Alloantigen-specific Tregs Research Articles

The Immunosuppressant Protosappanin A Promotes Dendritic Cell-Mediated Expansion of Alloantigen-Specific Tregs and Prolongs Allograft Survival in Rats

Protosappanin A (PrA), an immunosuppressive ingredient of the medicinal herb Caesalpinia sappan L, prolongs heart allograft survival in rats, possibly by impairing the function of antigen-presenting cells (APCs). We examined the effects of PrA on the maturation and function of dendritic cells (DCs), a potent class of APCs, and the downstream cell–cell and intracellular signaling pathways mediating the immunosuppressive activity of PrA. PrA inhibited LPS-stimulated maturation of Wistar rat DCs in vitro as reflected by reduced expression of costimulatory molecules (CD80 and CD86) and reduced expression of TLR4 and NF-κB, two critical signaling components for antigen recognition. PrA also enhanced the release of IL-10 and decreased the release of IL-12 from DCs, but had no effect on the production of TGF-ß. In mixed cultures, Wistar DCs pretreated with PrA impaired the proliferation of Sprague Dawley (SD) rat T cells while promoting the expansion of SD rat CD4+CD25+ regulatory T cells (Tregs). Both oral PrA treatment and infusion of PrA-pretreated Wistar DCs prolonged cardiac allograft survival (Wistar donor, SD recipient) and expanded recipient CD4+CD25+Foxp3+ Tregs. Donor spleen cells, but not spleen cells from a third rat strain (DA), supported the expansion of recipient CD4+CD25+Foxp3+ Tregs and suppressed recipient T cell proliferation. We conclude that PrA triggers a tolerogenic state in DCs that allows for the induction of alloantigen-specific Tregs and the suppression of allograft rejection in vivo.

Engineering antigen specific T regulatory cells for transplant tolerance (P2148)

Abstract T regulatory cells (Tregs) are critical for self-tolerance and are being pursued as a potential cell therapy in transplantation and autoimmunity. While polyclonal Tregs can prevent transplant rejection in mice, allo-antigen specific Tregs are more potent. A chimeric antigen receptor (CAR) may be an effective way to make ag-specific Tregs in suitable numbers for the clinic. CARs are created by combining antibody variable domains with T cell receptor signaling domains. However, CARs for Treg cell therapy must a) function within the distinct signaling network of Tregs and b) recognize a transplant-relevant antigen. CAR function in Tregs was assessed by transducing sorted cells with a highly-expressed, humanized HER2 CAR. Cells were labeled with proliferation dye and assessed for their ability to respond to target antigen. Two CARs specific for HLA-A2 were generated by cloning the variable portions of the anti-A2 antibody from a hybridoma, creating a single chain antibody and ligating it in frame in the humanized CAR. These CARs were assessed for expression and activity by flow cytometry, and cytotoxicity and proliferation assays. Preliminary data suggests that CARs can induce strong, ag-specific proliferation in human Tregs and that an ag-specific HLA-A2 CAR has been generated. Further work includes determining the effect of CAR stimulation on Treg phenotype via cytokine staining and suppression assays, and optimizing the surface expression of the HLA-A2 CAR via phage display.

5-Aminolevulinic acid induces permanent acceptance of murine cardiac allografts via induction of regulator dendritic cells and expansion of alloantigen-specific Tregs. (P2176)

Abstract 5-aminolevulinic acid (5-ALA), an intermediate in heme synthesis is fundamentally important in aerobic energy metabolism. Hemeoxygenase-1 (HO-1) cleaves heme to form biliverdin, carbon monoxide (CO) and iron (Fe2+), and the pathway is accelerated by 5-ALA. In the present study, we examined the effects of 5-ALA on the maturation and function of dendritic cells (DCs), a potent class of APCs, and the downstream cell-cell mediating immunosuppressive activity. 5-ALA inhibited LPS-stimulated maturation of murine bone marrow DCs in vitro as reflected by reduced expression of MHC class-II (I-A/I-E), co-stimulatory molecules (CD80, CD40) and proinflammatory cytokines, IFN-g, IL-12 mRNA expression. In mixed cultures, DCs pretreated with 5-ALA impaired the proliferation of allogeneic T cells while promoting the expansion of CD4+CD25+ regulatory T cells (Tregs). Using an in vitro mixed leukocyte reaction (MLR) assay system, we found that the DCs pretreated with 5-ALA have more regulatory function to suppress the both CD4+ and CD8+ T cells proliferation. Furthermore, 5-ALA treatment impaired T cells proliferation in response to alloantigens and increased Tregs, resulting in permanent acceptance of murine cardiac allografts. Thus we conclude that 5-ALA triggers a tolerogenic state in DCs that allows increasing alloantigen-specific Tregs and the suppression of allograft rejection in vivo.

