Enhancement of Graft-Versus-Host Disease Control Efficacy by Adoptive Transfer of Type 1 Regulatory T Cells in Bone Marrow Transplant Model

Abstract

Interleukin (IL)-10-producing type 1 regulatory T (Tr1) cells, which are Foxp3⁻memory T lymphocytes, play important roles in peripheral immune tolerance. We investigated whether Tr1 cells exert immunoregulatory effects in a mouse model of acute graft-versus-host disease (GVHD). Mouse CD4⁺ T cells were induced to differentiate in vitro into Tr1 cells using vitamin D3 and dexamethasone, and these donor-derived Tr1 cells were infused on the day of bone marrow transplantation. The Tr1 cell-transferred group showed less weight-loss and a twofold higher survival rate than the GVHD group, together with markedly decreased histopathologic grades. It was associated with the expansion of CD4⁺IL-4⁺ type 2 T-helper (Th2) cells and CD4⁺CD25⁺Foxp3⁺ regulatory T (Treg) cells. Furthermore, Tr1 cells decreased the numbers of CD4⁺interferon-γ⁺ Th1 and CD4⁺IL-17⁺ Th17 cells. Recipient mice harbored some Foxp3⁺ Tregs due to adoptive transfer of Tr1 cells, together with the upregulated expression of costimulatory molecules, including cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) and inducible T-cell costimulator (ICOS); however, the Treg cells did not show the plasticity. Therefore, adoptive Tr1 cell therapy may be effective against manifestations of GVHD, exert immunomodulatory effects in a manner dependent on CTLA-4 and ICOS, and induce differentiation of the transferred Tr1 cells into Foxp3⁺ Treg cells.

Introduction

Regulatory T cells are a component of the immune system involved in modulating immune reactions and in inducing tolerance. Among the various CD4⁺ regulatory T cell subsets, CD4⁺CD25⁺Foxp3⁺ regulatory T (Foxp3⁺ Treg) cells and interleukin (IL)-10-producing type 1 regulatory T (Tr1) cells have been the subject of in vivo and in vitro studies [1]. Among the several roles of Foxp3⁺ Treg cells, when there is enough Foxp3 cells functionally, they play an essential role in suppressing with effector T cells proliferation, cytokine production, and immune response to alloantigens in models of autoimmunity and organ transplantation. And, they can function to inhibit T cells of an irrelevant specificity and appear to preferentially localized to sites of inflammation where the antigen is expressed. Thus, the nonspecific suppressor functions are likely to be limited to localized areas of the graft or draining lymph node and not resulting in pan-suppression. These results support a basic concept that Treg cells could be used for therapeutic purpose to manage graft-versus-host disease (GVHD) [1 –3].

Conversely, under an environment where Foxp3 cells are insufficient, the deletion of Foxp3 gene leads to a loss of Treg-suppressive activities and the development of a lethal autoimmune syndrome in mice model and a fatal multisystem autoimmune disorder known as immune dysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX) syndrome, patients who suffer from IPEX syndrome have defective Treg function [4,5]. Indeed, adoptive transfer of Foxp3⁺ Treg cells has been reported to be effective in animal models of immune-related diseases and in a few prospective clinical trials [6 –8]. Despite these advances, the loss of Foxp3 transcription factor occurs, likely due to destabilization of Foxp3 expression in Foxp3⁺ Treg cells instead of outgrowth of a few contaminating conventional T cells. The cellular and molecular basis of Foxp3⁺ Treg destabilization, their low numbers in circulation, and poorly defined antigen specificity [9] during in vitro stimulation remain unclear.

Compared with Foxp3⁺ Treg cells, Tr1 cells do not constitutively express CD25 or Foxp3. Tr1 cells are distinct from Foxp3⁺ Treg cells because of their unique cytokine expression profile, denoted as IL-10⁺ transforming growth factor beta (TGF-β)⁺ interferon (IFN)-γ⁺IL-5⁺IL-4⁻IL-2^low/neg [10]. In addition to CD49b and lymphocyte-activation gene 3 (LAG-3) [11], Tr1 cells express inhibitory receptors. When activated via stimulation of the T cell receptor, Tr1 cells produce normal levels of molecular markers, such as cytotoxic T lymphocyte-associated antigen-4 (CTLA-4/CD152) [12,13], programmed cell death protein 1 (PD-1) [13], and inducible T cell costimulator (ICOS) [14]. These inhibitory receptors are expressed on Foxp3⁺ Treg cells and are negative regulators of T cell activation [15]. Moreover, the major mechanisms by which Tr1 cells control immune responses are secretion of high levels of IL-10 and TGF-β and killing of myeloid cells [3,10].

