Mesenchymal stromal cells induce regulatory T cells via epigenetic conversion of human conventional CD4 T cells in vitro

BackgroundChronic rejection characterized by chronic allograft vasculopathy (CAV) remains a major obstacle to long-term graft survival. Due to multiple complicated mechanisms involved, a novel therapy for CAV remains exploration. Although mesenchymal stromal cells (MSCs) have been ubiquitously applied to various refractory immune-related diseases, rare research makes a thorough inquiry in CAV. Meanwhile, melatonin (MT), a wide spectrum of immunomodulator, plays a non-negligible role in transplantation immunity. Here, we have investigated the synergistic effects of MT in combination with MSCs in attenuation of CAV.MethodsC57BL/6 (B6) mouse recipients receiving BALB/c mouse donor aorta transplantation have been treated with MT and/or adipose-derived MSCs. Graft pathological changes, intragraft immunocyte infiltration, splenic immune cell populations, circulating donor-specific antibodies levels, cytokine profiles were detected on post-operative day 40. The proliferation capacity of CD4+ and CD8+ T cells, populations of Th1, Th17, and Tregs were also assessed in vitro.ResultsGrafts in untreated recipients developed a typical pathological feature of CAV characterized by intimal thickening 40 days after transplantation. Compared to untreated and monotherapy groups, MT in combination with MSCs effectively ameliorated pathological changes of aorta grafts indicated by markedly decreased levels of intimal hyperplasia and the infiltration of CD4+ cells, CD8+ cells, and macrophages, but elevated infiltration of Foxp3+ cells. MT either alone or in combination with MSCs effectively inhibited the proliferation of T cells, decreased populations of Th1 and Th17 cells, but increased the proportion of Tregs in vitro. MT synergized with MSCs displayed much fewer splenic populations of CD4+ and CD8+ T cells, Th1 cells, Th17 cells, CD4+ central memory T cells (Tcm), as well as effector memory T cells (Tem) in aorta transplant recipients. In addition, the percentage of splenic Tregs was substantially increased in the combination therapy group. Furthermore, MT combined with MSCs markedly reduced serum levels of circulating allospecific IgG and IgM, as well as decreased the levels of pro-inflammatory IFN-γ, TNF-α, IL-1β, IL-6, IL-17A, and MCP-1, but increased the level of IL-10 in the recipients.ConclusionsThese data suggest that MT has synergy with MSCs to markedly attenuate CAV and provide a novel therapeutic strategy to improve the long-term allograft acceptance in transplant recipients.

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Follicular Lymphoma Infiltrating CD4+CD25+GITR+ Regulatory T-Cells Are Potent Suppressors of CD3/CD28 Co-Stimulated Autologous and Allogeneic CD8+CD25− and CD4+CD25− T-Cells.

Blood ◽

10.1182/blood.v108.11.3681.3681 ◽

2006 ◽

Vol 108 (11) ◽

pp. 3681-3681 ◽

Cited By ~ 1

Author(s):

Shannon P. Hilchey ◽

Richard B. Bankert ◽

Lisa M. Rimsza ◽

Steven H. Bernstein

Keyword(s):

