather unfavorable effect on the proliferation of Tregs. Newer substances with more specific effects on the immune system would be desirable.

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.

T Regulatory Cell Adoptive Therapy for Tolerance Induction in Autoimmunity and Transplantation

T Regulatory Cell Adoptive Therapy for Tolerance Induction in Autoimmunity and Transplantation

Combination of ATG and Rapamycin Prolongs the Persistence of Adoptively-Transferred, Ex Vivo-Expanded Third Party Treg in Cynomolgus Monkeys.

Background: It has been shown that ex vivo-expanded regulatory T cells (Treg) can promote transplant tolerance in small animal models as well as in phase I clinical trials of hematopoietic stem cell transplantation. The issue has been raised of establishing Treg banks for adoptive cell therapy of organ transplant rejection and autoimmune disease. However, little is known about the persistence, stability and fate of infused Treg, especially third-party allogeneic Treg, and the influence of T cell-depleting and other immunosuppressive agents on their survival and therapeutic efficacy. Methods and Results: In this study, we flow-sorted CD4+CD25+CD127- Treg from normal juvenile cynomolgus monkey PBMC and expanded these cells using a protocol with artificial antigen-presenting cells. Three rounds of stimulation expanded the cells over 1000-fold and consistently generated over 108 Treg from 30 ml peripheral blood, while maintaining expression of Foxp3, CD25, CTLA-4, Helios; potent capacity to suppress T cell proliferation in vitro; demethylation status at the Treg-Specific Demethylation Region (TSDR), and down-regulated expression of CD45RA, CCR7, CD62L and upregulation of CXCR3 suggesting that these cells are likely to migrate to inflamed sites immediately after infusion in vivo. More importantly, when CFSE-labeled autologous (auto) and VPD450-labeled allogeneic (allo) ex vivo expanded Tregs were transferred simultaneously into monkeys, the number of auto-Treg was up to 100 per ml peripheral blood for at least 6 days (ie. for the duration of the experiment) in both control and ATG/rapamycin-treated (IS) monkeys. In contrast, by day 3 after their infusion, allo-Treg could not be detected in control monkeys, while they were maintained up to 100 cells per ml peripheral blood for at least 15 days in IS monkeys. These Tregs maintain high expression of Foxp3, CD25, and CXCR3 expression, and low expression of CD127, CCR7, and CD62L. Conclusion: The finding that allo-Treg can survive in IS-treated monkeys and maintain Treg phenotype supports the concept and potential utility of Treg banks for clinical application in transplantation and autoimmune disease and provides valuable information required to take allo-Treg therapy towards phase II/III clinical trials for efficacy assessment.

Combination of ATG and rapamycin prolongs the persistence of ex vivo expanded allogeneic Treg transferred in cynomolgus monkeys (TRAN3P.904)

Abstract It has been shown that ex vivo-expanded regulatory T cells (Treg) can promote transplant tolerance in small animal models as well as in phase I clinical trials of hematopoietic stem cell transplantation. The issue has been raised of establishing Treg banks for cell therapy of organ transplant rejection and autoimmune disease. However, little is known about the persistence, stability and fate of infused Treg, especially third-party allogeneic Treg. In this study, we massively expanded flow-sorted monkey Tregs using artificial APCs. The expanded Tregs maintained expression of Treg signature markers, in vitro suppressive capacity, and demethylation status at the Treg-Specific Demethylation Region. When autologous (auto) and allogeneic (allo) ex vivo expanded Tregs were simultaneously transferred into monkeys, the number of auto-Treg was up to 100 per ml blood for at least 6 days in both control and ATG/rapamycin-treated (IS) monkeys. In contrast, by day 3 after their infusion, allo-Treg could not be detected in control monkeys, while they were maintained up to 100 cells per ml blood for at least 15 days in IS monkeys with the expression of Foxp3 and CD25. The finding that allo-Treg can survive in IS-treated monkeys and maintain Treg phenotype supports the concept and potential utility of Treg banks for clinical application in transplantation and autoimmune disease.