Endogenous T Cell Receptors Drive Non-Specific Suppression by Foxp3 MHC Class I-Restricted T Cell Receptor-Transduced Polyclonal CD4+ T Cells in Models of Graft-Versus-Host Disease.

AbstractAbstract 3152Alloreactive immune responses directed against malignant cells in recipients of allogeneic hematopoietic stem cell transplants are able to cure patients with hematological cancers. However, such immune responses may cause severe morbidity when directed against healthy recipient tissue, resulting in graft-versus-host disease (GvHD).Naturally occurring regulatory T cells (Tregs) are CD4+ T cells characterized by their expression of the transcription factor Foxp3. Whilst adoptively transferred polyclonal Tregs suppress GvHD in several murine models, their lack of specificity may compromise beneficial immunity against malignancy or infection. The generation of MHC class I-restricted, alloantigen-specific Tregs would allow them to recognize antigen presented directly on GvHD target tissues, concentrating their sites of activation at these tissues and possibly reducing the potential for non-specific immune suppression.We have generated ‘converted' Tregs through retroviral transfer of genes encoding Foxp3 and specific MHC class I-restricted T cell receptors (TCRs) into polyclonal conventional CD4+ T cells. We used the 2C-TCR, which recognizes the MHC class I molecule H-2Ld, expressed in Balb/c and other H-2d mice, in complex with the ubiquitously expressed peptide p2Ca; and the MH-TCR, which recognizes the MHC class I molecule H-2Db, expressed in B6 and other H-2b mice, in complex with the male peptide WMHHNMDLI.In vitro, Foxp3 2C-TCR-transduced B6 polyclonal CD4+ T cells were hyporesponsive to stimulation and suppressed the alloreactive proliferative response of B6 CD4+ and CD8+ T cells to Balb/c splenocytes, consistent with the acquisition of regulatory function. When adoptively transferred to lethally irradiated Balb/c recipients of MHC-mismatched B6 bone marrow and conventional T cells, Foxp3 2C-TCR-transduced B6 polyclonal CD4+ T cells significantly reduced early proliferation of donor T cells, weight loss and GvHD score in the recipients. Similarly, polyclonal CD4+ T cells transduced with Foxp3 and the MH-TCR caused marked suppression of allogeneic responses both in vitro and in vivo. However, while both the 2C-TCR and the MH-TCR conferred specificity to their cognate antigens in vitro, the potent suppression in these in vivo models was independent of the cognate antigen for the transduced TCRs. This non-specific suppression was markedly reduced when class I-restricted TCRs were transduced into OT-II Rag1-/- CD4+ T cells that are transgenic for a single endogenous TCR.These findings demonstrate an important role for the endogenous TCRs in driving non-specific suppression by polyclonal CD4+ T cells transduced with Foxp3 and class I-restricted TCRs, and suggest that strategies to downregulate endogenous TCRs will be required to achieve antigen-specific suppression in TCR gene-modified regulatory T cells. Disclosures:No relevant conflicts of interest to declare.

Comparative study of regulatory T cells expanded ex vivo from cord blood and adult peripheral blood

In this study, we expanded regulatory T cells (Tregs) ex vivo from CD4(+) CD25(+) T cells from cord blood (CB) and CD4(+) CD25(+) CD127(-) T cells from adult peripheral blood (APB) and compared the suppressive functions of the newly generated Tregs. The Tregs from CB and APB were expanded either in two cycles with a polyclonal stimulus or in two cycles with an alloantigen stimulus in the first cycle and a polyclonal stimulus in the second cycle. Cell yield after Treg expansion with polyclonal stimulation was greater than that of Tregs expanded with combined alloantigen and polyclonal stimulation. The expanded Tregs expressed high levels of Foxp3, CD39 and cytotoxic T-lymphocyte antigen-4 and low levels of CD127, interleukin-2 and interferon-γ. After two cycles of expansion, the CB Tregs maintained expression of the GARP gene and showed greater suppressive function than APB Tregs. The CB Tregs that were expanded with two cycles of polyclonal stimulation suppressed not only the polyclonal antigen-driven responder T (T(resp)) cell proliferation but also the HLA mismatched dendritic cell-driven T(resp) cell proliferation. When CB and APB Tregs were expanded with a primary alloantigen stimulus followed by a secondary polyclonal stimulus, the Tregs showed a potent, antigen-specific suppressive capacity. The Tregs expanded with two cycles of polyclonal stimulation from both CB and APB alleviated acute graft-versus-host disease symptoms and prolonged survival in a murine model of graft-versus-host disease. In conclusion, CB Tregs expanded with two cycles of polyclonal stimulation had a stronger immunosuppressive function than APB Tregs. It is feasible to obtain human functional alloantigen-specific Tregs expanded ex vivo from CB and APB in large numbers.

