Host Treg Cells Research Articles

AML-260: Influence of Chimerism in T-Regulatory Cells on Relapse Rate in Acute Leukemia Patients after Allo-HSCT

<h3>Introduction:</h3> T-regulatory cells (Treg) prevent the development of graft-versus-host disease (GVHD) after allo-HSCT (Beres & Drobyski, 2013). At the same time, this cell population can limit the anti-tumor response (graft-versus-leukemia, GVL), which can cause relapses. We report data of chimerism in a Treg population and the frequency of relapses in acute leukemia patients (ALL and AML) after allo-HSCT. <h3>Objective:</h3> To evaluate a possible relationship between mixed chimerism in Treg cells and the rate of relapses in acute leukemia patients after allo-HSCT. <h3>Methods:</h3> The study included 31 patients after allo-HSCT (ALL n=6, AML n=25). The median age was 38 years (19–66). Peripheral blood samples for analysis were taken on day +30 after transplantation. Immunomagnetic separation (Miltenyi Biotec, Germany) was used to isolate cell population with CD4+CD25+ phenotype, which is predominantly associated with Treg cells. Extraction of DNA was performed from the obtained cells. Chimerism in DNA samples was determined using the STR-PCR method. The percentage of donor chimerism was calculated using standard procedures (Nollet <i>et al.,</i> 2001). Statistical analysis of the data was carried out using SPSS ver 23 (IBM, Chicago, IL, USA). Fisher's exact test was used to analyze the 2 × 2 contingency tables. <h3>Results:</h3> In the group of patients with less than 11% of cells with host genotype (more than 89% of cells of donor origin), the relapse rate was significantly higher: 52.6% (10 of 19) than in the other group of patients with 11% and more cells with host genotype: 8.3% (1 of 12; p=0.02). At the same time, donor chimerism in the unselected bone marrow did not differ significantly from the groups (p = 0.36) and reached 100% (75–100%) and 97.5% (90–100%). The study groups were balanced for all other factors that could affect the relapse rate: disease status, graft source, and GVHD. <h3>Conclusions:</h3> According to our data, we proposed that host Treg cells are not capable to suppress GVL, which explains significant differences in relapse frequencies in patients with acute leukemia after allo-HSCT. Predominance of host Treg cells may be a favorable prognostic sign, but this hypothesis needs to be confirmed on the larger cohort of patients.

Trichinella spiralis Excretory–Secretory Products Stimulate Host Regulatory T Cell Differentiation through Activating Dendritic Cells

Trichinella spiralis maintains chronic infections within its host, involving a variety of immunomodulatory properties, the mechanisms of which have not been completely elucidated. In this study, we found that T. spiralis infection induced strong regulatory T cell responses through parasite excretory–secretory (ES) products, characterized by increase of CD4+CD25+Foxp3+ and CD4+CD25−Foxp3+ Treg cells accompanied by high levels of IL-10 and TGF-β. T. spiralis adult worm excretory–secretory products (AES) and muscle larvae excretory–secretory products (MES) were both able to activate BMDCs in vitro to facilitate their maturation and to create regulatory cytokines IL-10 and TGF-β. The T. spiralis AES- and MES-pulsed dendritic cells (DCs) possessed abilities not only to present antigens to sensitized CD4+ T cell to stimulate their proliferation but also to induce naive CD4+ T cells to differentiate to Treg cells secreting IL-10 and TGF-β. The passive transfer of T. spiralis AES- and MES-pulsed bone marrow-derived dendritic cells (BMDCs) conferred the naive mice to acquire the differentiation of Treg cells. T. spiralis AES possesses a better ability to induce Treg cells than did MES, although the latter has the ability to induce CD4+CD25−Foxp3+ Treg cells. The results obtained in this study suggested that T. spiralis ES products stimulate the differentiation of host Treg cells possibly through activating dendritic cells to create a regulatory environment that benefits the survival of the parasite in the host.