Tr1 cells play important roles in organ transplantation and the prevention and treatment of autoimmune and chronic inflammatory diseases by suppressing the immune response of effector and memory T cells and regulating immune tolerance in the periphery [6,16 –18]. And recently, Bacchetta et al. [19] demonstrated the feasibility of host-specific Tr1 cells in the only clinical trials. In particular, Tr1 cells contribute to immune tolerance in transplanted organs. For example, Asiedu et al. demonstrated that peritransplant treatment of diabetic nonhuman primates (NHPs) with anti-CD3 immunotoxin and deoxyspergualin induces stable rejection-free tolerance to allogeneic pancreatic islets transplant, which is associated with sustained elevation in serum IL-10 levels, whereas deoxyspergualin arrests the production of proinflammatory cytokines and the maturation of dendritic cells. In addition to the increased number of Tr1 cells, tolerant NHPs exhibit an almost threefold increase in the number of Foxp3⁺ Treg cells [20]. However, the administration of IL-10 alone does not protect against allograft rejection and is not sufficient to induce tolerance. IL-10 administration reduces inflammation and generates Tr1 cells in this model and seems insufficient to counteract the expansion and function of effector T cells.

We reported previously that co-infused Tr1 cells exert a synergistic immunoregulatory effect with mesenchymal stem cells (MSCs) in an experimental animal model of rheumatoid arthritis [21]. Therefore, we hypothesize that adoptive transfer of Tr1 cells also will exert immunoregulatory effects in an animal model of GVHD after allogeneic hematopoietic stem cell transplantation (HSCT). We found that Tr1 cells exerted immunoregulatory effects on the Th1/Th2 response and differentiated into Foxp3⁺ Treg cells in a CTLA-4 and ICOS-dependent manner, and thus, adoptive transfer of Tr1 cells may be effective against GVHD following allogeneic HSCT.

Materials and Methods

Mice

This animal work and the protocol used were approved by the Institutional Animal Care and Use Committee at the School of Medicine, The Catholic University of Korea in accordance with the Laboratory Animals Welfare Act (approval no.: CUMC-2015-0097-02).

BALB/c (H-2d) mice (8–10 weeks old) were purchased from OrientBio (Sungnam, Korea). C57BL/6 or C57BL/6-tg (CAG-EGFP; H-2b) mice were purchased from Japan SLC (Shizuoka, Japan). The mice were maintained under specific pathogen-free conditions in an animal facility with controlled humidity (55% ± 5%), light (12/12 h light/dark), and temperature (22°C ± 1°C). The air in the facility was passed through a high-efficiency particulate air filter system. Animals were provided mouse chow and tap water ad libitum. For blood collection, mice were anesthetized with 2.5% isoflurane in oxygen and euthanized by exposure to CO₂.

Tr1 and Foxp3⁺ Treg cell generation in vitro

To obtain Tr1 cells, CD4⁺ T cells (5 × 10⁵) isolated from C57BL/6 or C57BL/6-tg (CAG-EGFP) mice's spleen were cultured with plate-bound anti-CD3 (1 μg/mL; BD Pharmingen, San Jose, CA), soluble anti-CD28 (1 μg/mL; BioLegend, San Diego, CA), dexamethasone (5 × 10⁻⁸ M; Sigma–Aldrich, St. Louis, MO), and vitamin D3 (10⁻⁷ M; Sigma–Aldrich) for 3 days [21,22].

Previously, we reported a method of generating Tregs using all-trans-retinal (Retinal) [2]. To produce Foxp3⁺ Treg cells, CD4⁺ T cells isolated from C57BL/6 or C57BL/6-tg (CAG-EGFP) mice were cultured with plate-bound anti-CD3 (1 μg/mL; BD Pharmingen), soluble anti-CD28 (1 μg/mL; BioLegend), anti-IFN-γ (5 μg/mL; R&D Systems, Minneapolis, MN), anti-IL-4 (5 μg/mL; R&D Systems), human recombinant TGF-β (5 ng/mL; PeproTech, London, United Kingdom), and retinoic acid (0.1 μM; Sigma–Aldrich) for 3 days.