Relevance of regulatory T cell promotion of donor-specific tolerance in solid organ transplantation

Current clinical strategies to control the alloimmune response after transplantation do not fully prevent induction of the immunological processes which lead to acute and chronic immune-mediated graft rejection, and as such the survival of a solid organ allograft is limited. Experimental research on naturally occurring CD4+CD25highFoxP3+ Regulatory T cells (Tregs) has indicated their potential to establish stable long-term graft acceptance, with the promise of providing a more effective therapy for transplant recipients. Current approaches for clinical use are based on the infusion of freshly isolated or ex vivo polyclonally expanded Tregs into graft recipients with an aim to redress the in vivo balance of T effector cells to Tregs. However mounting evidence suggests that regulation of donor-specific immunity may be central to achieving immunological tolerance. Therefore, the next stages in optimizing translation of Tregs to organ transplantation will be through the refinement and development of donor alloantigen-specific Treg therapy. The altering kinetics and intensity of alloantigen presentation pathways and alloimmune priming following transplantation may indeed influence the specificity of the Treg required and the timing or frequency at which it needs to be administered. Here we review and discuss the relevance of antigen-specific regulation of alloreactivity by Tregs in experimental and clinical studies of tolerance and explore the concept of delivering an optimal Treg for the induction and maintenance phases of achieving transplantation tolerance.

Adoptive Immunotherapy with Tregs and Tcons Ensures Low TRM and a Low Incidence of Post Transplant Leukaemia Relapse After HLA Haploidentical Transplants for Acute Leukemia

Abstract 154In full haplotype mismatched (HLA-haploidentical) stem cell transplantation we showed adoptive transfer of freshly isolated donor CD4+CD25+ FoxP3+ T regulatory cells (Tregs) followed by donor T cells (Tcons) prevented acute and chronic GvHD without any post-transplant immunosuppression, promoted lymphoid reconstitution and improved immunity against opportunistic pathogens (Di Ianni et al., Blood 2011). The major drawback was the extra-haematological toxicity of the conditioning regimen which included TBI, thiotepa, fludarabine and cyclophosphamide. To reduce regimen related toxicity we replaced cyclophosphamide with alemtuzumab, given 22 days before the Treg infusion to prevent it from interfering with adoptive T cell immunotherapy (Fig 1). The graft consisted of immunoselected Tregs (median 2×106/kg; range 1.6–4.8; FoxP3+ cells 92% ± 8 SD;), CD34+ cells (median 9.1×106/kg; range 8.1–10.9) and Tcons (median 1×106/kg; range 0.5–3). No post-transplant prophylaxis against GvHD was given. [Display omitted] Since May 2010 18 patients (median age 43 years, range 23–61) with high risk acute leukaemia (16 AML, 2 ALL) have been transplanted. All sustained full donor-type engraftment. Neutrophils reached 0.5×109/L at a median of 12 days (range 9–28 days). Platelets reached 20×109/L and 50×109/L at median of 12 and 15 days, respectively (range 10–36 days and 11–55 days). CD4+ and CD8+ peripheral blood counts reached, respectively, 50/μL medianly on days 36 (range 27 – 120 days) and 34 (range 15– 85); 100/μL medianly on days 55 (range 27 – 147 days) and 48 (range 27 – 114); 200/μL on days 62 (range 37 – 177 days) and 49 (range 28 – 147). We observed a rapid development of a wide T-cell repertoire with specific CD4+ and CD8+ T cells for opportunistic pathogen antigen such as Aspergillus, Candida, CMV, ADV, HSV, VZV, Toxoplasma. Treg immunotherapy did not compromise post-transplant generation of donor-vs-recipient alloreactive natural killer (NK) cell repertoires in patients who received transplants from NK alloreactive donors (Ruggeri et al., Science 2002).Three of 16 valuable patients developed acute GvHD. Two responded to a short course of immunosuppressive therapy and at present (288 and 360 days after transplant) are alive and well with very good immunological reconstitution. The 3rd patient died of infectious complications. Two other patients died of non-leukemic causes (1 fulminant hepatitis 17 days post-transplant, 1 pneumonia 14 days post-transplant). The incidence of TRM is 17% (3/18). As hoped, extra-haematological toxicity was mild. One AML patient, who received a transplant from a non-NK alloreactive donor, relapsed 77 days post-transplant. Fourteen of the 18 patients are alive and well at a minimum follow-up of 3 months.This study shows adoptive immunotherapy with freshly isolated, naturally occurring Tregs is a feasible option in HLA-haploidentical stem cell transplantation since alloantigen-specific Tregs were efficiently activated in vivo and controlled alloreactivity of at least 1×106/kg Tcons without clinically significant inhibition of general immunity.Moreover Treg infusion did not weaken the GvL effect. The incidence of post-transplant leukaemia relapse was surprisingly low as only 1 patient has relapsed to date and even in our previous series no patient who was transplanted in CR has relapsed at a median follow-up of 25 months. Infusion of high numbers of Tcons in the absence of post-transplant immunosuppression can be hypothesized to exert a GvL effect. In addition, in patients who were transplanted from NK alloreactive donors, preservation of alloreactive NK cell repertoires played a key role in reducing the incidence of relapse. Disclosures:No relevant conflicts of interest to declare.

