Prevention of graft-versus-host disease by adoptive T regulatory therapy is associated with active repression of peripheral blood Toll-like receptor 5 mRNA expression.
ABSTRACT: Acute graft-versus-host disease (GVHD) occurs in 40% to 60% of recipients of partially matched umbilical cord blood transplantation (UCBT). In a phase I study, adoptive transfer of expanded CD4(+)CD25(+)Foxp3(+) natural regulatory T cells (nTregs) resulted in a reduced incidence of grade II-IV acute GVHD. To investigate potential mechanisms responsible for the reduced GVHD risk, we analyzed peripheral blood mononuclear cell mRNA expression of a tolerance gene set previously identified in operation- tolerant kidney transplant recipients, comparing healthy controls and patients who received nTregs and those who did not receive nTregs with and without experiencing GVHD. Samples from patients receiving nTregs regardless of GVHD status showed increased expression of Foxp3 expression, as well as B cell-related tolerance marker. This was correlated with early B cell recovery, predominately of naïve B cells, and nearly normal T cell reconstitution. CD8(+) T cells showed reduced signs of activation (HLA-DR(+) expression) compared with conventionally treated patients developing GVHD. In contrast, patients with GVHD had significantly increased TLR5 mRNA expression, whereas nTreg-treated patients without GVHD had reduced TLR5 mRNA expression. We identified Lin(-)HLADR(-)CD33(+)CD16(+) cells and CD14(++)CD16(-) monocytes as the main TLR5 producers, especially in samples of conventionally treated patients developing GVHD. Taken together, these data reveal interesting similarities and differences between tolerant organ and nTreg-treated hematopoietic stem cell transplantation recipients.
Project description:We showed previously that nonmyeloablative total lymphoid irradiation/rabbit anti-thymocyte serum (TLI/ATS) conditioning facilitates potent donor-recipient immune tolerance following bone marrow transplantation (BMT) across MHC barriers via recipient invariant NKT (iNKT) cell-derived IL-4-dependent expansion of donor Foxp3(+) naturally occurring regulatory T cells (nTregs). In this study, we report a more specific mechanism. Wild-type (WT) BALB/c (H-2(d)) hosts were administered TLI/ATS and BMT from WT or STAT6(-/-) C57BL/6 (H-2(b)) donors. Following STAT6(-/-) BMT, donor nTregs demonstrated no loss of proliferation in vivo, indicating that an IL-4-responsive population in the recipient, rather than the donor, drives donor nTreg proliferation. In graft-versus-host disease (GVHD) target organs, three recipient CD11b(+) cell subsets (Gr-1(high)CD11c(-), Gr-1(int)CD11c(-), and Gr-1(low)CD11c(+)) were enriched early after TLI/ATS + BMT versus total body irradiation/ATS + BMT. Gr-1(low)CD11c(+) cells induced potent H-2K(b+)CD4(+)Foxp3(+) nTreg proliferation in vitro in 72-h MLRs. Gr-1(low)CD11c(+) cells were reduced significantly in STAT6(-/-) and iNKT cell-deficient J?18(-/-) BALB/c recipients after TLI/ATS + BMT. Depletion of CD11b(+) cells resulted in severe acute GVHD, and adoptive transfer of WT Gr-1(low)CD11c(+) cells to J?18(-/-) BALB/c recipients of TLI/ATS + BMT restored day-6 donor Foxp3(+) nTreg proliferation and protection from CD8 effector T cell-mediated GVHD. Blockade of programmed death ligand 1 and 2, but not CD40, TGF-? signaling, arginase 1, or iNOS, inhibited nTreg proliferation in cocultures of recipient-derived Gr-1(low)CD11c(+) cells with donor nTregs. Through iNKT-dependent Th2 polarization, myeloid-derived immunomodulatory dendritic cells are expanded after nonmyeloablative TLI/ATS conditioning and allogeneic BMT, induce PD-1 ligand-dependent donor nTreg proliferation, and maintain potent graft-versus-host immune tolerance.
Project description:Background: Treg cells represent important viral reservoirs during chronic HIV infection. CD39 is closely involved in Treg-mediated immunosuppressive effects. However, CD39 expression on nTregs and mTregs and a relationship with HIV DNA levels during HIV infection is still unclear. In this study, we analyzed the distribution of HIV DNA in Treg subsets and the association between HIV DNA and CD39 expression on Treg subsets. Methods: Sixty-two HIV-infected patients with different HIV stages and 14 uninfected individuals were enrolled. nTregs (CD4+CD25+CD127lowCD45RO-) and mTregs (CD4+CD25+CD127lowCD45RO+) were isolated by magnetic selection and flow cytometric sorting. HIV DNA was quantified by real-time polymerase chain reaction (PCR). CD39 expression on nTregs and mTregs was analyzed by flow cytometry. Results: Higher levels of HIV DNA were detected in mTregs than those in nTregs during chronic HIV infection. The frequency of CD39+ nTregs and HIV DNA levels in nTregs were increased in patients with advanced HIV infection. Furthermore, HIV DNA levels in nTregs correlated positively with CD39+ nTreg frequency. CD39+ nTreg frequency was also increased in immune non-responders. Conclusions: mTregs and nTregs are both important reservoirs of virus during chronic HIV infection and HIV DNA levels increase in nTregs in patients with advanced HIV infection. We observed increased frequency of CD39+ nTregs and HIV DNA levels in nTregs in patients with advanced HIV infection. HIV DNA levels in nTregs correlated positively with CD39+ nTreg frequency.