Toll-like receptor agonist therapy augments the antitumor activity of adoptively transferred CD8+ T cells without host preconditioning

Abstract Lymphodepletion enhances adoptive T cell transfer (ACT) therapy by activating the innate immune system via microbes released from the radiation-injured gut. Microbial LPS is a key mediator of lymphodepletion enhancement, but our ability to use these TLR agonists to bolster the potency of T cell-based cancer therapies remains elusive. Herein, we used LPS as a tool to address how and when to use TLR agonists to improve cancer immunotherapy. We utilized the pmel-1 melanoma mouse model. B16F10-bearing mice were lymphodepleted with 5Gy total body irradiation (TBI) and given a tripartite ACT therapy (consisting of transferred pmel-1 CD8+ T cells, vaccination with fowlpox encoding gp100, and IL-2) along with TLR4 agonist LPS. The timing of LPS administration and the requirement of individual components of the tripartite therapy were evaluated. We discovered that while exogenous administration of LPS was able to enhance CD8+ T cells’ tumor destruction, LPS treatment alone did not replace individual components of the tripartite regimen. Interestingly, administering LPS one day before ACT compromised tumor regression. Conversely, administering LPS after ACT potentiated the antitumor effectiveness of the regimen, thereby supporting the expansion of transferred CD8+ T cells over host Treg cells. Non-toxic TLR agonists MPL and CpG also improved ACT therapy. Finally, TBI preconditioning was no longer needed to regress tumors in mice depleted of host CD4+ T cells, given a tripartite ACT regimen and then treated with a TLR agonist. Collectively, our results identify how and when to administer TLR agonists to augment ACT in the absence of host preconditioning. These findings have implications for the design of next generation T cell therapies.

A Selective TNFR2 Agonist Expands Host Treg Cells in Vivo to Protect from Acute Graft-Versus-Host Disease

▪Donor CD4+Foxp3+ regulatory T cells (Tregs) suppress graft-versus-host disease (GVHD) after allogeneic hematopoietic stem cell transplantation (allo-HCT) while maintaining the anti-tumoral effect of transplanted conventional T cells in preclinical mouse models. Current clinical study protocols with donor Tregs for treatment or prophylaxis of GVHD rely on their ex vivo expansion and infusion in high numbers. Here we present a fundamentally novel strategy for inhibiting GVHD that is based on the in vivo expansion of recipient Tregs prior to allo-HCT, exploiting the crucial role of tumor necrosis factor receptor 2 (TNFR2) in Treg biology.To this end we constructed a recombinant nonameric TNFR2-specific agonist, mimicking the activity of murine membrane-bound TNF on TNFR2 without TNFR1 stimulation, thereby avoiding the inflammatory side effects observed with conventional TNF. In vitro, this TNFR2-agonist expanded natural Tregs from wild type but not from TNFR2 KO mice. Accordingly, a human variant of this TNFR2-specific agonist expanded human Tregsin vitro. In vivo treatment of healthy mice with the murine TNFR2-agonist significantly increased Treg numbers in secondary lymphoid organs and peripheral tissues, particularly in the gastrointestinal tract, a prime target of acute GVHD.Next, we pre-treated recipient mice with this novel TNFR2-agonist to expand host-type radiation resistant Tregs prior to of allo-HCT in two models across MHC barriers (C57BL/6, H-2b->Balb/c, H-2d and FVB/N, H-2q->C57BL/6, H-2b). TNFR2-agonist pre-treatment resulted in significantly prolonged survival and reduced GVHD severity when compared to TNFR2-deficient recipients or untreated allo-HCT recipients. This was accompanied by reduced donor T cell proliferation and infiltration into GVHD target organs as assessed by in vivo and ex vivo bioluminescence imaging, flow cytometry and immunofluorescence microscopy. While in vivo TNFR2-agonist pre-treatment protected allo-HCT recipients from GVHD, anti-tumor effects of transplanted T cells remained unaffected in two different murine B cell leukemia models. In vivo depletion of host derived Tregs completely abrogated the protective effect of TNFR2-agonist pre-treatment.Our study shows that the expansion of host Tregs by selective in vivo TNFR2-activation significantly improves the outcome after allo-HCT and results in prolonged tumor-free survival. DisclosuresNo relevant conflicts of interest to declare.