Post-Transplant Cyclophosphamide (PTC) Gvhd Prophylaxis: Kinetics of Proliferation of Donor T Cells Affects Susceptibility to PTC Administration,

Abstract 4029PTC has been found to diminish experimental GVHD and is under investigation in clinical studies (Luznik Blood 2010). An important question is whether PTC affects dividing cells equivalently irrespective of whether they are driven by antigen, i.e. allo or lymphopenic conditions. Cyclophosphamide (cyc) is a DNA alkylating agent which causes death of dividing cells via double strand breaks. We reasoned that if alloantigen drives more rapid proliferation vs. cytokine driven proliferation, a threshold level of DNA damage may enable greater survival by donor non-antigen specific cells expanding under lymphopenic conditions. The difference in the rates of proliferation and the effect on donor cell survival after PTC were investigated in both syngeneic and MiHA mismatched transplants. To establish a basal rate of homeostatic proliferation by donor B6 T cells (H2b, CD45.2+), a syngeneic myeloablative transplant was performed with CFSE labeled donor CD4+ and CD8+ T cells using congenic B6 (H2b, CD45.1+) recipients. Based on the clinical dosing schedule, PTC was administered on days 3 and 4 at the lowest concentration found to ameliorate GVHD following MiHA allo-HCT in previous experiments. At 5 days post-syngeneic transplant we observed the greatest amount of proliferation via CFSE dilution in the mesenteric lymph nodes (MLN), followed by peripheral LN and the spleen. Notably, we did not observe a significant diminution in the numbers of total cells recovered from lymphoid tissues in PTC recipients and or in the percentage of cells in later cell generations as ascertained by CFSE. Interestingly, the dividing cells in the MLN displayed a more activated phenotype (CD44/CD62L expression) compared with donor cells in the other lymphoid compartments consistent with more rapid expansion. The same protocol was performed in the B6 (H2b, Ly9.2+)->BALB.B (H2b, Ly9.1+) MiHA disparate strain combination. In this MiHA disparate transplant, we observed more rapid expansion kinetics in the MLN vs. other tissues and detected a greater cell recovery vs. the group that received PTC. Cell recovery in groups that received PTC, as well as number of CFSE divisions, was comparable to that of the syngeneic model of homeostatic proliferation. This result leads us to the conclusion that at this lowest effective concentration of cyc, cells undergoing homeostatic proliferation are comparatively spared. In order to further test this hypothesis and rule out the possibility that homeostatic proliferation could be affected by an ongoing allogeneic response, we included CFSE labeled OT.1 transgenic CD8+ T cells as a donor population which undergoes homeostatic, i.e. non-host antigen reactive proliferation with polyclonal B6 donor T cells (B6, Thy1.1+) transplanted into MiHA disparate recipients. At 5 days post-transplant, polyclonal donor B6 T cells in later divisions (>4) were substantially diminished after PTC. However, the percentage of OT.1 T cells in later generations was not affected by PTC. Taken together, these results suggest that cells undergoing homeostatic proliferation do so at a rate which permits the repair of damage induced by 33mg/kg PTC on two successive days. To extend these findings, we asked whether cells undergoing lymphopenic expansion in a syngeneic system were in fact susceptible to PTC induced death. A single PTC dose of 66mg/kg resulted in a decrease in later (>2) CFSE generations of both OT-I and polyclonal CD8+ T cells undergoing lymphopenic expansion in the mesenteric LN.We further employed CFSE labeled cells from Foxp3 FIR knock-in mice in these transplant models and followed the persistence of Tregs after syngeneic and allogeneic transplants. Results demonstrated that Tregs are required for successful PTC abrogation of GVHD in agreement with others (Luznik ASH 2010). Donor Tregs demonstrated more rapid proliferation in allogeneic transplants and were more susceptible to PTC deletion than CD4+FoxP3−cells. We therefore hypothesize that relatively few slowly proliferating non host allo-antigen specific Tregs are sufficient following PTC deletion of most anti-host alloreactive donor T cells to inhibit GVHD in myeloablative transplants and speculate that low numbers of Tregs transplanted after the administration of PTC should further improve clinical outcomes. Disclosures:No relevant conflicts of interest to declare.