Role of Lymphocyte Activation Gene-3 (Lag-3) in Conventional and Regulatory T Cell Function in Allogeneic Transplantation

Lag-3 has emerged as an important molecule in T cell biology. We investigated the role of Lag-3 in conventional T cell (Tcon) and regulatory T cell (Treg) function in murine GVHD with the hypothesis that Lag-3 engagement diminishes alloreactive T cell responses after bone marrow transplantation. We demonstrate that Lag-3 deficient Tcon (Lag-3−/− Tcon) induce significantly more severe GVHD than wild type (WT) Tcon and that the absence of Lag-3 on CD4 but not CD8 T cells is responsible for exacerbating GVHD. Lag-3−/− Tcon exhibited increased activation and proliferation as indicated by CFSE and bioluminescence imaging analyses and higher levels of activation markers such as CD69, CD107a, granzyme B, and Ki-67 as well as production of IL-10 and IFN-g early after transplantation. Lag-3−/− Tcon were less responsive to suppression by WT Treg as compared to WT Tcon. The absence of Lag-3, however, did not impair Treg function as both Lag-3−/− and WT Treg equally suppress the proliferation of Tcon in vitro and in vivo and protect against GVHD. Further, we demonstrate that allogeneic Treg acquire recipient MHC class II molecules through a process termed trogocytosis. As MHC class II is a ligand for Lag-3, we propose a novel suppression mechanism employed by Treg involving the acquisition of host MHC-II followed by the engagement of Lag-3 on T cells. These studies demonstrate for the first time the biologic function of Lag-3 expression on conventional and regulatory T cells in GVHD and identify Lag-3 as an important regulatory molecule involved in alloreactive T cell proliferation and activation after bone marrow transplantation.

Syngeneic and Allogeneic Hematopoietic Stem Cell Transplantation In Mice With Myelodysplastic Syndrome

The myelodysplastic syndromes (MDS) are clonal hematopoietic malignancies characterized by dysplasia, ineffective hematopoiesis and a propensity for progression to acute myeloid leukemia (AML). Allogeneic hematopoietic stem cell transplantation (HSCT) remains the only curative therapy for the majority of patients. However, overall survival (OS) of patients with MDS following allogeneic HSCT is only about 40%, due to both relapse and non-relapse mortality (NRM) including graft versus host disease (GVHD). Available data suggests that long-term survival following HSCT for MDS is due both to myeloablative therapy and a graft versus tumor (GVT) effect. Mice that express a NUP98-HOXD13 (NHD13) transgene develop MDS with virtually 100% penetrance. In order to develop a pre-clinical model for the study of MDS HSCT, we transplanted NHD13 mice, which are bred on a C57 Bl6 background, with bone marrow harvested from syngeneic C57Bl6 donors. Sub lethally irradiated (650 rad) recipient NHD13 mice transplanted with syngeneic donor cells relapsed early, with no therapeutic benefit in terms of hematologic parameters in peripheral blood or survival. However, lethally irradiated (1000 rad) recipient mice that were transplanted with syngeneic donor bone marrow (BM) showed complete normalization of peripheral blood counts significantly enhanced survival (median survival of 15 months) compared with non-transplanted NHD13 mice (median survival 10 months). Although there were no detectable MDS cells for up to 38 weeks post-transplant, all mice eventually relapsed and died. In order to determine if a GVT effect could enhance survival, we performed 3 types of allogeneic HSCT with donor BM that was mismatched at minor histocompatibility antigen loci (C3H.SW x C57Bl6 donors); donor BM only, donor BM with donor splenocytes (6 x 10E06 CD3+ T cells), and donor BM with donor regulatory T cells (Treg). None of these forms of allogeneic HSCT let to enhanced survival compared to that achieved with syngeneic HSCT. The early relapse rate for allogeneic HSCT with donor BM only was decreased compared to the syngeneic HSCT group (8.3% vs 28% at post-transplantation week 6 and 17% vs 43% at post-transplantation week 16); however, the relapse rate at 38 weeks was similar between the two groups (83.3% vs 85.7%). Adding donor splenocytes, containing reactive T-cells, dramatically decreased the relapse rate, such that the relapse rate was only 20% at post-transplantation week 38, suggesting a GVT effect. This GVT effect was accompanied by a severe GVH effect, and OS was not different between the allogeneic BM + splenocyte and the syngeneic HSCT groups. In an attempt to induce a GVT effect without a severe GVHD, we transplanted allogeneic Treg cells along with allogeneic BM, however, survival and relapse rates were similar to those with allogeneic BM only. Taken together, these findings suggest that a lethal dose of ionizing radiation (1000 rads) is insufficient to eradicate the MDS initiating cell, and that transplantation of donor CD3+ splenocytes leads to decreased relapse rates, but at the cost of severe GVHD. We suggest that the NHD13 mice are a feasible pre-clinical model for the study of HSCT for MDS. Disclosures:No relevant conflicts of interest to declare.