Bone marrow transplantation and GVHD induction and scoring

All the recipient (BALB/c, H-2d) mice were exposed to an 800 cGy dose of radiation from a Mevatron MXE-2 instrument (Siemens, New York, NY) at a distance of 100 cm and a rate of 70 cGy/min. In GVHD group (n = 10), recipient mice were then injected intravenously with 5 × 10⁶ T cell-depleted bone marrow (TCD-BM) cells and 5 × 10⁵ CD4⁺CD25⁻ T cells from donor mice spleen (C57BL/6, H-2b). Adoptive Tr1 cell group as experimental one (n = 10) was also infused 5 × 10⁶ TCD-BM cells plus 5 × 10⁵ CD4⁺CD25⁻ T cells from spleen with the additional cultured 5 × 10⁵ Tr1 cells on bone marrow transplant (BMT) day 0, which was generated by CD4⁺ T cells isolated from C57BL/6-tg (CAG-EGFP) mice's spleen. Control group (n = 10) composed of irradiated mice receiving 5 × 10⁶ TCD-BM cells, which do not induced GVHD. T cell depletion method was adopted from BM by using CD90.2 microbeads (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany) for negative selection. Survival after BMT was monitored daily, and the degree of clinically acute GVHD was assessed weekly using a scoring system that summed changes in five clinical parameters: weight loss, posture, activity, fur texture, and skin integrity [23].

Cultured Tr1 cells were transferred during BM transplantation

Mice were infused one time of 5 × 10⁵ Tr1 cells, which were generated by CAG-EGFP mice peri-BM transplantation period (transplant day 0). The TCD-BM control and GVHD group received intravenous injections of an equal volume of phosphate-buffered saline (PBS; Gibco) at the same time point.

Clinico-histopathology of GVHD

Mice were euthanized on day 80 after BMT for blinded histopathologic analysis of acute GVHD changes in the liver and small intestine. Organs were harvested, cryo-embedded, and sectioned. Tissue sections were fixed in 10% buffered formalin (Sigma–Aldrich) and stained with hematoxylin (Sigma–Aldrich) and eosin Y (1% solution; Muto Pure Chemical Co., Ltd., Tokyo, Japan) for histological examination. The GVHD histology scoring system was as follows: 0, normal; 0.5, focal and rare; 1, focal and mild; 2, diffuse and mild; 3, diffuse and moderate; and 4, diffuse and severe, in accordance with the previously reported GVHD histology [24].

Flow cytometric analysis

Single-cell suspensions of the spleen were immunostained by using various combinations of the following fluorophore-conjugated antibodies: CD4 (RM4-5; eBioscience), H-2kd (SF1-1.1; BD Pharmingen), LAG3 (C9B7W; BioLegend), CD49b (HMα2; BioLegend), ICOS (15F9; BioLegend), and PD-1 (J43; BD Pharmingen). The cells were also intracellularly stained using antibodies against IL-4 (11B11; BD Pharmingen), IL-6 (MP5-20F3; BioLegend), IL-10 (JES5-16E3; eBioscience), IL-17 (eBio17B7; eBioscience), IFN-γ (XMG1.2; eBioscience), Forkhead box protein 3 (Foxp3) (FJK-16s; eBioscience), and CTLA-4 (UC10-4B9; BioLegend). Before intracellular staining, cells were restimulated for 4 h with 25 ng/mL phorbol myristate acetate (Sigma–Aldrich) and 250 ng/mL ionomycin (Sigma–Aldrich) in the presence of GolgiStop (BD Pharmingen). Intracellular staining was conducted using an intracellular staining kit (eBioscience) according to the manufacturer's protocol. Flow cytometric analysis was performed on a FACS_LSR Fortessa instrument (BD Pharmingen).

Real-time reverse transcription–polymerase chain reaction

Total RNA was extracted using TRIzol-LS reagent (Invitrogen, Carlsbad, CA). Total RNA (2 μg) was reverse-transcribed at 50°C for 2 min, followed by 60°C for 30 min. Quantitative polymerase chain reaction was performed using the FastStart DNA Master SYBR Green I kit and a LightCycler 480 instrument (both from Bio-Rad, Hercules, CA), as specified by the manufacturer. The crossing point was defined as the maximum of the second derivative of the fluorescence curve. Negative controls were included and contained all components of the reaction mixture, except for template DNA. For quantification, relative mRNA levels of genes determined using the 2⁻ΔCt method are reported. β-Actin, a housekeeping gene, was used for normalization. The following gene-specific primers were used: β-actin (forward: 5′-GAAATCGTGCGTGACATCAA-3′; reverse: 5′-TGTAGTTTCATGGATGCCAC-3′), RORγ-T (forward: 5′-TGTCCTGGGCTACCCTACTG-3′; reverse: 5′-GTGCAGGAGTAGGCCACATT-3′), T-bet (forward: 5′-TGCCTGCAGTGCTTCTAACA-3′; reverse: 5′-GCTGTGAGATCATATCCTTGGG-3′), and GATA3 (forward: 5′-TCCAAGTGTGCGAAGAGTTCCT-3′; reverse: 5′-GTGCCCATTTGGACATCAGACT-3′).