Expansion of a restricted residual host Treg‐cell repertoire is dependent on IL‐2 following experimental autologous hematopoietic stem transplantation

We previously identified a population of residual T(reg) cells following autologous hematopoietic stem transplantation (HSCT), that rapidly undergoes significant expansion in lymphopenic transplant recipients prior to repopulation by donor de novo derived T(reg) cells. These CD4(+) Foxp3(+) T cells provide protection from the development of autoimmune disease. Although ablative conditioning results in excess IL-7 and IL-15, IL-2 is typically not found at high levels following autologous HSCT. We therefore examined the role of these three STAT-5 signaling cytokines in the expansion of residual T(reg) cells after autologous HSCT. The present study found that the residual T(reg) cell population included surviving peripheral host Foxp3(+) CD4(+) T cells whose expansion was critically dependent on IL-2, which could be solely provided by surviving host cells. IL-7 was found to contribute to T(reg) cell homeostasis, however, not as a growth factor but rather for their persistence. In conjunction with this expansion, TCR spectratype analyses revealed that the residual host T(reg) -cell compartment differed from that present in non-conditioned healthy mice since the residual host Treg cells exhibit a limited TCR diversity. Collectively, these data indicate that the proliferation of T(reg) and T effector (T(eff) ) cells post-HSCT utilize separate pools of cytokines which has important implications regarding the development of clinical strategies to elicit the desired immune responses in patients post-transplant.

Effect of in vitroexpanded CD4+CD25+Foxp3+ regulatory T cell therapy combined with lymphodepletion in murine skin allotransplantation

A promising approach for preventing allograft rejection involves shifting the balance between cytopathic and regulatory T cells to dominance of the latter cell type. Nonspecific lymphodepletion was conducted by administration of depleting anti-CD4 and anti-CD8 antibodies to reduce effector T cells and adoptive transfer of ex vivo-expanded host Treg cells by stimulation with donor dendritic cells to augment the Treg cell compartment. Evaluation of an MHC-mismatched skin allograft model revealed that combined therapy with these two protocols consistently induced modest prolongation of allograft survival, although all skin grafts were eventually rejected. The administration of IL-2/anti-IL-2 complexes significantly improved the efficacy of combination therapy via promoting the expansion of adoptively transferred Treg cells as well as endogenous recipient Treg cells. We conclude that Treg cell therapy combined with lymphodepletion is of practical benefit for the control of allograft rejection, but requires supplementary measures to promote immune tolerance.

IL-2 + Anti-IL2 Complex in Situ Stimulation of Host Tregs Combined with Absence of Donor B7.1 / B7.2: A Novel Approach to Facilitate Chimerism in RIC MHC-Matched Miha-Mismatched BMT Recipients.