Project description:Graft-versus-host disease (GVHD) is a major cause of morbidity and mortality in patients treated with allogeneic hematopoietic stem cell transplantation (HSCT). Posttransplant immunosuppressive drugs incompletely control GVHD and increase susceptibility to opportunistic infections. In this study, we used flagellin, a TLR5 agonist protein (?50 kDa) extracted from bacterial flagella, as a novel experimental treatment strategy to reduce both acute and chronic GVHD in allogeneic HSCT recipients. On the basis of the radioprotective effects of flagellin, we hypothesized that flagellin could ameliorate GVHD in lethally irradiated murine models of allogeneic HSCT. Two doses of highly purified flagellin (administered 3 h before irradiation and 24 h after HSCT) reduced GVHD and led to better survival in both H-2(b) ? CB6F1 and H-2(K) ? B6 allogeneic HSCT models while preserving >99% donor T cell chimerism. Flagellin treatment preserved long-term posttransplant immune reconstitution characterized by more donor thymic-derived CD4(+)CD25(+)Foxp3(+) regulatory T cells and significantly enhanced antiviral immunity after murine CMV infection. The proliferation index and activation status of donor spleen-derived T cells and serum concentration of proinflammatory cytokines in flagellin-treated recipients were reduced significantly within 4 d posttransplant compared with those of the PBS-treated control recipients. Allogeneic transplantation of radiation chimeras previously engrafted with TLR5 knockout hematopoietic cells showed that interactions between flagellin and TLR5 expressed on both donor hematopoietic and host nonhematopoietic cells were required to reduce GVHD. Thus, the peritransplant administration of flagellin is a novel therapeutic approach to control GVHD while preserving posttransplant donor immunity.
Project description:OBJECTIVE:To assess whether reshaping of the immune balance by infusion of autologous natural regulatory T cells (nTregs) in patients after kidney transplantation is safe, feasible, and enables the tapering of lifelong high dose immunosuppression, with its limited efficacy, adverse effects, and high direct and indirect costs, along with addressing several key challenges of nTreg treatment, such as easy and robust manufacturing, danger of over immunosuppression, interaction with standard care drugs, and functional stability in an inflammatory environment in a useful proof-of-concept disease model. DESIGN:Investigator initiated, monocentre, nTreg dose escalation, phase I/IIa clinical trial (ONEnTreg13). SETTING:Charité-University Hospital, Berlin, Germany, within the ONE study consortium (funded by the European Union). PARTICIPANTS:Recipients of living donor kidney transplant (ONEnTreg13, n=11) and corresponding reference group trial (ONErgt11-CHA, n=9). INTERVENTIONS:CD4+ CD25+ FoxP3+ nTreg products were given seven days after kidney transplantation as one intravenous dose of 0.5, 1.0, or 2.5-3.0×106 cells/kg body weight, with subsequent stepwise tapering of triple immunosuppression to low dose tacrolimus monotherapy until week 48. MAIN OUTCOME MEASURES:The primary clinical and safety endpoints were assessed by a composite endpoint at week 60 with further three year follow-up. The assessment included incidence of biopsy confirmed acute rejection, assessment of nTreg infusion related adverse effects, and signs of over immunosuppression. Secondary endpoints addressed allograft functions. Accompanying research included a comprehensive exploratory biomarker portfolio. RESULTS:For all patients, nTreg products with sufficient yield, purity, and functionality could be generated from 40-50 mL of peripheral blood taken two weeks before kidney transplantation. None of the three nTreg dose escalation groups had dose limiting toxicity. The nTreg and reference groups had 100% three year allograft survival and similar clinical and safety profiles. Stable monotherapy immunosuppression was achieved in eight of 11 (73%) patients receiving nTregs, while the reference group remained on standard dual or triple drug immunosuppression (P=0.002). Mechanistically, the activation of conventional T cells was reduced and nTregs shifted in vivo from a polyclonal to an oligoclonal T cell receptor repertoire. CONCLUSIONS:The application of autologous nTregs was safe and feasible even in patients who had a kidney transplant and were immunosuppressed. These results warrant further evaluation of Treg efficacy and serve as the basis for the development of next generation nTreg approaches in transplantation and any immunopathologies. TRIAL REGISTRATION:NCT02371434 (ONEnTreg13) and EudraCT:2011-004301-24 (ONErgt11).
