High Efficacy of Alloantigen-Specific Induced Regulatory T Cells in the Prevention of Acute Graft-Versus-Host Disease in Mice

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 4112-4112
Author(s):  
Jun Li ◽  
Kenrick Semple ◽  
Jessica Heinrichs ◽  
Anandharaman Veerapathran ◽  
Kelley M.K. Haarberg ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
Regulatory T Cells ◽  
Graft Versus Host Disease ◽  
Cell Response ◽  
T Cell Response ◽  
Suppressive Activity ◽  
Graft Versus Host ◽  
Mhc Ii

Abstract Abstract 4112 Introduction: Naturally occurring regulatory T cells (nTregs) may prevent graft-versus-host disease (GVHD) while preserving graft-versus-leukemia (GVL) activity. However, clinical application of nTregs has been severely hampered by their scarce availability and non-selective suppression. To overcome these limitations, we took an alternative approach to generate antigen-specific induced Tregs (iTregs), and tested their efficacy and selectivity in the prevention of GVHD in pre-clinical models of bone marrow transplantation (BMT). Methods: We selected HY as target antigen, because it is a naturally processed and ubiquitously expressed minor histocompatibility antigen (miHAg) with a proven role in GVHD and GVL effect. To generate HY-specific iTregs, naïve CD4+CD25− cells were isolated from MHC II-restricted HY-specific transgenic mice, and were stimulated with HY peptide and APCs, in the presence of TGFb and retinoic acid. Using similar protocol, we also generated polyclonal iTregs from CD4+CD25− cells of normal C57BL/6 (B6) mice with allogeneic dendritic cells (DCs). iTregs were isolated by positively selecting CD4+CD25hi cells 6–7 days after generation. Frequency of T cells in recognizing alloantigens was measured using a limited dilution assay. Various MHC-mismatched or matched murine BMT models were used, where polyclonal T cells (Teffs hereafter) were transplanted with donor bone marrow to induce GVHD in myeloablative allogeneic recipients. Results: We first assessed the effect of HY-specific iTregs on GVHD using an MHC II-mismatched B6 ® (B6 × bm12)F1 BMT model, and found that HY-specific iTregs prevented GVHD mortality in male (HY+) but not female (HY−) recipients. On the per-cell basis, HY-specific iTregs were significantly more potent than polyclonal Tregs in the prevention of GVHD. By measuring iTregs and Teffs in spleen and liver of the recipients, we found that HY-specific iTregs expanded extensively and significantly suppressed expansion, activation and infiltration of Teffs in male but not female recipients. To exclude the possibility the observation was model specific, we evaluated the efficacy of HY-specific iTregs and found that those iTregs were highly effective in the prevention of GVHD in two additional BMT models, including one MHC-matched but miHAg-mismatched B6 ® BALB.b model and one haplo-mismatched B6 ® B6D2F1 model. To increase translational potential of our approach, we extended our studies to alloantigen-specific polyclonal iTregs. After 7-day culture in iTreg- generating condition with BALB/c DCs, the frequency of B6 iTregs in recognizing BALB/c alloantigens was increased for 16-fold than that of naïve CD4+CD25− T cells. Furthermore, these iTregs were 64–128 fold more suppressive than nTregs to conventional T-cell response against BALB/c alloantigens. Their highly suppressive activity was antigen-specific, because the same alloreactive iTregs had significant lower suppressive activity to T-cell response against the third-party alloantigens. In vivo using an MHC-mismatched B6 ® BALB/c BMT model, we found that these alloreactive iTregs could effectively prevent GVHD at 2:1 ratio of Teff:Treg, at which polyclonal nTregs had a minimal effect. Conclusion: Using monoclonal HY-specific or polyclonal alloantigen-specific iTregs, these studies demonstrate that antigen-specific iTregs were highly effective in controlling GVHD in an activation-dependent manner. Because iTregs specific for a given miHAg (e.g. HY) can control polyclonal Teffs in response to multiple alloantigens and prevent GVHD in allogeneic recipient, these data also indicate that Tregs may control GVHD through a linked-suppression on Teffs in vivo. In conclusion, the current study presents a promising strategy to generate alloantigen-specific iTregs for effective GVHD prevention in human allogeneic hematopoietic cell transplantation. Disclosures: No relevant conflicts of interest to declare.