Abstract 2441Poster Board II-418Reducing and ultimately eliminating conditioning while enabling donor cell engraftment remains an elusive goal of allogeneic hematopoietic stem cell transplantation. The control of host resistance (HVG) to donor allogeneic transplantation antigens is pivotal for engraftment and the successful induction and maintenance of immune tolerance. Using a well characterized minor histocompatibility antigen (MiHA) matched allogeneic (HSCT) model, we have demonstrated a requirement for CD80/CD86 expression on donor APC in order to elicit resistance under reduced intensity conditioning (RIC) of 5.5Gy TBI. Based on this observation, we hypothesize that expansion of host Treg cells which may engage donor APC – the site of host anti-donor Tconv activation - is an advantageous strategy to effectively inhibit HVG responses. To address this hypothesis, we reduced TBI almost 30% to 4.0Gy, and found that even recipients transplanted with 4×106 CD80-/-CD86-/- T cell depleted donor BMC failed to engraft. Experiments were therefore initiated to target and rapidly expand host Tregs by administering IL-2 + anti-IL2 (IAC) complex to further strengthen suppression of HVG. C3H.SW (H2b) recipient mice were conditioned with 4.0Gy TBI (D-1) and B6-CD80-/-CD86-/- TCD-BM (H2b, 7×106) infused on the following day (D0). IAC was introduced on the day of conditioning (D-1) and then daily on days D 0, 1 and 2. We reported that anti-IL-2 + IL-2 complex can expand residual host Treg cells in RIC recipients (Blood, 113:733-743, 2009; Biol. Blood Marrow Transplantation. 15: 785-794, 2009). One month later, 1/3 of mice receiving complex – but not untreated controls demonstrated significant levels (15% B220+) of hematopoietic chimerism. These results, while encouraging, indicated that stimulation of the endogenous Treg compartment alone in 4.0 Gy recipients was not sufficient to regulate the HVG response. We then modified our approach by infusing 1×106 recipient Tregs obtained from normal C3H.SW mice one day following 4.0Gy RIC, i.e. at D0 and began expanding these cells with IAC injected early on D0 and again on D1 prior to the B6-CD80-/-CD86-/- TCD-BM graft – delayed one day and administered on D1. 100% of these recipients (n=3/gr) exhibited high donor chimerism (43.7+/-10.8 SD) 6 weeks post-HSCT, whereas all non-Treg infused control recipients rejected these marrow allografts. Results of 4 independent transplant experiments demonstrated 10/12 recipients contained similar levels of B220+ chimerism (mean donor: 53.9%) and significant T cell chimerism (mean donor: CD4, 19.8%; CD8, 35%). When both host Tregs and IAC were employed, strong chimerism was observed using 4 or 7×106 CD80-/-86-/- TCD-BM grafts. Notably, addition of 1×106 donor (i.e. B6-wt) Tregs and IAC treatment did not augment chimerism vs. IAC only in 4.0Gy conditioned C3H.SW recipients. These results suggest that the introduction of host Tregs 2 days prior to the BM graft allowed adequate time for IAC to mediate Treg expansion in the lymphopenic environment to generate sufficient Treg levels and together with the absence of donor APC B7 signaling to host Tconv cells overcome, i.e. suppressed HVG responses. Transplants were then performed using the identical Treg and IAC protocol with B6-wt TCD-BM which is readily rejected in 4.0 Gy TBI conditioned recipients. Despite the presence of B71/2 on B6-wt donor APC, one-third of the recipients receiving an infusion of host Tregs and IAC treatment exhibited chimerism, although lower (6.3%) than observed with B7.1/2 deficient donors. Transplants of wild type TCD-BM into MiHA mismatched recipients will determine how anti-B7.1/2 mab blockade, together with rapid in situ expansion of infused host Tregs post-TBI and pre-HSCT can be utilized to further diminish immunosuppressive TBI conditioning (<4.0 Gy) regimens. Disclosures:No relevant conflicts of interest to declare.

Bortezomib treatment and regulatory T-cell depletion enhance the antitumor effects of adoptively infused NK cells