High Efficiency Isolation of Alloantigen-Reactive Foxp3+ Natural Regulatory T Cells According to Specific Activation Marker Signatures

Abstract 1915Transplant tolerance induction and avoidance of exposure to destructive immunosuppressive drugs are still major objectives in transplantation medicine. Many preclinical animal models have proven the adoptive transfer of polyclonal CD4+CD25+Foxp3+ regulatory T (Treg) cells to be an important and effective tool for the prevention of graft rejection or graft-versus-host-disease. However, polyclonal Treg may also modulate immunity to foreign or tumor antigens. It is unclear yet, whether and how their application might lead to an unwanted general immunosuppression. Therefore the selective transfer of alloantigen-reactive Treg represents a very attractive therapeutic option. However, so far this strategy has been hampered so far by the lack of appropriate marker to assess and isolate antigen-reactive Treg cells with high efficiency.In order to get access to alloantigen-reactive Treg, we established cytometric methodologies allowing a clear dissection between (allo)antigen-specific Treg and Teff based on expression of 4-1BB and the lack of expression of CD40L. After allogeneic stimulation CD4+ T cells expressing 4-1BB but lacking CD40L expression were highly enriched in Foxp3+ T cells. Further molecular analysis of the Foxp3 gene locus revealed that only 4-1BB+CD40L− T cells were characterised by a completely demethylated TSDR (Treg specific demethylated region). 4-1BB+CD40L− T cells highly expressed the transcription factor HELIOS recently demonstrated to specify thymic-derived Treg. Alloantigen-reactive Treg isolated according to 4-1BB and CD40L were superior with respect to their alloantigen-specific in vitro suppression as compared their polyclonal Treg counterparts. Finally 4-1BB+CD40L− alloantigen-reactive Treg could be easily expanded in vitro up to 300 fold during 3 weeks culture, maintaining alloantigen-specific suppression. Our results offer the possibility to improve current approaches for adoptive cell-therapy with alloantigen-specific Treg to achieve transplantation tolerance aiming at a specific inhibition of pathology. Disclosures:Scheffold:Miltenyi Biotec GmbH: Employment.

Ex vivo expansion of human Tregs specific for alloantigens presented directly or indirectly

AbstractAdoptive transfer of regulatory T cells (Tregs) prevents GVHD in experimental animals. Because antigen activation drives Treg function, we measured the frequency, growth requirements, and function of alloantigen-specific (allospecific) Tregs from human blood. When alloantigen was presented directly, the precursor frequency of allo-specific Tregs in normal individuals was 1.02% (95% confidence interval [95% CI]: 0.65-1.59) and non-Tregs 1.56% (95% CI: 0.94-2.55). When alloantigen was presented indirectly, the frequency of specific Tregs was approximately 100-fold less. Purified Tregs were expanded with APCs, rapamycin, IL-2, and IL-15. In 12 days, allo-specific Tregs expanded 793-fold (95% CI: 480-1107), with duplication approximately every 24 hours. Purified allo-specific Tregs suppressed responses to specific alloantigen selectively and were approximately 100-fold more potent than polyspecific Tregs and nonexpanded Tregs. Allo-specific Tregs maintained high expression of Foxp3, Bcl-2, lymphoid homing receptor CD62L, and chemokine receptor CCR7, predicting sustained function and migration to lymphoid tissues in vivo. Allo-specific Tregs produced TGF-β and IL-10 and expressed more cytoplasmic CTLA-4 compared with non-Tregs. These data provide a platform for the selective expansion of Tregs against major and possibly minor histocompatibility antigens and predict the feasibility of adoptive immunotherapy trials using Tregs with indirect allo-recognition for preventing GVHD while sparing GVL effects.

Emerging role of innate immunity in organ transplantation part III: the quest for transplant tolerance via prevention of oxidative allograft injury and its consequences