Early Treatment with CD4+CD25+ Regulatory T Cells Provides Prolonged Suppressive Effects Which Control Evolving but Not Established Graft-Versus-Host-Disease.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1303-1303
Author(s):  
Vu H. Nguyen ◽  
Robert Zeiser ◽  
Daniel Dasilva ◽  
Christopher N. Contag ◽  
Robert S. Negrin
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
Regulatory T Cells ◽  
Graft Versus Host Disease ◽  
Signal Intensity ◽  
P Value ◽  
Host Disease ◽  
Graft Versus Host ◽  
Allogeneic Bmt

Abstract CD4+CD25+ Regulatory T cells (Treg) reduce the incidence and severity of acute graft-versus-host disease (GvHD) in murine models of allogeneic bone marrow transplantation (BMT) when given at the time of transplantation at a 1:1 ratio with donor effector T cells (Tcon). In our previous study with Treg trafficking, bioluminescene imaging (BLI) indicated a persistence of signal consistent with a prolonged survival of Treg in vivo following allogeneic BMT. In the current study, we evaluated the duration of Treg suppression and the impact of Treg on evolving and established GvHD. Lethally irradiated Balb/c (H2d) hosts received 5x106 T-cell depleted bone marrow (TCD-BM) cells from wild-type FVB mice (H2q) on day 0. On day 2, 3x106 splenocytes, or 1x106 purified CD4+/CD8+ T cells (Tcon) from luciferase+ transgenic mice (FVB) were infused to induce GvHD. Treg, purified from wt-FVB mice, in a 1:1 dose ratio with Tcon, were infused either on day 0, 2, 9, or 23 post-transplantation. Bioluminescence imaging was used to localize and quantitate the proliferation of Tcon in the absence or presence of Treg. Signal intensity, measured by photons/second/mouse, was significantly decreased in animals which received Treg at day 0, 2, or 9 (p-value < 0.05). More importantly, the greatest reduction in signal intensity occurred when Treg were given prior to the induction of GvHD by Tcon. This reduction was associated with a significantly lower clinical score for GvHD. Studies in which Treg are given up to 10 days prior to the addition of Tcon show similar findings. At day 23, when clinical GvHD was fully established in mice which received Tcon, the addition of Treg did not alter the increasing BLI signal level or the clinical course such that all animals died of GvHD (p-value=0.38). Lymphoid reconstitution was not affected by the addition of Treg prior to the induction of GvHD. In dose titration studies whereby Treg are given two days prior to the induction of GvHD, a 10-fold dose reduction in Treg was sufficient to significantly reduce Tcon proliferation and suppress clinical GvHD. We next assessed the duration of Treg suppressive effect by inducing GvHD on day 7, 14, 19 with luc+ Tcon following the infusion of Treg on day 0 of allogeneic BMT. Treg provided protection from the Tcon challenge at all 3 time points, leading to improved survival (p-value < 0.05). We conclude that Treg provide prolonged protection due to their ability to proliferate in vivo in an allogeneic setting. The capacity of Treg to proliferate in vivo permits a significant reduction in the number of Treg needed for adoptive transfer to induce a clinical response, a practical finding given the rarity of Treg. In addition, we conclude that Treg suppress the early proliferation of Tcon, allowing them to prevent and control evolving but not established GvHD.

Rapamycin and IL-2 Prevents Lethal Acute Graft-Versus Host Disease by Expansion of Donor Type CD4+CD25+Foxp3+ Regulatory T Cells.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1334-1334
Author(s):  
Ho-Jin Shin ◽  
Jeanette Baker ◽  
Dennis Leveson-Gower ◽  
Emanuela I Sega ◽  
Robert Negrin
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
Regulatory T Cells ◽  
Graft Versus Host Disease ◽  
Cd8 T Cells ◽  
Graft Versus Host ◽  
Donor Type ◽  
Lethal Irradiation ◽  
Acute Graft