AbstractLigation of inhibitory receptors renders natural killer (NK) cells inactive against autologous tumors. Recently, the proteasome inhibitor bortezomib was shown to sensitize tumors to autologous NK-cell cytotoxicity in vitro. Here, we show bortezomib augments the antitumor effects of syngeneic NK-cell infusions in tumor-bearing animals; this effect is further enhanced in regulatory T cell (Treg cell)–depleted hosts. In vitro, bortezomib-treated tumors had higher tumor necrosis factor–related apoptosis-inducing ligand (TRAIL) and perforin/granzyme-mediated caspase-8 activity, which enhanced their susceptibility to NK-cell lysis. Bioluminescence imaging of mice with established tumors showed treatment with bortezomib and syngeneic NK cells reduced tumor growth and prolonged survival compared with controls receiving bortezomib or NK cells alone. In contrast, tumor progression was not delayed when animals received bortezomib and perforin-deficient NK cells, showing drug-induced augmentation in NK-cell cytotoxicity was mediated through perforin/granzyme. Furthermore, tumor growth was slower in bortezomib-treated recipients when host Treg cells were eradicated with anti-CD25 antibody before infusing NK cells compared with mice without Treg-cell ablation (tumor doubling time, 16.7 vs 4.9 days, respectively; P = .02). These findings suggest that depletion of Treg cells followed by bortezomib-induced tumor sensitization to autologous NK cells could be used as a novel strategy to treat cancer.

Administration of IL-2-Anti-IL2mAb Complex Post-Allogeneic HCT: a New Approach to Facilitate Rapid and Stable Hematopoietic Chimerism Following Reduced Intensity Conditioning and Experimental HCT

Engraftment and the induction of tolerance to donor antigens is a central aim of allogeneic hematopoietic cell transplantation (HCT). Currently, the administration of Treg cells is being examined as a new approach to support these processes. Based upon the reported ability of IL-2 anti-IL-2 mAb complex (IACX) to expand CD4+CD25+ FoxP3+ (Tregs) in vivo, we hypothesized that the administration of this complex to allogeneic HCT recipients may expand host Treg cells and inhibit host anti-donor responses, thereby effectively facilitating hematopoietic engraftment. To begin to address these questions, the effect of IACX treatment on the frequency of circulating Tregs was examined. Two days following the second injection of IACX in normal or reduced intensity conditioned (RIC) mice, the % of circulating Tregs in IACX treated mice was strikingly increased vs. PBS treated controls. Notably, the expression of Treg CD25 (MFI) was also significantly elevated (p<0.05) in IACX treated mice, consistent with the expansion and activation of Tregs following IACX treatment. Varying regimens of the complex were then administered to RIC MHC-matched allogeneic recipients. B6 mice (RIC, 5.5 Gy TBI) transplanted with 4 × 106 BALB. B TCD-BM (day 0) were administered IACX (rIL-2/JES6-1) at days −5 and −3 (pre-HCT) or days +3 and +5 (post-HCT). In multiple independent studies, IACX treatment post-HCT consistently induced markedly superior expansion of host Treg populations at 1 wk post-HCT. Analysis of donor cells (Ly9.1+) 1 wk post-HCT clearly demonstrated that recipients treated with IACX post-HCT had markedly increased levels of circulating donor cells compared to the pre-HCT and PBS treated recipients (p<0.0001). To address the hypothesis that Treg expansion and activation following IACX treatment could inhibit host anti-donor CD8 T cell reactivity, host CD8+ T cell responses to the immunodominant donor epitope (H60) were analyzed by tetramer staining during the first 3 wks post-HCT. The frequency of these cells in recipients administered IACX post-HCT was greatly reduced compared to pre-HCT and PBS treated groups. Finally, to determine the effect of IACX treatment on engraftment, long-term chimerism was examined. Analysis for donor chimerism at 25 weeks post-HCT demonstrated: that only recipients administered IACX post-HCT were chimeric, containing high levels of donor cells vs. IACX pre-HCT and PBS controls, i.e. 36.1 ± 10.9% SE, 1.2 ± 0.785 and 0.78 ± 0.18, respectively. These findings demonstrate that infusion of IL-2 anti-IL-2mAb IACX in RIC allogeneic HCT can induce stable, long-term donor hematopoietic chimerism. Notably, the timing of IACX administration was found to be crucial with respect to conditioning and hematopoietic progenitor cell transplantation. At least part of the mechanism appears to involve the suppression of host anti-donor T cell responses, which presumably facilitates the initiation of early chimerism during the process of engraftment by donor cells. This approach may provide a new strategy to promote successful hematopoietic engraftment by manipulating endogenous Treg cells and thereby obviating the need for Treg infusions in certain transplant settings.