Regulatory T Cells (Tregs) in Neonatal Tolerance: Allogeneic Tregs Regulate the Neonatal Immune System and Prolong Heart Graft Survival

Purpose Semi-allogeneic (F1; H-2k/b) splenocytes (SC) injected into newborn mice (H-2k) induce immunological tolerance whereas allogeneic SC (H-2b) lead to graft- vs -host disease (GVHD). This study tested injection of purified allogeneic Tregs in our model of neonatal tolerance to cardiac allografts. Methods and Materials CD4 + CD25 + Tregs were isolated using StemCell Technologies EasySep® purification kits. Injected labeled syngeneic, F1 or allogeneic SC, or purified allogeneic Tregs, were tracked in newborn mice for 6 days by OV100 small animal imaging system. Treg interactions and fates were studied by confocal microscopy, and their ability to prolong heart allograft survival in adults. Results By whole body and organ imaging syngeneic and F1 SC tracked to lymphoid organs while allogeneic SC initially tracked there but then proliferated and distributed throughout the mice consistent with GVHD. Injected Tregs also trafficked to lymphoid organs but unlike allogeneic SC did not proliferate. Allogeneic Tregs survived post-injection in spleen suggesting they were not being attacked by the host immune system. In neonatal spleen, allogeneic Tregs interacted with host Tregs either directly or via host dendritic cells indicating a possible role in iTreg formation. Injected allogeneic Tregs prolonged survival of both donor-type and third-party heart grafts to 100 days in 50% of transplanted mice (n=6). A >100 day functioning donor-type heart graft showed mild-moderate cellular infiltrate of macrophages and T cells including CD8+ T cells, with focal regions of myocyte damage. Conclusions Injection of neonates with purified allogeneic Tregs prolongs survival of donor-type and third party heart grafts transplanted as adults. Prolonged third-party graft survival suggests suppression of the host immune system in a localized “non-specific” manner; alternatively given the presumed clonal diversity of allogeneic Tregs, some form of specific/cross-reactive suppression may be occurring.

Functional Characterization of CD4+ T-Cells in Aplastic Anemia (AA)