T Cells ◽

Lymph Node ◽

Regulatory T Cells ◽

Follicular Lymphoma ◽

Cd4 T Cells ◽

Critical Role ◽

Response To Treatment ◽

Target Cells ◽

Normal Donor

Abstract Regulatory T-cells (Tregs) play a critical role in the inhibition of self-reactive immune responses and as such have been implicated in the suppression of tumor reactive effector T-cells. We demonstrate that follicular lymphoma (FL) T-cells are hypo-responsive to CD3/CD28 costimulation, as assessed by proliferation of CFSE (5-(and-6)-carboxyfluorescein diacetate succinimidyl ester) labeled cells, with only 3.11% ± 2.38 and 2.26% ± 1.76 of the CD8+ and CD4+ T-cells proliferating upon stimulation, respectively (n=7). In contrast, both normal lymph node (NLN), and reactive lymph node (RLN, lymphoid hyperplasia) T-cells proliferate significantly in response to costimulation. Specifically, NLN CD8+ and CD4+ T-cells demonstrate 35.2% ± 31.1 and 18.1% ± 15.9 cells proliferating upon stimulation, respectively (n=7). Similarly, upon stimulation, RLN CD8+ and CD4+ T-cells demonstrate 40.6% ± 22.6 and 40.3% ± 30.3 cells proliferating, respectively (n=5). We identify a population of FL infiltrating CD4+CD25+GITR+ T-cells that are significantly overrepresented within FL, 9.86% ± 6.70 (n=11) of the CD4+ T-cells, as compared to that seen in NLN, 0.70% ± 0.29 (n=13), or RLN, 1.40% ± 1.04 (n=5). These cells actively suppress the proliferation of autologous nodal CD8+ and CD4+ T-cells after costimulation, as CD25+ magnetic bead depletion of these cells in vitro restores proliferation of the remaining CD25− T-cells. Specifically, proliferation of FL CD8+CD25− and CD4+CD25− T-cells increases to 24.05% ± 11.46 and 10.53% ± 6.47, respectively, upon costimulation (n=4). The CD25+ enriched cell fraction contains functionally suppressive cells since add back of unlabelled CD25+ enriched cells to CFSE labeled CD25− cells results in a decrease in proliferation of the costimulated CD8+CD25− and CD4+CD25− T-cells, namely 7.59% ± 3.86 and 4.16% ± 1.79, respectively (n=4). These cells also suppress cytokine production (IFN-g, TNF-a and IL-2) from autologous nodal T-cells as assessed by multiplex analysis of culture supernatants. In addition to suppressing autologous nodal T-cells, the FL CD25+ enriched cells are also capable of suppressing proliferation of allogeneic CD8+CD25− and CD4+CD25− T-cells from NLN as well as normal donor peripheral blood lymphocytes (PBL), regardless of very robust stimulation of the target cells with plate bound anti-CD3 and anti-CD28 antibodies. The allogeneic suppression is not reciprocal, since CD25+ enriched cells derived from either NLN or normal donor PBL, used at the same ratio, are not capable of suppressing allogeneic CD8+CD25− and CD4+CD25− T-cells derived from FL and in fact, are less suppressive against autologous T-cells than are the FL derived CD4+CD25+ cells. Whether this is due to a higher proportion of functionally suppressive T cells within the FL derived CD25+ enriched cells, compared to that of NLN or normal donor PBL, or to an increased suppressive capacity of the FL derived CD25+ T cells is currently being investigated. These data show that FL infiltrating CD4+CD25+GITR+ T-cells have a phenotype and function consistent with Tregs and are very potent suppressors of lymphoma associated-CD8+ and CD4+ T-cells, and therefore may play an important role in lymphoma development, progression and response to treatment.

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Dendritic Cell-Induced T Cell Non-Responsiveness Is Mediated by FOXP3+ and TGF-beta+IL-10+ CD4+ T Cells.

Blood ◽

10.1182/blood.v108.11.3891.3891 ◽

2006 ◽

Vol 108 (11) ◽

pp. 3891-3891

Author(s):

Zwi N. Berneman ◽

Nathalie Cools ◽

Viggo F.I. Van Tendeloo ◽

Marc Lenjou ◽

Griet Nijs ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Regulatory T Cells ◽

T Cell Activation ◽

Cd4 T Cells ◽

Cell Activation ◽

T Cell Immunity ◽

Tgf Beta ◽

Cell Immunity

Abstract Dendritic cells (DC), the professional antigen presenting cells of the immune system, exert important functions both in induction of T cell immunity as well as of tolerance. Previously, it was accepted that the main function of immature DC (iDC) in their in vivo steady state condition is to maintain peripheral tolerance to self-antigens and that these iDC mature upon encounter of so-called danger signals and subsequently promote T cell immunity. However, a growing body of experimental evidence now indicates that traditional DC maturation can no longer be used to distinguish between tolerogenic and immunogenic properties of DC. In this study, we compared the in vitro stimulatory capacity of immature DC (iDC), cytokine cocktail-matured DC (CC-mDC) and poly I:C-matured DC (pIC-mDC) in the absence and presence of antigen. All investigated DC types could induce at least 2 subsets of regulatory T cells. We observed a significant increase in both the number of functionally suppressive transforming growth factor (TGF)-beta+ interleukin (IL)-10+ T cells as well as of CD4+CD25+FOXP3+ T cells within DC/T cell co-cultures as compared to T cell cultures without DC. The induction of these regulatory T cells correlates with in vitro T cell non-responsiveness after co-culture with iDC and CC-mDC, while stimulation with pIC-mDC resulted in reproducible cytomegalovirus pp65 or influenza M1 matrix peptide-specific T cell activation as compared to control cultures in the absence of DC. In addition, the T cell non-responsiveness after stimulation with iDC was shown to be mediated by TGF-beta and IL-10. Moreover, the suppressive capacity of CD4+ T cells activated by iDC and CC-mDC was shown to be transferable when these CD4+ T cells were added to an established T cell response. In contrast, addition of CD4+ T cells stimulated by pIC-mDC made responder T cells refractory to their suppressive activity. In conclusion, we hypothesize that DC have a complementary role in inducing both regulatory T cells and effector T cells, where the final result of antigen-specific T cell activation will depend on the activation state of the DC. This emphasizes the need for proper DC activation when T cell immunity is the desired effect, especially when used in clinical trials.