Project description:Adoptive transfer of thymus-derived natural regulatory T cells (nTregs) effectively suppresses disease in murine models of autoimmunity and graft-versus-host disease (GVHD). TGFß induces Foxp3 expression and suppressive function in stimulated murine CD4+25- T cells, and these induced Treg (iTregs), like nTreg, suppress auto- and allo-reactivity in vivo. However, while TGFß induces Foxp3 expression in stimulated human T cells, the expanded cells lack suppressor cell function. Here we show that Rapamycin (Rapa) enhances TGFß-dependent Foxp3 expression and induces a potent suppressor function in naive (CD4+ 25-45RA+) T cells. Rapa/TGFß iTregs are anergic, express CD25 at levels higher than expanded nTregs and few cells secrete IL-2, IFN? or IL-17 even after PMA and Ionomycin stimulation in vitro. Unlike other published methods of inducing Treg function, Rapa/TGFß induces suppressive function even in the presence of memory CD4+ T cells. A single apheresis unit of blood yields an average ~240 × 10? (range ~ 70-560 × 10?) iTregs from CD4+25- T cells in ? 2 weeks of culture. Most importantly, Rapa/TGFß iTregs suppress disease in a xenogeneic model of GVHD. This study opens the door for iTreg cellular therapy for human diseases.
Project description:FoxP3(+) regulatory T cells (Tregs) suppress GVHD while preserving graft-versus-tumor effects, making them an attractive target for GVHD therapy. The donor-derived Treg pool can potentially be derived from the expansion of preexisting natural Tregs (nTregs) or from de novo generation of inducible Tregs (iTregs) from donor Tconvs in the transplantation recipient. Using an MHC-mismatched model of acute GVHD, in the present study we found that the Treg pool was comprised equally of donor-derived nTregs and iTregs. Experiments using various combinations of T cells from wild-type and FoxP3-deficient mice suggested that both preexisting donor nTregs and the generation of iTregs in the recipient mice contribute to protection against GVHD. Surprisingly, CD8(+)FoxP3(+) T cells represented approximately 70% of the iTreg pool. These CD8(+)FoxP3(+) T cells shared phenotypic markers with their CD4(+) counterparts and displayed suppressive activity, suggesting that they were bona fide iTregs. Both CD4(+) and CD8(+) Tregs appeared to be protective against GVHD-induced lethality and required IL-2 and TGF? receptor expression for their generation. These data illustrate the complex makeup of the donor-derived FoxP3(+) Treg pool in allogeneic recipients and their potential role in protection against GVHD.
Project description:The relationship between the TCR repertoires of natural regulatory T cells (nTregs) and conventional CD4(+) T cells (Tconv) capable of responding to the same antigenic epitope is unknown. In this study, we used TCR?-chain transgenic mice to generate polyclonal nTreg and Tconv populations specific for a foreign Ag. CD4(+) T cells from immunized 3.L2?(+/-) TCR?(+/-) Foxp3(EGFP) mice were restimulated in culture to yield nTregs (EGFP(+)) and Tconv (EGFP(-)) defined by their antigenic reactivity. Relative to Tconv, nTreg expansion was delayed, although a higher proportion of viable nTregs had divided after 72 h. Spectratype analysis revealed that both the nTreg and Tconv responses were different and characterized by skewed distributions of CDR3 lengths. CDR3 sequences from nTregs displayed a divergent pattern of J? usage, minimal CDR3 overlap (3.4%), and less diversity than did CDR3 sequences derived from Tconv. These data indicate that foreign Ag-specific nTregs and Tconv are clonally distinct and that foreign Ag-specific nTreg populations are constrained by a limited TCR repertoire.