Quantitation of cytokine levels by enzyme-linked immunosorbent assay

IL-10 concentrations were measured by sandwich enzyme-linked immunosorbent assay. An anti-mouse IL-10 monoclonal antibody (R&D Systems) was added to a 96-well plate (Nunc, Roskilde, Denmark) and incubated overnight at 4°C. Wells were blocked with blocking solution (PBS containing 1% bovine serum albumin and 0.05% Tween 20) for 2 h at room temperature. Test samples and standard recombinant mouse IL-10 (R&D Systems) were added to separate wells; the plate was incubated at room temperature for 2 h and washed. A biotinylated anti-IL-10 polyclonal antibody (R&D Systems) was added, and the reaction was allowed to proceed for 2 h at room temperature. The plate was washed, ExtrAvidin^®-Alkaline Phosphatase (1:2,000 dilution; Sigma–Aldrich) was added, and the reaction was allowed to proceed for a further 2 h. The plate was washed and 50 μL p-nitrophenyl phosphate disodium salt (Pierce Chemical Company, Rockford, IL), diluted to 1 mg/mL in diethanolamine buffer, was applied. Procedures were performed according to the manufacturer's instructions.

Statistical analyses

Statistical analyses were performed using GraphPad Prism (ver. 5.01) and SAS version 9.2 (SAS Institute, Inc., Cary, NC), and all statistical tests were two-sided, and values of P < 0.05 were considered statistically significant. Comparisons between groups were analyzed statistically using the Kruskal–Wallis test. Pairwise group comparisons used the Mann–Whitney U-test, and P values were adjusted for multiple comparisons using Bonferroni's method to determine the statistical significance of these comparisons. Survival analyses were performed with the Kaplan–Meier and Log-rank (Mantel–Cox) method of survival distribution. In all analyses, P values <0.05 were considered to indicate statistical significance.

Results

Immunophenotypes of culture-expanded Tr1 cells in vitro

First, we conducted an in vitro analysis with cultured Tr1 cells to verify if our culture-expanded Tr1 cells from C57BL/6 mice (CAG-EGFP) were similar to the immunophenotypic characterization of the well-known Tr1 cells [11]; Tr1 cells generally represent the co-expression of CD49b and LAG3 and the ability to secrete IL-10. Although other molecules, including of ICOS, CD39, CD73, PD-1, and GITR [25], have been related to surface markers as Tr1 cells, their expressions have a limitation in considering as Tr1-specific markers due to the frequent expression on other cell types too.

In our results, >45% portion showed a co-expression of LAG-3 and CD49b in CD4⁺ cells (Fig. 1A) and high expression of IL-10 (Fig. 1B). Based on these results, our generated cells had fulfilled the characteristics of general Tr1 cells. Although the absence of specific surface markers that uniquely identify Tr1 cell, Gagliani et al. showed a highly representative method for Tr1 cell counts by using the co-expression of LAG3 and CD49b [11]. Tr1 cells, which were >45% of the purity and highly expressed IL-10, were consequently used for this study.

FIG. 1.