44: The CD4 +CD25 +Foxp3 + Compartment Following Conditioning and Transplant: Host Treg Cells Expand and Comprise the Predominant Component for Several Months During Reconstitution post-HCT

The capacity of CD4+CD25+Foxp3+ (Treg) cells to regulate adaptive and innate immune responses has led to studies investigating their use in novel strategies to regulate allogeneic T cell responses during hematopoietic stem cell transplants (HCT). A fundamental clinical concern post-HCT is the reconstitution of the lymphoid compartment, particularly T cells which can be exceptionally delayed. We have previously found that host Treg cells can regulate resistance to engraftment following HCT, demonstrating that such cells survive and function at least transiently in recipients. The present studies investigated the residual host Treg compartment following varying levels of conditioning (3.0 – 14Gy TBI), and transplant. We found that recipient CD4+CD25+Foxp3+ cells: 1) can survive ablative as well as reduced intensity conditioning, 2) undergo expansion (BrdU uptake/cell numbers) and 3) contribute greatly to the Treg compartment for several months post-HCT during which time donor derived Treg cells gradually arise and cede this compartment. Within the first 3 weeks post-lethal conditioning and HCT, 95% of the splenic CD4+FoxP3+ cells are positive for BrdU, vs. ∼40% in normal mice. Using Thy1.1 congenic mice, the vast majority of these cells were found to be resistant (host) Tregs. Two months post-HCT, almost 30% of the compartment was still of host origin. To assess the functional capacity of the residual Treg cell compartment, we examined development of autoimmune disease following transplant of IL-2Rβ−/− BM into syngeneic recipients. Autoimmune disease was prevented in B6-wt but not T cell deficient recipients. Interestingly, the failure to transfer autoimmune disease following IL-2Rβ−/− HCT into B6-CD4-/- recipients was associated with the presence of a peripheral CD8+FoxP3+ population not detected in B6-wt mice. This finding suggests that in the genetic absence of CD4+ T cells, a CD8 regulatory population may emerge. In total, our observations support the notion that functioning host Tregs initially occupy a niche in the transplant recipient permitting lymphopenic expansion and an extended period of contribution to this compartment. Notably, this contribution reflected much greater levels than that by other lymphoid cell populations. Overall, the findings imply that host Treg cells may be important to consider with respect to eliciting anti-tumor and vaccination responses in recipients during the early period post-hematopoietic cell transplantation. The capacity of CD4+CD25+Foxp3+ (Treg) cells to regulate adaptive and innate immune responses has led to studies investigating their use in novel strategies to regulate allogeneic T cell responses during hematopoietic stem cell transplants (HCT). A fundamental clinical concern post-HCT is the reconstitution of the lymphoid compartment, particularly T cells which can be exceptionally delayed. We have previously found that host Treg cells can regulate resistance to engraftment following HCT, demonstrating that such cells survive and function at least transiently in recipients. The present studies investigated the residual host Treg compartment following varying levels of conditioning (3.0 – 14Gy TBI), and transplant. We found that recipient CD4+CD25+Foxp3+ cells: 1) can survive ablative as well as reduced intensity conditioning, 2) undergo expansion (BrdU uptake/cell numbers) and 3) contribute greatly to the Treg compartment for several months post-HCT during which time donor derived Treg cells gradually arise and cede this compartment. Within the first 3 weeks post-lethal conditioning and HCT, 95% of the splenic CD4+FoxP3+ cells are positive for BrdU, vs. ∼40% in normal mice. Using Thy1.1 congenic mice, the vast majority of these cells were found to be resistant (host) Tregs. Two months post-HCT, almost 30% of the compartment was still of host origin. To assess the functional capacity of the residual Treg cell compartment, we examined development of autoimmune disease following transplant of IL-2Rβ−/− BM into syngeneic recipients. Autoimmune disease was prevented in B6-wt but not T cell deficient recipients. Interestingly, the failure to transfer autoimmune disease following IL-2Rβ−/− HCT into B6-CD4-/- recipients was associated with the presence of a peripheral CD8+FoxP3+ population not detected in B6-wt mice. This finding suggests that in the genetic absence of CD4+ T cells, a CD8 regulatory population may emerge. In total, our observations support the notion that functioning host Tregs initially occupy a niche in the transplant recipient permitting lymphopenic expansion and an extended period of contribution to this compartment. Notably, this contribution reflected much greater levels than that by other lymphoid cell populations. Overall, the findings imply that host Treg cells may be important to consider with respect to eliciting anti-tumor and vaccination responses in recipients during the early period post-hematopoietic cell transplantation.