In parts I and II of this tripartite review, the innate immune system was briefly described, focusing on its emerging role in organ transplantation and by emphasizing the oxidative injury-induced allograft inflammation that promotes the generation of immunostimulatory donor- and recipient-derived dendritic cells (DCs) translating innate immune events into adaptive alloimmunity. This part III of the review discusses the possibility to induce adaptive antigen-specific T regulatory cells (Tregs) in the clinical situation by harnessing donor- and recipient-derived tolerogenic DCs (tolDCs) as inducers for alloantigen-specific adaptive Tregs, an effort that follows current trends to harnessing DCs for immunotherapy. This challenge is based on accumulating evidence from basic immunological work in support of the notion that presentation of antigens, including weak transplantation antigens under subimmunogenic conditions within a noninflammatory microenvironment, promotes generation of tolDCs. With respect to these basic immunological studies, which are expressively reviewed, prevention of oxidative allograft injury can be regarded as an efficient tool in the clinical situation to present alloantigens under subimmunogenic conditions within an intragraft noninflammatory milieu, thereby potentially generating tolDCs able to induce Tregs-mediated innate allotolerance. Various therapeutic strategies to prevent oxidative allograft injury occurring in both the brain-dead donor before/during organ removal and the recipient during/after donor organ reperfusion as well as therapeutic options to inhibit injury-induced molecular and cellular consequences are finally discussed.

Generation of adaptive regulatory T cells by alloantigen is required for some but not all transplant tolerance protocols.

Because CD4CD25Foxp3 regulatory T cells (Tregs) are essential for the maintenance of self-tolerance, significant interest surrounds the developmental cues for thymic-derived natural Tregs (nTregs) and periphery-generated adaptive Tregs (aTregs). In the transplant setting, the allograft may play a role in the generation of alloantigen-specific Tregs, but this role remains undefined. We examined whether the immune response to a transplant allograft results in the peripheral generation of aTregs. To identify generation of aTregs, purified graft-reactive CD4CD25 T cells were adoptively transferred to mice-bearing skin allograft. To demonstrate that aTregs are necessary for tolerance, DBA/2 skin was transplanted onto C57BL/6-RAG-1-deficient recipients adoptively transferred with purified sorted CD4CD25 T cells; half of the recipients undergo tolerance induction treatment. By tracking adoptively transferred cells, we show that purified graft-reactive CD4CD25 T lymphocytes up-regulate Foxp3 in mice receiving skin allografts in the absence of any treatment. Interestingly, cotransfer of antigen-specific nTregs suppresses the up-regulation of Foxp3 by inhibiting the proliferation of allograft-responsive T cells. In vitro data are consistent with our in vivo data-Foxp3 cells are generated on antigen activation, and this generation is suppressed on coculture with antigen-specific nTregs. Finally, blocking aTreg generation in grafted, rapamycin-treated mice disrupts alloantigen-specific tolerance induction. In contrast, blocking aTreg generation in grafted mice treated with nondepleting anti-CD4 plus anti-CD40L antibodies does not disrupt graft tolerance. We conclude that graft alloantigen stimulates the de novo generation of aTregs, and this generation may represent a necessary step in some but not all protocols of tolerance induction.

High Frequency of Alloantigen-Specific T-Regs in Human Blood Allow Rapid Ex Vivo Expansion for Adoptive Therapy

High Frequency of Alloantigen-Specific T-Regs in Human Blood Allow Rapid Ex Vivo Expansion for Adoptive Therapy

Adoptive transfer of CD4 +CD25 + regulatory cells combined with low-dose sirolimus and anti-thymocyte globulin delays acute rejection of renal allografts in Cynomolgus monkeys

Although donor alloantigen specific Treg cells play an important role in transplant tolerance, therapeutic applications are limited by their low frequency. In this study, isolated Tregs from Cynomolgus monkeys were efficiently expanded by a co-culture system, and maintained suppressive function on the proliferation of CD4 + effector cells in vitro. Adoptive transfer of expanded donor alloantigen specific Tregs without any immunosuppressants could prolong survival of MHC-mismatched allografts in Cynomolgus monkeys. To reach the feasibility of clinical transplantation, our objectives focused on whether exposure of monkey Tregs to immunosuppressants could preserve suppressive function in vitro and in vivo. The results showed that low-dose sirolimus selectively expanded Tregs, increased the expression of CD25 bright and Foxp3 markers, and suppressed TCR- or allo-antigens induced CD4 + T cell proliferation in vitro. In vivo, after pre-treated with anti-thymocyte globulin (ATG) for consecutive 3 days, a 14-day therapy of adoptive infusion of donor alloantigen-specific Tregs combined with low-dose sirolimus delayed acute rejection of renal allografts in Cynomolgus monkeys, showing an MST of 48.5 days as compared with those of untreated and sirolimus-treated monkeys (7 days and 22 days). The frequencies of CD4 +CD25 bright T cells were significantly elevated in mesenteric lymph nodes vs. those in inguino lymph nodes and peripheral blood. In summary, expanded donor alloantigen specific Tregs exposed to sirolimus can preserve inhibition in vitro and in vivo. Tregs are more resistant to sirolimus than other T cells. Expanded donor alloantigen specific Tregs combined with sirolimus and ATG prolongs renal allograft survival in monkeys, suggesting that sirolimus might be one of the best synergists for Tregs therapy.