Characterization of culture-expanded Tr1 and Treg cells. Treg and Tr1 cell culture conditions were as follows: Treg CD4⁺Foxp3⁺IL-10⁻ cells (anti-IFN-γ, anti-IL-4, TGF-β, retinoic acid, anti-CD3, and anti-CD28); Tr1 CD4⁺Foxp3⁻IL-10⁺ cells (dexamethasone, vitamin D3, anti-CD3, and anti-CD28). (A) Tr1 and Treg cells in gated CD4⁺ T cell populations were analyzed by flow cytometry for intracellular LAG-3 and surface CD49b expression. The purity of Tr1 was >45% in CD4⁺LAG-3⁺CD49b⁺ T cells. (B) Culture-expanded Tr1 and Treg cells were distinguishable from CD4⁺ T cell populations by their increased expression of IL-10 and Foxp3. The histogram showed that Tr1 cells expressed high IL-10 level and very low IL-10 in Treg cells, whereas Foxp3 expression was very low in Tr1 cells and very high in Treg cells. (C) To assess the stability of adoptive-transferred Tr1 cells, Tr1 and Treg cells were treated with IL-17, IL-12p70, IL-6, IL-1β, and IFN-γ; Tr1 cells expressed a higher level of IL-10 than Treg cells. (D, E) The number of Foxp3⁺CTLA-4⁺ and Foxp3⁺ICOS⁺ decreased rapidly over time in Treg cells, but the number of Foxp3⁺CTLA-4⁺ and Foxp3⁺ICOS⁺ increased in Tr1 cells. Means ± SEM are shown; results are representative of three independent experiments. Statistical significance was determined by Student's two-tailed t-test and ANOVA with Bonferroni correction for multiple comparisons; **P < 0.01, ***P < 0.001. IL, interleukin; Tr1, type 1 regulatory T; Treg, regulatory T; IFN, interferon; CTLA, cytotoxic T lymphocyte-associated antigen; ICOS, inducible T cell costimulator; LAG-3, lymphocyte-activation gene 3; TGF-β, transforming growth factor beta; SEM, standard error of the mean; ANOVA, analysis of variance.

Tr1 cells could maintain stable status under the inflammatory condition

To confirm the stability of each regulatory T cells under the inflammatory circumstance, Tr1 and Foxp3⁺ Treg cells were subjected to severe inflammatory conditions in vitro. Our institute and others previously reported that Treg cells could be generated in the presence of IFN-γ, anti-IL-4, TGF-β, and retinoic acid; ∼70%–80% of these cells presented CD4⁺Foxp3⁺ T cells with low production of CD4⁺IL-10⁺ T cells [21,26].

Each Tr1 and Foxp3⁺ Treg cells were cultured with a mixture of IL-17, IL12p70, IL-6, IL-1β, and IFN-β in vitro. IL-10 level of Tr1 cells has remained stably high than Foxp3⁺ Treg cells even in the excessive inflammatory environment (Fig. 1C). Interestingly, Tr1 cells showed transiently increased expression of CD4⁺Foxp3⁺CTLA-4⁺ (expression ratios were 1.54%, 1.06%, and 3.09% on day 0, 1, and 3, respectively, under inflammatory condition; Fig. 1D, lower panel) and CD4⁺Foxp3⁺ICOS⁺ (expression ratios were 0.15%, 0.45%, and 5.11% on day 0, 1, and 3, respectively; Fig. 1E, lower panel). However, Treg cells revealed that CD4⁺Foxp3⁺CTLA-4⁺ (expression ratios were 44.1%, 38.0%, and 11.1% on day 0, 1, and 3, respectively, under inflammatory condition; Fig. 1D, upper panel) and CD4⁺Foxp3⁺ICOS⁺ expressions (expression ratios were 15.8%, 3.25%, and 3.09% on day 0, 1, and 3, respectively; Fig. 1E, upper panel) were decreased over time. These results suggest that Tr1 cells were relatively stable even under severe inflammatory in vivo condition. And we assumed that this stability might be one of the significant reasons for the immunomodulatory effect of Tr1 cells.

Adoptive transfer of Tr1 cells ameliorates clinical manifestations of GVHD

To explore the GVHD control efficacy of adoptive-transferred Tr1 cells, we used acute GVHD mouse model; TCD-BM cells-infused group was used as a control group, which did not induce GVHD. And the GVHD group was injected 5 × 10⁶ CD4⁺CD25⁻ T cells from donor mice spleen (C57BL/6, H-2b). Adoptive Tr1 cell group as experimental one was the additional infusion of the culture Tr1 cells using the enhanced fluorescent protein tagging mice's CD4⁺ T cells. Each group consisted of five mice; three independent experiments were performed for deriving all results.

After the enhanced fluorescent protein tagging, Tr1 cells from CAG-EGFP mice were infused during the BM transplantation period, and clinical manifestations were observed. As a control group, all recipients of adoptive transfer of Tr1 cells survived significantly longer than the GVHD group (P < 0.05, Fig. 2A) and showed an increase in body weight (Fig. 2B) and decrease in clinical GVHD score (Fig. 2C) compared with the GVHD group as well.