Surviving Host CD4+CD25+Foxp3+ Cells Following Ablative Conditioning Expand and Comprise the Major Component of the Treg Compartment during the Lymphoid Reconstitution Period Following HCT.

The capacity of CD4+CD25+Foxp3+ (Treg) cells to regulate adaptive and innate immune responses has led to studies investigating their application to regulate allogeneic T cell responses arising during hematopoietic stem cell transplants (HCT). With respect to HCT, a fundamental clinical concern is the reconstitution of the lymphoid compartment in recipients, particularly T cells since this process can be exceedingly delayed. We have previously found that host Treg cells can regulate resistance to engraftment following HCT, demonstrating that such cells survive and function at least transiently in recipients. The present studies investigated the residual host Treg compartment following HCT. Utilizing varying levels of total body irradiation (5.0 – 14Gy), we observed:1.recipient CD4+CD25+Foxp3+ cells can survive ablative as well as reduced intensity conditioning,2.the surviving, i.e. residual Treg cells undergo expansion as assessed by BrdU uptake and cell numbers, and3.these cells comprise the majority of the Treg compartment in recipients for several months post-HCT during which time donor derived Treg cells gradually arise and cede this compartment.Residual Tregs also dominated the compartment following allogeneic HCT of MHC-matched bone marrow depleted of T cells. To assess the functional capacity of the residual Treg cell compartment, the development of autoimmune disease following transplant of IL-2Rβ −/− (CD122−/−) bone marrow into syngeneic recipients with and without residual Tregs was examined. Autoimmune disease symptoms and T cell alterations were prevented in B6-wt but not T cell deficient recipients. Interestingly, the failure to transfer autoimmune disease following IL-2Rβ −/− HCT into lethally conditioned B6-CD4−/− recipients was associated with the presence of a peripheral CD8+FoxP3+ population not detected in B6-wt mice or the B6-wt mice transplanted with IL-2Rβ −/− BM. This finding indicates that in the genetic absence of CD4+ T cells, a CD8 regulatory population appears to emerge. In total, our observations support the notion that functioning host Tregs initially occupy a niche in the transplant recipient permitting lymphopenic expansion and an extended period of contribution to this compartment. Notably, this contribution reflected much greater levels than conventional T cell populations - even in aggressively conditioned recipients. Finally, these findings imply that the presence of host regulatory cells may be important to consider with respect to eliciting anti-tumor responses and vaccination in recipients during the early period post -hematopoietic cell transplantation.

Suppression of natural killer cell-mediated bone marrow cell rejection by CD4+CD25+ regulatory T cells.