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Unique Role of mTOR Inhibitor, Everolimus in Inducible Regulatory T Cells

Blood ◽

10.1182/blood.v120.21.4349.4349 ◽

2012 ◽

Vol 120 (21) ◽

pp. 4349-4349

Author(s):

Tokiko Nagamura-Inoue ◽

Yuki Yamamoto ◽

Seiichiro Kobayashi ◽

Kazuo Ogami ◽

Kiyoko Izawa ◽

...

Keyword(s):

T Cells ◽

Regulatory T Cells ◽

Cd4 T Cells ◽

Mtor Inhibitor ◽

Conflicts Of Interest ◽

Ex Vivo ◽

Foxp3 Expression ◽

Induction Rate ◽

The Impact

Abstract Abstract 4349 Background: Regulatory T cells (Tregs) play an important role in immune-tolerance to allograft. Unbalance between Tregs and effector T cells is involved in graft-versus-host disease (GvHD) and other autoimmune disorders. Adoptive use of inducible Tregs (iTregs) is a candidate immunosuppressive therapy, and major concern has been focused on sustained expression of Foxp3 in iTregs. We previously reported that iTregs can be efficiently expanded from cord blood (CB)-derived CD4+ T cells in the presence of IL2, TGFb and a mTOR inhibitor, Everolimus (Eve). However, the effect of Eve on in vitro induction of iTreg remains to be elucidated. Here we studied the impact of Eve on CB-CD4+ T cells. Methods: CD4+ T cells were prepared from CB with a purity of >95% and put into the flask coated with anti-CD3/CD28 MAb. For Treg induction, these cultures were supplemented with IL2, IL-2/TGFb, IL2/TGFb/Eve, or IL2/Eve and kept for two weeks. The resulting CD4+ T cells including variable proportion of iTregs were subjected to mixed lymphocyte reaction (MLR) along with CFSE-labeled autologous responder T cells and allogeneic dendritic cells (DCs) as stimulator. Results: The basal proportion of CD25+Foxp3+ cells in CB-CD4+ T cells was 0.60 ± 0.59%. After two weeks, the induction rate of CD25+Foxp3+CD4+ T cells was higher in the culture with IL2/TGFb/Eve than that with IL2/TGFb, but Eve itself could not significantly induce iTregs in the absence of TGFb (Figure1.). The iTreg ratio (CD25+Foxp3+ cells/total CD4+ T cells) was 79.3 ± 17.4% in the culture with IL2/TGFb/Eve, 53.1 ± 23.8% with IL2/TGFb, 35.5±18.6% with IL2/Eve and 22.7 ± 18.6% with IL2, respectively. There was no significant relationship between the dose of Eve and the iTreg ratio, but the highest ratio and induction rate of iTregs were observed at 10nM Eve. Thus, an average of 2.95 ± 2.8 ×107 iTregs was obtained from 5 ×104 CB-CD4+ T cells after two weeks of culture with IL2/TGFb/Eve. The iTreg-rich population cultured with IL2/TGFb/Eve and IL2/TGFb, but not IL2 alone, efficiently inhibited MLR triggered by allogeneic DCs (Figure 2.). These iTregs were also active in MLR using allogeneic responder T cells. Interestingly, IL2/Eve-treated CB-CD4+ T cells also inhibited MLR, irrespective of the low or moderate iTreg ratio. The inhibitory effect on MLR was much less observed by another mTOR inhibitor, rapamycin, rather than Eve (Figure2). Expression of CD26 on CD4+ T cells was inversely correlated to Foxp3 expression and significantly down-regulated by TGFb with or without Eve. Discussion: Treatment of CB-CD4+ T cells with IL2/TGFb/Eve results in the efficient ex vivo expansion of functional iTregs. Eve enhanced TGFb induction of Foxp3 expression, but did not induce Foxp3 expression by itself. mTOR is a complex of TORC1 and 2. Rapamycin is reported to inhibit TORC1, while Eve inhibits both of them, at general dose. In recent report, mTOR-deficient T cells (TORC1/2, not TORC1 alone) displayed normal activation and IL-2 production upon initial stimulation, but failed to differentiate into effecter T cells, instead, differentiated into Tregs. Although the direct mechanism to inhibit MLR by CB-CD4+ T cells treated with Eve remained to be elucidated, these results suggested the aberrant pathways of immunological inhibition. Disclosures: No relevant conflicts of interest to declare.