FIG. 2.

Survival was prolonged and GVHD score was improved in the adoptive-transfer group following BMT. All recipient (BALB/c, H-2d) mice were exposed to an 800 cGy dose of radiation. In the GVHD group (n = 10), recipient mice were then injected intravenously with 5 × 10⁶ TCD-BM cells and 5 × 10⁵ CD4⁺CD25⁻ T cells from donor mice spleen (C57BL/6, H-2b). Adoptive-transferred Tr1 cell group as experimental one (n = 10) was also infused 5 × 10⁶ TCD-BM cells and 5 × 10⁵ CD4⁺CD25⁻ T cells from the spleen with the additional cultured 5 × 105 Tr1 cells on BMT day 0, which was generated by CD4⁺ T cells isolated from C57BL/6-tg (CAG-EGFP) mice's spleen. Control group (n = 10) composed of irradiated mice receiving 5 × 10⁶ TCD-BM cells, which do not induced GVHD. (A) Survival was prolonged in the adoptive-transferred group. (B, C) Acute GVHD was scored weekly. Weight loss and clinical score were improved at a late stage after allo-BMT in the adoptive-transfer group. Means ± SEM are shown; results are representative of three independent experiments. Statistical significance was determined by Student's two-tailed t-test and ANOVA with Bonferroni correction for multiple comparisons. Survival analyses were performed with the Kaplan–Meier and Log-rank (Mantel–Cox) method of survival distribution. In all analyses, P values <0.05 were considered to indicate statistical significance; GVHD, graft-versus-host disease; TCD-BM, T cell-depleted bone marrow; BMT, bone marrow transplant; BM, bone marrow.

Up to 25 days after allogeneic BMT, clinical features, such as survival rate, change in body weight, and clinical GVHD score, were not different statistically between the adoptive-transfer and GVHD groups. In contrast, at >30 days after allo-BMT, generally the adoptive-transfer group showed more favorable survival outcomes; a longer survival duration (38%–50% vs. 10%–20% in Tr1 cell-infused group vs. GVHD group, respectively, P < 0.05, Fig. 2A), less weight loss (81%–92% weight of initial vs. 66%–72% in Tr1 cell-infused group vs. GVHD group, respectively, P < 0.001, Fig. 2B), and improved clinical GVHD score (score of 0.5–1.5 vs. 1.5–4.5 in Tr1 cell-infused group vs. GVHD group, respectively, P < 0.001, Fig. 2C).

Also, when we examined the immunohistochemistry on major organs, it showed markedly reduced lymphocyte infiltration on the liver, intestine, and skin (Fig. 3A). Using the GVHD histological grading system, it was revealed that decreased GVHD histopathologic grade in the periportal triad area of the liver (mean 1.0 vs. 3.3 in Tr1 cell-infused group vs. GVHD group, respectively, P < 0.05), intestinal tissue (mean 1.2 vs. 2.3 in Tr1 cell-infused group vs. GVHD group, respectively, P < 0.05), and subcutaneous skin lesions (1.6 vs. 2.6 in Tr1 cell-infused group vs. GVHD group, respectively, P < 0.05) compared with the GVHD group (Fig. 3B). Therefore, these data showed that the transfer of Tr1 cells was effective for the control of acute GVHD-related manifestations.

FIG. 3.

Histopathologic scores in the adoptive-transfer group at day 80 after allo-BMT. Histological scores were assessed in the liver (magnification, × 200), intestine ( × 200), and skin ( × 200 and × 400). (A, B) The adoptive-transferred group showed markedly reduced lymphocyte infiltration and GVHD histopathologic scores in intestinal tissue, the periportal triad area of the liver, and subcutaneous skin lesions compared with the GVHD group. Means ± SEM are shown; results are representative of two independent experiments. Statistical significance was determined by Student's two-tailed t-test and ANOVA with Bonferroni correction for multiple comparisons; *P < 0.05. Color images available online at www.liebertpub.com/scd

Adoptive transfer of Tr1 cells modulates acute GVHD-related Th1 and Th2 responses