Abstract 1340We have examined the role of CD4+ T-cells in the pathogenesis of AA in 63 patients, 48 of whom were analyzed at diagnosis and 15 following immunosuppressive therapy (IST). Absolute numbers of CD4+ regulatory T cells (Tregs, defined as CD3+CD4+CD25highCD27+Foxp3+) were lower in pre-treatment AA patients compared to 10 healthy donors (HDs) (5.5 × 106 v 1.4 × 107)(p=0.01). In patients with severe (SAA) and very severe AA (VSAA), the absolute number and frequency of Tregs were lower than non-severe AA (NSAA) (4.4 × 106/L v 1 × 107/L)(p=0.01) and HDs (4.4 × 106/L v 3 × 107/L) (p<0.001). Absolute numbers of Th1 and Th2 cells in all pre-treatment patients were higher compared to HDs (6.4 × 107/L v 1.8 × 107/L)(p=0.03) for Th1 and (2.6 × 107/L v 2.4 × 106/L)(p=0.006) Th2 cells. Although mean percentages of AA Th17 cells were higher than in HDs (1.5% v 0.15%)(p=0.04), differences in absolute numbers were not significant. Absolute numbers of Th2 and Th17 cells were increased in SAA (1.3 × 107/L v 7.4 × 106/L for Th2)(p=0.01) compared to NSAA (5.7 × 106/L v 2.15 × 106/L for Th17)(p=0.02). Ratios of Th1/Tregs (p=0.003), Th2/Tregs (p=0.02), and Th17/Tregs (p=0.001) were higher in SAA and VSAA compared to NSAA. Percentage of both activated (CD4+CD45RA−CD25highFoxp3high) and resting (CD4+CD45RA+ CD25highFoxp3low) Tregs was decreased in AA patients, compared to HDs (p=0.004 and p=0.01), whereas cytokine secreting Tregs (CD4+CD45RA−CD25high Foxp3low) were increased in AA (p<0.003). Sorted Tregs from AA patients did not suppress cytokine secretion by autologous or HD T effectors (Te) cells in 1:1 co-cultures, whereas IL-2 and IFN-γ secretion by AA Te (CD4+CD25lowCD127high) was suppressible by allogeneic Tregs from HDs, confirming Tregs dysfunction. AA Tregs did not inhibit either CD154 or CD69 expression on Te cells. Tregs from AA patients secreted significantly more IFN-γ, TNF-α and IL-17 (p=0.02, p=0.02 and p=0.01, respectively) after 4 hours stimulation with PMA/Ionomycine compared to HDs. Expression levels of FoxP3, ROR□c and T-bet in AA Tregs was normal. IFN-γ secreting cells (Th1) were enriched using enrichment kit then further enriched by FACS sorting. CDR3 region products of TCR Vβ-chain were amplified using Vβ specific forward and Cβ reverse primers. CDR3 PCR products from AA patients and HDs were subjected 454 sequencing (Roche GS FLX titanium). Sequencing was performed to yield an average ‘depth' in excess of 1000 clonally reads (1000x) for each sample specific CDR3 PCR amp icon. Reads were processed using Roche Amp icon Variant Analyzer software (AVA). Diversity of TCR receptors (measured by spectratyping and confirmed by high throughput deep sequencing) in AA Th1 cells was lower than HDs (p=0.037), as shown by the percentage and number of consensus clusters in total sequence reads. Interestingly, percentages of the most dominant CDR3 clones, revealed by high throughput sequencing, were higher in AA compared to HDs, regardless of spectratyping pattern. Global gene expression of Tregs was compared in 3 pre-IST AA patients and 5 HDs. A unique gene signature consisting of 86 genes that were significant was identified. There were 8 down regulated genes (fold change) in the pre-treatment group; PIN4 (−4.1), OR2T12 (−3.3), AMAC1 (−2.73), PERP (−2.69), UTS2 (−2.27), RNF139 (−2.13), COMMD9 (−2.09) and LOC100128356 (−2.01). The top 10 of 78 up-regulated genes in the pre-treatment group were HBB (19.5), PSME2 (13.8), CSDA (13.07), FAM127A (7.78), EXOSC1 (7.73), BPGM (7.43), CYSLTR1 (7.17), CHPT1 (6.96) and PLAC8 (6.71). qPCR analysis for CSDA, HBB, PSMiE2, PERP, PIN4, and UTS2 confirmed a similar trend to the microarray results. Interestingly absolute number of Tregs, and Th2/Treg ratio were higher in 10 IST responsive patients compared to 5 non-responsive patients (p=0.005 and 0.02, respectively).We show that expansion of Th1, Th2, Th17, and decreased/skewed Tregs immunophenotype and function are a consistent and defining feature of SAA and VSAA. Clonal expansion of Th1 cells is likely to be antigen driven and the presence of dysfunctional Tregs aggravates this autoimmune response.Increases of Tregs, and Th2/Treg ratios following IST predicts a favourable response to this treatment. Disclosures:No relevant conflicts of interest to declare.