Combined Administration of a Mutant TGF-β1/Fc and Rapamycin Promotes Induction of Regulatory T Cells and Islet Allograft Tolerance

The critical roles of TGF-β in the reciprocal differentiation of tolerance-promoting CD4(+)Foxp3(+) regulatory T cells (Tregs) and proinflammatory Th17 effector cells affect alloimmune reactivity and transplant outcome. We reasoned that a strategy to harness TGF-β and block proinflammatory cytokines would inhibit the differentiation of Th17 cells and strengthen the cadre of Tregs to promote tolerance induction and long-term allograft survival. In this study, we report the development of a long-lasting autoactive human mutant TGF-β1/Fc fusion protein that acts in conjunction with rapamycin to inhibit T cell proliferation and induce the de novo generation of Foxp3(+) Treg in the periphery, while at the same time inhibiting IL-6-mediated Th17 cell differentiation. Short-term combined treatment with TGF-β1/Fc and rapamycin achieved long-term pancreatic islet allograft survival and donor-specific tolerance in a mouse model. This effect was accompanied by expansion of Foxp3(+) Tregs, enhanced alloantigen-specific Treg function, and modulation of transcript levels of Foxp3, IL-6, and IL-17. Our strategy of combined TGF-β1/Fc and rapamycin to target the IL-6-related Tregs and Th17 signaling pathways provides a promising approach for inducing transplant tolerance and its clinical application.

CD4+CD25+TREGS ACTIVATED IN VITRO WITH IL-2 AND IL-12P70 SUPPRESS REJECTION OF FULLY ALLOGENEIC GRAFTS IN ANIMALS WITH NO OTHER IMMUNOSUPPRESSION.

Hall, B. M.1; Verma, N. D.1; Boyd, R.1; Robinson, C. M.1; Tran, G. T.1; Wang, C.2; Bishop, G. A.3; Hodgkinson, S. J.1 Author Information

Intrinsic IFN-gamma-T-bet pathway mediates the generation of Th1-like regulatory T cells induced by CD40-activated B cells (145.11)

Abstract The plasticity of CD4+ T cell is found to be higher than previously thought and might be modified by the cross-talking among different signal pathways. On the other hand, the role of CD40-activated B cells in the differentiation of CD4+ T cell is still on debate and their underlying mechanism for inducing Tregs remains unknown. In this study, using coculture of human naïve CD4+CD25- T cells with allogeneic CD40-activated B cells without any exogenous cytokines, we have demonstrated that CD40-activated B cells could induce alloantigen-specific CD4hiCD25hi Tregs and Th1 cells from their naïve precursors under weak and optimal activation respectively. Interestingly, the induced CD4hiCD25hi Tregs had Th1 properties with high level of T-bet expression, and some of them produced IFN-gamma. Both IFN-gamma+ and IFN-gamma-CD4hiCD25hi Tregs had a similar alloantigen-specific suppressive capacity. Importantly, we further found that the endogenously produced IFN-gamma and intrinsic T-bet expression were involved in the generation of these CD4hiCD25hi Tregs but did not mediate their suppression by neutralization of IFN-gamma and interference of T-bet expression with siRNA. We concluded that the generation of these Th1-like Tregs was mediated by IFN-gamma-T-bet pathway and the stimulatory or tolerogenic role of CD40-activated B cells in CD4+ T-cell differentiation was dependent on the strength of the activation to T cells.

Mammalian Target of Rapamycin Inhibition and Alloantigen-Specific Regulatory T Cells Synergize To Promote Long-Term Graft Survival in Immunocompetent Recipients

Minimization of immunosuppression and donor-specific tolerance to MHC-mismatched organ grafts are important clinical goals. The therapeutic potential of regulatory T cells (Tregs) has been demonstrated, but conditions for optimizing their in vivo function posttransplant in nonlymphocyte-depleted hosts remain undefined. In this study, we address mechanisms through which inhibition of the mammalian target of rapamycin (Rapa) synergizes with alloantigen-specific Treg (AAsTreg) to permit long-term, donor-specific heart graft survival in immunocompetent hosts. Crucially, immature allogeneic dendritic cells allowed AAsTreg selection in vitro, with minimal expansion of unwanted (Th17) cells. The rendered Treg potently inhibited T cell proliferation in an Ag-specific manner. However, these AAsTreg remained unable to control T cells stimulated by allogeneic mature dendritic cells, a phenomenon dependent on the release of proinflammatory cytokines. In vivo, Rapa administration reduced danger-associated IL-6 production, T cell proliferation, and graft infiltration. Based on these observations, AAsTreg were administered posttransplant (day 7) in combination with a short course of Rapa and rendered >80% long-term (>150 d) graft survival, a result superior to that achieved with polyclonal Treg. Moreover, graft protection was alloantigen-specific. Significantly, long-term graft survival was associated with alloreactive T cell anergy. These findings delineate combination of transient mammalian target of Rapa inhibition with appropriate AAsTreg selection as an effective approach to promote long-term organ graft survival.