Project description:Azacitidine (AzaC) mitigates Graft vs. Host Disease (GVHD) in both murine preclinical transplant models and in human clinical trials while maintaining a robust Graft vs. Leukemia (GVL) effect. Previous studies have failed to investigate the role of natural Tregs on the mitigation of GVHD by AzaC; instead focusing on the generation of suppressive regulatory T cells (Tregs, CD4+CD25+FOXP3+) through the in vivo conversion of alloreactive donor T effectors (Teff CD4+ CD25-, FOXP3-) and the direct anti-proliferative effects of AzaC on allogeneic T cells. Using B6.Foxp3DTR/GFP mice in which Tregs can be specifically ablated through administration of Diphtheria toxin, we demonstrate that nTregs are required in the donor graft for AzaC to optimally protect against GvHD and that nTregs, unlike T effectors (Teff, CD3+FOXP3-), are resistant to the anti-proliferative effects of AzaC. Gene expression analysis identified the potent cell cycle inhibitor, p21, was significantly upregulated in Teff but not nTregs after treatment with AzaC. Furthermore, we demonstrate that Teff cells deficient in p21 are less sensitive to the antiproliferative effects of AzaC. These results, demonstrate that nTregs are essential for AzaC to fully protect against GvHD and have important clinical implications for future clinical trials testing AzaC as a novel method of GVHD prophylaxis in man. Overall design: n=3 per group nTregs and AzaC generated Tregs, nTregs were purified from the spleens of B6.Foxp3GFP mice and Treg depleted T cells were obtained from B6. Foxp3GFP x B6.CAGDSRED. T Cells were co-cultured at a 1:10 ratio of nTreg to Teff and were activated for 2 days in the presence of anti-CD3/CD28 beads (bead:cell 1:1; Invitrogen) and Xcyte medium supplemented with L-glutamine (4mM), penicillin (100 U/mL), streptomycin (100 µg/mL), and human recombinant IL-2 (hIL-2; 500 U/mL). The activated T cells were incubated in the presence of AzaC (1µM) (Sigma-Aldrich) or PBS for an additional 2 days. Cells were sorted using FACS Aria II (BD) to isolate nTreg (CD4+DSRED-FOXP3GFP+), CD4+ Teff (CD4+DSRed+FOXP3GFP-), CD8+ Teff (CD8+DSRed+FOXP3GFP-).
Project description:BACKGROUND: In humans and mice naturally occurring CD4(+)CD25(+) regulatory T cells (nTregs) are a thymus-derived subset of T cells, crucial for the maintenance of peripheral tolerance by controlling not only potentially autoreactive T cells but virtually all cells of the adaptive and innate immune system. Recent work using Dicer-deficient mice irrevocably demonstrated the importance of miRNAs for nTreg cell-mediated tolerance. PRINCIPAL FINDINGS: DNA-Microarray analyses of human as well as murine conventional CD4(+) Th cells and nTregs revealed a strong up-regulation of mature miR-155 (microRNA-155) upon activation in both populations. Studying miR-155 expression in FoxP3-deficient scurfy mice and performing FoxP3 ChIP-Seq experiments using activated human T lymphocytes, we show that the expression and maturation of miR-155 seem to be not necessarily regulated by FoxP3. In order to address the functional relevance of elevated miR-155 levels, we transfected miR-155 inhibitors or mature miR-155 RNAs into freshly-isolated human and mouse primary CD4(+) Th cells and nTregs and investigated the resulting phenotype in nTreg suppression assays. Whereas miR-155 inhibition in conventional CD4(+) Th cells strengthened nTreg cell-mediated suppression, overexpression of mature miR-155 rendered these cells unresponsive to nTreg cell-mediated suppression. CONCLUSION: Investigation of FoxP3 downstream targets, certainly of bound and regulated miRNAs revealed the associated function between the master regulator FoxP3 and miRNAs as regulators itself. miR-155 is shown to be crucially involved in nTreg cell mediated tolerance by regulating the susceptibility of conventional human as well as murine CD4(+) Th cells to nTreg cell-mediated suppression.
Project description:Prior work using allogeneic bone marrow transplantation (allo-BMT) models showed that peritransplant administration of flagellin, a toll-like receptor 5 (TLR5) agonist protected murine allo-BMT recipients from CMV infection while limiting graft-vs-host disease (GvHD). However, the mechanism by which flagellin-TLR5 interaction promotes anti-CMV immunity was not defined. Here, we investigated the anti-CMV immunity of NK cells in C57BL/6 (B6) mice treated with a highly purified cGMP grade recombinant flagellin variant CBLB502 (rflagellin) followed by murine CMV (mCMV) infection. A single dose of rflagellin administered to mice between 48 to 72 hours prior to MCMV infection resulted in optimal protection from mCMV lethality. Anti-mCMV immunity in rflagellin-treated mice correlated with a significantly reduced liver viral load and increased numbers of Ly49H+ and Ly49D+ activated cytotoxic NK cells. Additionally, the increased anti-mCMV immunity of NK cells was directly correlated with increased numbers of IFN-?, granzyme B- and CD107a producing NK cells following mCMV infection. rFlagellin-induced anti-mCMV immunity was TLR5-dependent as rflagellin-treated TLR5 KO mice had ?10-fold increased liver viral load compared with rflagellin-treated WT B6 mice. However, the increased anti-mCMV immunity of NK cells in rflagellin-treated mice is regulated indirectly as mouse NK cells do not express TLR5. Collectively, these data suggest that rflagellin treatment indirectly leads to activation of NK cells, which may be an important adjunct benefit of administering rflagellin in allo-BMT recipients.