Abstract Abstract 1334 Poster Board I-356 Previous work has demonstrated that both rapamycin (RAPA) and IL-2 enhance CD4+CD25+Foxp3+ regulatory T cells (Treg) proliferation and function. We investigated whether the combination of RAPA and IL-2 administered in vivo could reduce acute graft-versus-host disease (aGVHD) induction and increase survival after bone marrow transplantation (BMT) by induction of Treg. T cell depleted bone marrow (TCD-BM) cells from wild type C57BL/6 mice were injected after lethal irradiation with (800 cGy) into Balb/c recipients on day 0. To induce aGVHD and evaluate the proliferation of donor T cells by in vivo bioluminescence imaging, 1 × 106 conventional CD4+ and CD8+ T cells (Tcon) from luciferase-expressing (luc+) transgenic C57BL/6 were injected intravenously on the same day. RAPA was administered intraperitoneally (dose of 0.5 mg/kg/day) for 14 days and IL-2 (dose of 5 × 104 IU) for 3 days (twice a day). RAPA plus IL-2 significantly reduced the expansion of luc+ Tcon more than RAPA (P=0.04) or IL-2 alone (P=0.002) or no treatment (P = 0.01). Weight loss and aGVHD score were significantly reduced in the mice which were injected with the combination of RAPA and IL-2 compared with those animals injected with RAPA (P = 0.03) or IL-2 alone (P = 0.05). The percentage of donor type CD4+CD25+ cells was increased and CD4+CD25− T cells were reduced from thymic and extrathymic tissues after treatment with RAPA plus IL-2 on day 7 after BMT. The combination of RAPA and IL-2 resulted in a 2.4 fold (mesenteric LN), 2.7 fold (peripheral LN), 4.2 fold (spleen) and 2.1 fold (thymus) increase in the percentage of donor type CD4+CD25+Foxp3+ Treg. In animals receiving the combination of RAPA and IL-2, the percentage of CD4+CD25+Foxp3+ T cells increased from 8.98% (RAPA alone) to 24.2% (RAPA plus IL-2) at 7 days after BMT, representing a 2.7 fold expansion. The cell numbers of CD4+ and CD8+ T cells and CD4+CD25− T cells were reduced and CD4+CD25+Foxp3+ Treg were expanded in the thymus and extrathymic tissues after administration RAPA and IL-2. To study the origin of the expanded Treg in RAPA plus IL-2, BALB/c recipient mice were injected with purified donor CD4+CD25highFoxP3+ Treg and CD4+CD25− and CD8+CD25− (both Foxp3−) Tcon after lethal irradiation followed by RAPA plus IL-2 for 7 days. The combination of RAPA and IL-2 increased the expansion of donor type CD4+CD25+Foxp3+ Treg, but did not result in an increase in the conversion of Foxp3+ Treg from donor CD25− Tcon. We also evaluated whether Foxp3+ non-Treg were present in the expansion of Treg by RAPA plus IL-2 by evaluating interferon-γ- and IL-2-production which was minimally detected in the Foxp3+ cells. In conclusion, the combination of RAPA and IL-2 after BMT synergistically promoted the expansion of donor CD4+CD25+Foxp3+ Treg resulting in prevention of lethal aGVHD. Disclosures No relevant conflicts of interest to declare.

Characterization of in vitro antimurine thymocyte globulin–induced regulatory T cells that inhibit graft-versus-host disease in vivo

Blood ◽  
2008 ◽  
Vol 111 (3) ◽  
pp. 1726-1734 ◽  
Author(s):  
Melanie C. Ruzek ◽  
James S. Waire ◽  
Deborah Hopkins ◽  
Gina LaCorcia ◽  
Jennifer Sullivan ◽  
...  
Keyword(s):  
T Cells ◽  
Regulatory T Cells ◽  
Graft Versus Host Disease ◽  
Antithymocyte Globulin ◽  
Mrna Level ◽  
Regulatory Cells ◽  
Host Disease ◽  
Graft Versus Host

Abstract Antithymocyte/antilymphocyte globulins are polyclonal antihuman T-cell antibodies used clinically to treat acute transplant rejection. These reagents deplete T cells, but a rabbit antihuman thymocyte globulin has also been shown to induce regulatory T cells in vitro. To examine whether antithymocyte globulin–induced regulatory cells might be functional in vivo, we generated a corresponding rabbit antimurine thymocyte globulin (mATG) and tested its ability to induce regulatory cells in vitro and whether those cells can inhibit acute graft-versus-host disease (GVHD) in vivo upon adoptive transfer. In vitro, mATG induces a population of CD4+CD25+ T cells that express several cell surface molecules representative of regulatory T cells. These cells do not express Foxp3 at either the protein or mRNA level, but do show suppressive function both in vitro and in vivo when adoptively transferred into a model of GVHD. These results demonstrate that in a murine system, antithymocyte globulin induces cells with suppressive activity that also function in vivo to protect against acute GVHD. Thus, in both murine and human systems, antithymocyte globulins not only deplete T cells, but also appear to generate regulatory cells. The in vitro generation of regulatory cells by anti-thymocyte globulins could provide ad-ditional therapeutic modalities for immune-mediated disease.