Integrin Alpha E (CD103) Beta 7 Expression Marks A Subset of Human Bone Marrow-Derived CD207+ Dendritic Cells Which Induce T Regulatory Cells Via Indoleamine 2,3-Dioxygenase,

Abstract 3233Indoleamine 2,3-dioxygenase (IDO) is the rate-limiting enzyme in tryptophan catabolism along the kynurenine pathway. IDO expressed by different cell subsets inhibits T-cell activation, proliferation and survival and induces regulatory T cells (Tregs), thus mediating immunological tolerance. Although human monocyte-derived dendritic cells (DCs) have been shown to express IDO, little is known about its expression in other subsets of human DCs, including those generated from CD34+ hematopoietic progenitors (CD34+-derived DCs). In particular, no data are currently available for IDO expression in CD34+-derived DC subsets. CD34+-derived DCs were generated from healthy donors from purified CD34+ cells after 7 days of culture with GM-CSF and TNF-a. Then, DCs were separated into CD1a−CD14+ and CD1a+CD14− cells. DCs subsets were analyzed for IDO expression by real-time PCR and western immunoblot, kynurenine production, inhibition of allogeneic proliferation and Tregs induction. CD34+ cells did not express IDO mRNA expression regardless of the progenitor cell sources (cord blood, mobilized peripheral blood, bone marrow). During DC differentiation, IDO expression and function, evaluated by enzymatic and immunological tests, was markedly induced at day 7. Interestingly, the expression of IDO was shown to be 10 times higher in the CD1a+ compartment as compared to CD1a- cell fraction. IDO expression resulted in increased production of kynurenine and in reduced allostimulatory capacity of T-cell proliferation. Moreover, CD1a+ cells were shown to induce a population of CD4+CD25+Foxp3+ which acted as Tregs by inhibiting allogeneic T cell proliferation. This effect was abrogated by the addition of the IDO inhibitor 1-methyl tryptophan. Phenotypically, IDO-expressing CD1a+ cells expressed typical marker of Langerhans cells such as CD207, CD11b, CD1c, as well as CD103, which has been recently identified as a marker for tolerogenic DCs. Importantly, IDO expression was mainly detected in the CD103+ CD207+ fraction, which induces Tregs through an IDO-dependent manner. In conclusion, DC differentiation of CD34+ cells results in the expression of a functionally active IDO protein in CD103-expressing DCs. Given the role of IDO in regulating immune tolerance, a subset of bone marrow-derived DCs, expressing CD103, may be intrinsically committed to function as regulatory DCs. These data point toward IDO expression as part of a tolerogenic signature during DC development. Disclosures:No relevant conflicts of interest to declare.

IL-2 Stimulated Treg Inhibit in Vitro Expansion of Haploidentical Natural Killer (NK) Cells, Which Is Partially Overcome with An IL-2-Diphtheria Toxin Fusion Protein In Vivo,