T helper 1 (Th1), Th2, Th17, and Treg cells are critical in the allogeneic immune response in the presence of immune dysregulation, and an increased number of Th1 cells and a decreased number of Th2 cells are important in the pathogenesis of acute GVHD after allo-BMT [27]. To confirm the immunomodulatory effect of adoptive transfer of Tr1 cells after allo-BMT, we determined the Th2/Th1 cell ratio in spleen cells at day 80 after BMT by flow cytometry. IFN-γ secretion by Th1 cells was increased in the GVHD group and decreased in the adoptive-transfer group. Also, IL-4 secretion by Th2 cells was decreased in the GVHD group and increased in the adoptive-transfer group. The number of CD4⁺IFN-γ⁺ cells was decreased, and that of CD4⁺IL-4⁺ cells increased, in the adoptive-transfer group (Fig. 4A). The Th2/Th1 cell ratio was significantly increased in the adoptive-transfer group compared with the GVHD group (Fig. 4B). Furthermore, the expression of T-bet (which is the master regulator of Th1 cell differentiation of naive CD4⁺ T cells) and GATA3 (a master regulator of Th2 cell differentiation of CD4⁺ T cells) [28,29] was downregulated and upregulated, reciprocally, in the adoptive-transfer group (Fig. 4C). It suggested the upregulated Th2/Th1 response after adoptive Tr1 cells. And this restored Th2/Th1 ratio exerted an immunomodulatory effect and thus ameliorated GVHD.

FIG. 4.

Adoptive transfer of Tr1 cells modulates the Th2/Th1 ratio after allo-BMT. (A) Th1 and Th2 cells in the spleen of recipient mice were examined by flow cytometry at day 25 and at day 80 after allo-BMT. At the first 25 days after transplant, there is no statistical difference in cytokines between the GVHD group (n = 6) and adoptive-transferred Tr1 cells group (n = 6); however, there was a rather favorable tendency toward the adoptive-transferred Tr1 cell group than the GVHD group. At 80 days after transplant, IFN-γ secretion by Th1 cells was increased with statistical significance in the GVHD group (n = 4) but decreased in the adoptive-transfer group (n = 6). Also, IL-4 expression by Th2 cells was decreased in the GVHD group but increased in the adoptive-transfer group statistically. (B) The Th2/Th1 cell ratio was significantly decreased in the GVHD group but not in the adoptive-transfer group at post-BMT +80 days. (C) Expression of the transcription factor T-bet in spleen cells was decreased and that of GATA3 was increased in the adoptive-transfer group compared with the GVHD group at post-BMT + 80 days. Means ± SEM are shown; results are representative of three independent experiments. Statistical significance was determined by Student's two-tailed t-test and ANOVA with Bonferroni correction for multiple comparisons; *P < 0.05, **P < 0.01.

The Treg/Th17 ratio is increased by adoptive transfer of Tr1 cells

Generally, it is well known that the overexpression of Th17 with the regressed function of Treg cells plays a central role in the progression of acute GVHD. In this context, considerable heterogeneity or developmental plasticity in the Treg lineage has been reported [30 –32]. Th17-inducing conditions (IL-1β, IL-6, IL-23, and a transient Foxp3⁺RORγ-t⁺ population) can induce Treg cells to differentiate into Foxp3⁻RORγ-t⁺ Th17 cells [30,33]. Therefore, we examined IL-17 and IL-6 expressions in spleen cells of recipient mice at the early and delayed periods (BMT +25 days and BMT +80 days). IL-17 expression was significantly increased (Fig. 5A), and IL-6 expression showed an increasing trend in the GVHD group (Fig. 5B). The transcription factor RORγ-t is required for the differentiation of Th17 cells [34]; indeed, its expression was upregulated in the GVHD group (Fig. 5C). And these changes have been maintained for the delayed period.

FIG. 5.

The Treg/Th17 ratio was increased by adoptive transfer of Tr1 cells. The number of Th17 cells decreased significantly and that of Treg cells increased significantly in the spleens of recipient mice at day 80 after allo-BMT. (A, B) IL-17 and IL-6 expression levels were decreased in the adoptive-transfer group. (C) The expression level of RORγ-t, a transcription factor involved in Th17 cell differentiation, was decreased in the adoptive-transfer group. (D) The number of CD4⁺CD25⁺Foxp3⁺ cells was increased in the adoptive-transfer group. (E) The Treg/Th17 ratio was increased in the adoptive-transfer group. Means ± SEM are shown; results are representative of three independent experiments. Statistical significance was determined by Student's two-tailed t-test and ANOVA with Bonferroni correction for multiple comparisons; *P < 0.05, **P < 0.01, ***P < 0.001.