Naturally occurring CD4(+)CD25(+) T regulatory (Treg) cells have been shown to inhibit adaptive responses by T cells. Natural killer (NK) cells represent an important component of innate immunity in both cancer and infectious disease states. We investigated whether CD4(+)CD25(+) Treg cells could affect NK cell function in vivo by using allogeneic (full H2-disparate) bone marrow (BM) transplantation and the model of hybrid resistance, in which parental marrow grafts are rejected solely by the NK cells of irradiated (BALB/c x C57BL/6) F(1) recipients. We demonstrate that the prior removal of host Treg cells, but not CD8(+) T cells, significantly enhanced NK cell-mediated BM rejection in both models. The inhibitory role of Treg cells on NK cells was confirmed in vivo with adoptive transfer studies in which transferred CD4(+)CD25(+) cells could abrogate NK cell-mediated hybrid resistance. Anti-TGF-beta mAb treatment also increased NK cell-mediated BM graft rejection, suggesting that the NK cell suppression is exerted through TGF-beta. Thus, CD4(+)CD25(+) Treg cells can potently inhibit NK cell function in vivo, and their depletion may have therapeutic ramifications for NK cell function in BM transplantation and cancer therapy.

Overcoming Rejection of Bone Marrow Allografts by Treg Cells in a Stringent Mouse Model for T Cell Mediated Rejection: Tolerance Induction by Third Party “Off-the-Shelf” Cells.

Recent reports have shown that donor or host CD4+CD25+ Treg cells can be used to control GVHD or graft rejection following allogeneic BMT in mice. More recent data suggests that in the context of T cell depleted BM allografting, engraftment was only mildly improved by Treg cells alone, or by Rapamycin (RAPA) alone, but it was markedly enhanced by using Treg cells in conjunction with RAPA. These studies were carried out in a mouse model specifically designed to measure T cell mediated graft rejection. In this model, lethally irradiated (11Gy) C3H mice were infused with 1x104 purified host type T cells (HTC) and were transplanted one day later with 2x106 BM cells from Balb-Nude donors, which are markedly depleted of T cells and do not induce GVHD. Rejection mediated by the HTC is manifested by severe aplasia and lethality within 21 days posttransplant. In 10 independent experiments none of the mice in the irradiation control survived (0/62), the majority of the mice receiving BM survived (58/63) while marked rejection, associated with poor survival (2/62) was found in the group receiving purified HTC prior to the BM transplant. In the present study we further tested in this model whether third party Treg cells could be used instead of donor or host Treg cells to overcome rejection of BM allografts. We initially tested freshly isolated lymph node CD4+CD25+ cells. C3H (H2k) recipients received BM from Balb- Nude (H2d) donors and the Treg cells were obtained from Balb/c or FVB (H2q) donors. As in our previous study, while none of the recipients survived upon treatment with RAPA alone, using third party or donor type Treg cells in conjunction with RAPA led to survival of 9 of 13 and 7 of 10 mice respectively. Thus, the third party fresh Treg cells were as effective as the donor type cells in preventing graft rejection (P>0.05). Considering the low levels of CD4+CD25+ cells in peripheral blood or spleen, new strategies for growing these cells ex-vivo have been developed. Although, Treg cells exhibit low proliferative potential in-vitro upon TCR stimulation, the feasibility of growing mouse or human regulatory cells has been demonstrated mainly using the combination of TCR stimulation (either with an anti-TCR antibody or with allogeneic stimulator cells), costimulatory signals and high doses of IL-2. When tested in the same model, Treg cells ex-vivo expanded by stimulation against 4th party allogeneic cells, exhibited effective enhancement of engraftment of Balb-Nude BM. Thus, in four independent experiments, when assessing treatment with expanded Treg cells, of third party or donor type origin, the survival rate was 19 of 35 (54%) and 25 of 40 (62%) mice, respectively. Again, in both instances the marked potential of Treg cells to overcome T cell mediated rejection was exhibited only when co-administered with RAPA. In conclusion, our data strongly indicate that, at least in the bone marrow transplantation setting, third party Treg cells could afford a new viable ‘off-the-shelf' source for tolerance induction. The use of third party Treg cells in contrast to donor type cells could allow advanced preparation of a large bank of Treg cells, with all the appropriate quality controls required for cell therapy. Further studies with human Treg cells in-vitro are required to ascertain the potential of third party cells as a valuable source for clinical transplantation.