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Generation of Functional Regulatory T Cells By FOXP3 Gene Transfer into CD4 T Cells from Scurfy Mice and IPEX Patients

Blood ◽

10.1182/blood.v128.22.2526.2526 ◽

2016 ◽

Vol 128 (22) ◽

pp. 2526-2526 ◽

Cited By ~ 2

Author(s):

Marianne Delville ◽

Emmanuelle Six ◽

Florence Bellier ◽

Nelly Sigrist ◽

David Zemmour ◽

...

Keyword(s):

T Cells ◽

Gene Transfer ◽

Regulatory T Cells ◽

Immune Tolerance ◽

Adoptive Transfer ◽

Cd4 T Cells ◽

Hematopoietic Stem ◽

Foxp3 Gene ◽

Human Pathology

Abstract IPEX (Immunodysregulation Polyendocrinopathy Enteropathy X-linked) syndrome is the prototype of primary immunodeficiency with prevailing autoimmunity. The disease is caused by mutations in the gene encoding the transcription factor forkhead box P3 (FOXP3), which leads to the loss of function of thymus-derived CD4+CD25+ regulatory T (tTreg) cells. In IPEX patients, the absence of a functional Treg cell compartment leads to the development of multiple autoimmune manifestations (including severe enteropathy, type 1 diabetes and eczema) usually in the first months or years of life. The current treatments for IPEX syndrome include immunosuppressive, hormone replacement therapies. Unfortunately, immunosuppressive treatments are usually only partially effective and their dose is often limited because of the occurrence of infectious complications and toxicity. Currently, the only curative treatment for IPEX syndrome is allogeneic hematopoietic stem cell transplantation (HSCT). The absence of an HLA-compatible donor for all patients and their poor clinical condition particularly expose them to a risk of mortality when HLA partially compatible donors are used. For all these reasons, effective alternative therapeutic approaches are urgently needed. Various preclinical studies have shown that partial donor chimerism is sufficient for complete remission meaning that a small number of functional natural Treg is sufficient to restore immune tolerance. This suggests that a gene therapy approach designed to selectively induce a Treg program in T cells by expressing FOXP3 could be a promising potential cure for IPEX. However, several issues might compromise the success of this strategy: (i) will the introduction of FOXP3 alone be sufficient to induce a stable Treg program or will it require additional transcription factors to lock the Treg function and sustain the stability of transduced cells? (ii) Targeting effector CD4+ T cells might be an issue in terms of T-cell receptor repertoire, since the TCR repertoire of nTregs is different from the one of effector CD4+ T cells, (iii) will FOXP3-transduced T cells be able to migrate to appropriate tissues to control auto-immune reactions?, (iv) infusion of nTreg prevents the appearance of some autoimmune manifestations in murine models, however the infusion was done in prophylaxis before the appearance of the symptoms. In order to address these questions, we have developed a mouse scurfy model to evaluate the functional and stability of the correction in vivo in parallel to the characterization of gene corrected human CD4 T cells from IPEX patients. Scurfy mice develop a disease very close to human pathology due to a spontaneous mutation of Foxp3 gene. We improved Scurfy mice model to improve animal production and increase the timeline of treatement. We demonstrated that FOXP3 gene transfer into murine CD4+ T cells enable the generation of potent regulatory T cells. Indeed we showed the functional suppressive properties of the generated CD4-FOXP3 cells in an optimized flow-cytometry-based in vitro suppression assay. The ability of CD4-FOXP3 to prevent Scurfy disease by adoptive transfer in the first days of life is currently under evaluation. Similarly in humans, we demonstrated that FOXP3 gene transfer into CD4+ T cells from IPEX patients enable the generation of potent regulatory T cells, as shown through the functional in vitro suppressive properties of the generated CD4IPEX-FOXP3. Moreover comparison of the transcriptional profile of these regulatory CD4IPEX-FOXP3 cells to natural Treg by RNA-seq analysis demonstrated a good repression of cytokine transcripts (IL4/5/13/CSF2, CD40L), a strong repression of IL7R, a strong induction of IL1R2, and a moderate activation of typical Treg genes (IL2RA, IKZF2, CTLA4). Therefore, the introduction of a functional copy of the FOXP3 gene into an IPEX patient's T cells may be enough to restore immune tolerance and thus avoid the complications of allogenic HSCT. We will also discuss the challenge of generating a large, homogenous and stable population of cells in vitro for adoptive transfer and whether it can ensure long-term disease correction without generating a context of generalized immunosuppression. Disclosures No relevant conflicts of interest to declare.

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