Abstract 3611Adoptive transfer of haploidentical natural killer (NK) cells can induce remissions in patients with refractory myeloid leukemia (AML). However, NK cells do not expand and persist in all patients despite lymphodepleting chemotherapy. In trials of adoptive NK cell therapy in solid tumors or lymphoma, host regulatory T cells (Treg) often expand in response to IL-2 given to stimulate donor NK cell expansion. Although murine studies report that Tregs inhibit NK cells, the influence of human Treg on NK cell proliferation and function is not well characterized. We studied the effect of allogeneic Tregs that were derived from human umbilical cord blood (UCB) as described by our group. Resting CFSE labelled NK cells or Teff were purified from healthy donors, and mixed with UCB Treg at various ratios. Unstimulated NK cells did not proliferate and thus IL-2 or IL-15 were added to the media at concentrations of 0.1, 0.25 and 0.5 ng/ml. In the absence of Treg, both cytokines induced equal NK cell proliferation at 5 days as measured by CFSE dilution in a concentration dependent manner. CFSE dilution was inhibited by Treg at a 1:1 ratio, especially at low cytokine concentrations. There were marked differences between the two cytokine conditions. Following IL-15 induced stimulation, the reduction in NK cell proliferation by Treg ranged from 1–35% (at different concentrations tested), whereas the inhibition of IL-2 stimulated NK cell proliferation ranged from 65–85%. Treg inhibition of NK cell proliferation could be measured at ratios as low as 1:8 in the presence of IL-2, but not IL-15. This inhibitory effect was partially explained by competition from CD25+ Tregs for IL-2. We measured Treg utilization of IL-2 by incubating NK cells with or without Treg in 0.5 ng/ml IL-2 for 4 days. The level of IL-2 with NK cells alone was 40 pg/ml vs. 17 pg/ml with Treg (compared to 330 pg/ml in IL-2-supplemented media without cells). Based on this data, we have incorporated host Treg depletion to enhance NK expansion after adoptive transfer to treat patients with refractory AML. As murine data from Blazar’s group shows that CTL therapy is enhanced by Treg depletion, we added one dose of denileukin diftitox (ONTAK®, Eisai Inc) at 12 mg/kg to our lymphodepleting preparative regimen of fludarabine 25 mg/m2 × 5 days, cyclophosphamide 60 mg/kg × 2 days for 12 AML patients. Haploidentical NK cells (CD3- and CD19-depleted PBMCs and overnight activated with IL-2 1000 U/ml) were infused on Day 0, followed by 6 doses subcutaneous IL-2 (9 million units) given every other day to promote in vivo NK cell expansion. Eleven of 12 patients were evaluable, having received at least 4 of 6 planned doses of IL-2. Blood and marrow were collected 7 and 14 days after infusion to assess NK cell and Treg expansion, as well as leukemia clearance. Of the 10 patients with interpretable day 7 chimerism data, 9 had detectable donor DNA (median 68% donor DNA). At day 14, 4 of the 12 patients (33%) had successfully expanded NK cells in vivo, with absolute donor derived NK cell counts of 480, 530, 1470 and 12390 cells/μL blood, improving on our previous 10% rate of in vivo NK cell expansion which was observed with the same regimen, without Treg depletion. In the 4 patients who expanded NK cells in vivo, there were no detectable Treg (defined as a CD25+CD4+FoxP3+ lymphocyte population) at either day 7 or day 14. In contrast, the presence of a bona fide Treg population at either day 7 [range 9.5–53%] or day 14 [27–71%] correlated with a lack of in vivo NK cell expansion at day 14. Clinically, 8 of the 11 evaluable subjects cleared leukemia (72%), 7 of whom recovered neutrophils (63% CRp) and 6 of whom went on to best donor transplant (45%). In summary, we demonstrate in vitro and in vivo suppression of NK cell proliferation by IL-2 stimulated Treg. This effect is not seen in vitro with IL-15. We have shown that the absence of host Treg correlates with in vivo NK cells expansion. Although an increased rate of donor NK expansion was observed with a single dose of denileukin diftitox, it did not completely overcome the IL-2 induced host Treg expansion. Future trials testing additional doses of denileukin difitox or other methods of Treg depletion, as well as the use of IL-15 are planned. Disclosures:No relevant conflicts of interest to declare.

Janus kinase 2 offers a precise therapeutic target against DC stimulated alloreactivity in transplantation (169.21)

Abstract JAK2 is critical to cytokine signaling and has a specific role in mediating alloreactivity. We have demonstrated that specific JAK2 inhibition with TG101348 suppresses alloimmunity stimulated by human dendritic cells in vitro, while maintaining immunity against viral antigens. JAK2 inhibition also extends survival in a mouse graft-versus-host disease (GvHD) model. Methods: Major MHC-mismatch models were used in human and mouse experiments. Human moDC and moDC:T cell cultures were treated with either TG101348 (1uM) or vehicle. Mice underwent 850 cGy irradiation followed by T cell-depleted marrow and splenic T cells. TG101348 (60mg/kg) or vehicle was administered per oral gavage daily. Results: JAK2 inhibition reduced the expression of CD83 and CCR7 in 2-day cytokine-matured moDCs (p&amp;lt;0.05) and suppressed T cell proliferation in a 5-day alloMLR (p&amp;lt;0.005). JAK2 blockade preserved allogeneic Tregs (p=NS), and enhanced the Treg:CD8+CD25+ T cell ratio (p&amp;lt;0.05). T cells exposed to JAK2-inhibited DCs maintained tolerance to alloantigen restimulation, with preserved response to flu-loaded autologous moDCs. JAK2 inhibition delayed the onset of GvHD and improved survival in BALB/c mice that received B6 marrow and T cells (median survival JAK2 inhibitor 35 days vs vehicle 25 days, p&amp;lt;0.005). All mice engrafted. Conclusions: JAK2 offers a novel, specific therapeutic target against GvHD and allograft rejection by suppressing DC allostimulation of T cells, without broader immune impairment.