Impact of Regulatory T Cells on Antigen Specific T Cell Response Using Recombinant Chimeric T Cell Receptors In Vivo.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 5475-5475
Author(s):  
David M. Kofler ◽  
Markus Chmielewski ◽  
Heike Koehler ◽  
Tobias Riet ◽  
Patrick Schmidt ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Regulatory T Cells ◽  
Tumor Cells ◽  
T Cell Receptors ◽  
Cell Response ◽  
Effector T Cells ◽  
T Cell Response ◽  
Cell Receptors

Abstract Recombinant T cell receptors with defined specificity against tumor cells are a promising experimental approach in the elimination of residual leukemia and lymphoma cells. It is so far unresolved whether regulatory T cells with suppressor activities impair the efficiency of cytolytic T cells grafted with a recombinant immunoreceptor. The frequency of regulatory T cells is highly increased in tumor patients and their suppressive function seems to play a role in the fail of an autologous T cell response against the malignant cells. In this study we analyzed the antigen-triggered, specific activation of receptor grafted T cells in the presence or absence of regulatory CD4+CD25high T cells. CD3+ T cells were grafted with CEA-specific immunoreceptors containing the CD3-zeta signaling domain for T cell activation. Co-cultivation of receptor grafted effector T cells together with regulatory T cells repressed proliferation of the effector cells and decreased IL-2 secretion. Secretion of IFN-gamma and IL-10 was not impaired. Interestingly, the cytotoxicity of grafted effector T cells towards CEA-expressing tumor cells was not impaired by regulatory T cells in vitro. To evaluate the relevance in vivo, we used a Crl:CD1 Nu/Nu mouse model to assess growth of CEA+ tumor cells in the presence of receptor grafted effector T cells and of regulatory T cells. Mice inoculated with tumor cells together with CD3+ effector T cells without immunoreceptor and regulatory T cells developed earlier tumors with faster growth kinetics compared to mice that were inoculated with tumor cells, CD3+ T cells and CD4+CD25- control T cells. Using effector T cells that were equipped with a recombinant CEA-specific CD3-zeta immunoreceptor, 2 of 5 mice developed a tumor in the presence of regulatory T cells while none of the mice developed a tumor in the absence of regulatory T cells. Taken together, regulatory T cells obviously impair an antigen-specific, anti-tumor T cell attack in vivo. This seems to be due to repression of proliferation of the effector T cells and not to diminished cytotoxicity. These findings have major impact on the design of clinical studies involving adoptively transferred effector T cells.

Cell Autonomous Role of TGFβ and IL-2 Receptor in the In Vivo Generation of CD4 and CD8 Inducible Regulatory T Cells During Graft-Versus-Host Disease,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 4015-4015
Author(s):  
Atsushi Satake ◽  
Norifumi Sawamukai ◽  
Taku Kambayashi
Keyword(s):  
Weight Loss ◽  
T Cells ◽  
Regulatory T Cells ◽  
Graft Versus Host Disease ◽  
Acute Gvhd ◽  
Receptor Expression ◽  
Host Disease ◽  
Graft Versus Host ◽  
Tgfβ Receptor