Preparation of Human Alloantigen-Specific Natural Regulatory T-Cells for the Control of Graft Versus Host Disease.

Abstract 47Adoptive transfer of natural regulatory T-cells (Tregs) may have a great therapeutic potential for the induction of tolerance in transplantation patients. This concept was demonstrated in murine models of graft versus host disease (GVHD) where alloantigen-specific Tregs (sTregs) were always more efficient to control GVHD than polyclonal Tregs (pTregs). Recently, we reported a procedure for expanding human pTregs in the presence of anti-CD3/anti-CD28 coated beads, cells being cultured in RPMI medium containing 10% human serum, interleukin (IL)-2 and rapamycin (namely TCM for Tregs Culture Medium) during 3 weeks. Whether or not alloantigen sTregs could be generated for the control of GVHD was investigated. In a first set of experiments, we compared the activation of Tregs when stimulated by allogeneic monocyte-derived dendritic cells (DCs) under different conditions. To follow cell divisions, Tregs (CD4+CD25high T-cells) were purified by FACS, stained with CFSE and co-cultured in TCM ± IL-15 in the presence of either immature (i) or mature (m) allogeneic DCs. Data showed that: i) mDCs yielded higher Treg divisions than iDCs; ii) combination of IL-2 + IL-15 triggered better cell division than IL-2 or IL-15 alone; iii) Tregs divided from day-3 to day-10 when stimulated by mDCs and cultured in TCM + IL-15. Next, the alloantigen specificity of divided Tregs was evaluated: Tregs stimulated by allogeneic mDCs (mDC1) were FACS-sorted at day-4 on the basis of CFSE-staining. CFSElow, CFSEintermediate (int) and CFSEhigh cell fractions were separated and expanded in TCM medium IL-15 in the presence anti-CD3/anti-CD28 coated beads. At day-21, they were assayed for their ability to inhibit within 4 days the proliferation of autologous effector T-cells (Teff) stimulated by either mDC1 or a third party of allogeneic mDCs (mDC2), used as control of specificity. In these mixed leukocyte cultures, different Tregs/Teff cell ratio were tested. Results showed that the CFSElow fraction (highly dividing Tregs) was greatly enriched in sTregs, by contrast to the CFSEhigh and CFSEint fractions. From these data, further experiments that are more suitable for clinical application were performed. Tregs selected by FACS or by immunomagnetic selection (MACs), based on CD25 expression, were cultured in TCM + IL-15 and stimulated twice (day-0 and day-10) by allogeneic mDCs (mDC1). At day-21, the alloantigen-specific suppression of sTregs was studied and compared to the suppression activity of pTregs. Results showed that sTregs better suppressed the proliferation of mDC1-stimulated Teff than pTregs. In addition, sTregs better maintained their suppressive activity than pTregs at low Tregs/Teff cell ratio. Under these culture conditions, sTregs can be expanded ∼12 fold within 3-weeks. Our data showed that human sTregs can be generated under clinical grade conditions. Further experiments using a xenogeneic GVHD model are now envisaged to compare the respective capacity for sTregs and pTregs to control GVHD. Disclosures:No relevant conflicts of interest to declare.

Naturally acquired tolerance and sensitization to minor histocompatibility antigens in healthy family members

AbstractBidirectional cell transfer during pregnancy frequently leads to postpartum persistence of allogeneic cells and alloimmune responses in both the mother and in her offspring. The life-long consequences of naturally acquired alloimmune reactivity are probably of importance for the outcome of allogeneic stem cell transplantation. We investigated the presence of CD8pos minor histocompatibility (H) antigen–specific cytotoxic T lymphocytes (TCTL) and CD8pos minor H antigen–specific T regulator cells (TREG) in peripheral blood cells obtained from 17 minor H antigen–disparate mother-offspring pairs. Absence of minor H antigen–specific TREG, as marked by the feasibility to expand TCTL from isolated tetramerpos populations, was observed in 6 mothers and 1 son. The presence of minor H alloantigen–specific TREG was observed in 4 mothers and 5 sons. These TREG were detected within isolated tetramerdim staining fractions and functioned in a CTLA-4–dependent fashion. Our study indicates that both TCTL and TREG mediated alloimmunity against minor H antigens may be present in healthy female and male hematopoietic stem cell donors, potentially influencing graft-versus-host reactivity in different ways.