Abstract Abstract 4015 FoxP3+ regulatory T cells (Tregs) suppress graft-versus-host disease (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 expansion of pre-existing natural Tregs (nTregs) or from de novo generation of inducible Tregs (iTregs) from donor conventional T cells (Tconvs) in the transplant recipient. Although the co-adoptive transfer of nTregs or in vitro -derived iTregs has been shown to prevent the development of GVHD, the relative contribution of these two Treg subsets in protection against GVHD has been unclear. To investigate the contribution of the different FoxP3+ Treg subsets, we used a MHC-mismatched mouse model of acute GVHD. Lethally irradiated (500cGy × 2) B6D2F1.SJL (H-2bxd) host mice were injected with T cell-depleted bone marrow cells and FACS-sorted Tconvs (WT or Foxp3-deficient) with or without FACS-sorted Tregs of C57BL/6 (H-2b) mouse origin. Weight loss in mice receiving Foxp3-deficient Tconvs alone was significantly more pronounced compared to other groups. The presence of either donor-derived nTregs or iTregs alone protected against GVHD-induced weight loss but was suboptimal compared to the presence of both donor-derived nTregs and iTregs. Next, we sought to determine how the donor-derived Treg pool was established during acute GVHD and tracked the appearance of Tregs in the secondary lymphoid organs at different time points post transplant. On Day 8 post GVHD induction, ∼5% of the donor-derived CD4+ T cells in the spleen were FoxP3+. We found that the Treg pool was comprised equally of donor-derived nTregs and iTregs. Unexpectedly, we found a significant fraction of CD8+FoxP3+ T cells (1–3% of all CD8+ T cells) in the spleen and in the lymph nodes. These CD8+FoxP3+ T cells representing ∼70% of the iTreg pool on Day 8 post GVHD induction. 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 expanded in vivo with IL-2 treatment and required IL-2 and TGFβ receptor expression for their generation. In summary, we found that donor derived-iTregs are generated during GVHD and contribute to suppression of acute GVHD induced-weight loss. Surprisingly, CD8+Foxp3+T cells were a major contributor to the donor derived-iTreg pool after transplantation. The generation of CD8+ and CD4+ iTregs occurred at least in part by a cell autonomous IL-2 and TGFβ receptor-dependent mechanism. Thus, our data suggest that in addition to increasing nTregs, concomitant strategies aimed at enhancing the conversion of donor-derived Tconvs to iTregs for example by engaging the IL-2 and TGFβ signaling pathways might be beneficial for the treatment of GVHD. Disclosures: No relevant conflicts of interest to declare.

Characterization of the T-cell repertoire in autologous graft-versus-host disease (GVHD): evidence for the involvement of antigen-driven T-cell response in the development of autologous GVHD

Blood ◽  
2001 ◽  
Vol 98 (3) ◽  
pp. 868-876 ◽  
Author(s):  
Yuji Miura ◽  
Christopher J. Thoburn ◽  
Emilie C. Bright ◽  
Matthias Sommer ◽  
Susan Lefell ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Mhc Class Ii ◽  
Graft Versus Host Disease ◽  
Cell Response ◽  
Effector T Cells ◽  
T Cell Response ◽  
T Cell Repertoire ◽  
Cell Repertoire ◽  
Graft Versus Host

Abstract Administration of cyclosporine A (CsA) after autologous stem cell transplantation elicits an autoimmune syndrome with pathology similar to graft-versus-host disease (GVHD). This syndrome, termed autologous GVHD, is associated with the appearance of autoreactive T cells directed at major histocompatibility class (MHC) class II antigens. In the rat model of autologous GVHD, clonal analysis reveals that the effector T cells are highly conserved and recognize a peptide from the invariant chain peptide presented by MHC class II. Although human autologous GVHD effector T cells share a similar phenotypic specificity, clonality of the response in humans has not been determined. To examine the human effector T-cell response, the T-cell repertoire of peripheral blood lymphocytes was assessed by complementarity-determining region 3 (CDR3) size distribution analysis and T-cell clonotype analysis in 26 patients treated with CsA after transplantation. Autologous GVHD developed in 3 of 4 patients with human leukocyte antigen (HLA)-DRB1*0701, and clonal expansions of β-chain variable region (BV)16+ T cells were shared. Clonal expansions within BV15+ and BV22+ T cells were also detected in 4 of 6 patients with HLA-DRB1*1501 and in 3 of 4 patients with HLA-DRB1*0401, respectively. Sequencing of BV16 cDNA for which the CDR3 size pattern exhibited apparent clone predominance revealed an identical CDR3 peptide sequence in 2 different patients, one with HLA-DRB1*0701 and the other with HLA-DRB1*1502. These findings indicate that the discrete antigen-driven expansion of T cells is involved in autologous GVHD.

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