In the adoptive-transfer group, the numbers of CD4⁺IL-6⁺ and CD4⁺IL-17⁺ T cells were decreased and that of CD4⁺CD25⁺Foxp3⁺ T cells was increased (Fig. 5D), and RORγ-t was downregulated (Fig. 5C). The Treg/Th17 ratio was increased in the adoptive-transfer group (Fig. 5E). These results indicated that adoptive transfer of Tr1 cells increases the Treg/Th17 ratio, which also leads to the amelioration of GVHD.

Some adoptive-transferred Tr1 cells differentiated into CD4⁺Foxp3⁺ Treg cells after BMT

CD4⁺Foxp3⁺ Treg cells inhibit the formation of GVHD-causing Th1 effector cells by suppressing T cell activation in lymph nodes, and so, the modulation of CD4⁺Foxp3⁺ Treg cells may be effective against GVHD after allo-BMT [27,35,36]. Because the CD4⁺CD25⁺Foxp3⁺ T cell population was increased in the adoptive-transfer group (Fig. 5), the percentage of enhanced green fluorescent protein (eGFP)⁺H-2d⁺ cells among CD4⁺Foxp3⁺ Treg cells was examined by flow cytometry to determine the origin of the CD4⁺Foxp3⁺ Treg cells. On day 5 after allo-BMT, Foxp3 expression was detected in the adoptive-transfer group but not in the GVHD group. However, a portion of CD4⁺Foxp3⁺ cells originated from adoptive cells in the adoptive-transfer group, whereas most CD4⁺Foxp3⁺ cells in the GVHD group and TCD-BM control group were host-derived (Fig. 6A). Also, CD4⁺eGFP⁺ cells were detected in the adoptive-transfer group, and Foxp3⁺-expressing CD4⁺eGFP⁺ cells were detected in the adoptive-transfer group but not in the GVHD group or TCD-BM control group (Fig. 6B).

FIG. 6.

Origin of CD4⁺Foxp3⁺ Treg cells in recipient mice after BMT. (A) Foxp3 expression was high in the adoptive-transfer group and low in the GVHD group. However, a portion of CD4⁺Foxp3⁺ cells originated from Tr1 cells in the adoptive-transferred Tr1 cell group; in contrast, the vast majority of CD4⁺Foxp3⁺ cells were host-derived in the GVHD group. (B) CD4⁺Foxp3⁺eGFP⁺ cells were detected only in the adoptive-transfer group. (C–E) The costimulatory molecules associated with Treg cells were examined, including CTLA-4, ICOS, and PD-1. CD4⁺eGFP⁺CTLA-4⁺ and CD4⁺eGFP⁺ICOS⁺ cells (white bar in each figure), which originated from adoptive Tr1 cells, were slightly increased in the adoptive-transfer group, whereas CD4⁺eGFP⁺PD-1⁺ cells were not detected in any of the groups. PD-1, programmed cell death protein 1.

The expression of costimulatory molecules, such as CTLA-4, ICOS, and PD-1, was evaluated next. CTLA-4 expression was detected in the adoptive-transfer group but not in the GVHD group or TCD-BM control group (10.2% vs. 0.04% in Tr1 cell therapy group vs. GVHD group, Fig. 6C). Also, the expression of ICOS, which is correlated with differentiation of adoptive-transferred Tr1 cells into CD4⁺Foxp3⁺ Treg cells, was detected in the adoptive-transfer group but not in the GVHD or TCD-BM control groups (5.29% vs. 0% in Tr1 cell therapy group vs. GVHD group, Fig. 6D). In contrast, PD-1 expression was not detected in any of the three groups (0.36% vs. 0% in Tr1 cell therapy group vs. GVHD group, Fig. 6E). Therefore, a portion of adoptive-transferred Tr1 cells differentiated into CD4⁺Foxp3⁺Treg cells. Through this process, the upregulated CTLA-4 and ICOS might be associated with one of the immunoregulatory effects.

Discussion

Induction of peripheral immune tolerance is essential for maintaining the stability of the immune system and involves a variety of regulatory immune cells [37]. Tr1 cell therapy modulates the immune response in transplantation recipients and patients with autoimmune or chronic inflammatory diseases [10,38]. In this study, we show that Tr1 cells can be induced by dexamethasone and vitamin D3 and that adoptive transfer of Tr1 cells regulates the Th1 and Th2 responses, as well as the functions of Th17 and Treg cells, resulting in the amelioration of acute GVHD following allo-BMT.

Roncarolo and colleagues reported that a subset of CD4⁺ T cells suppresses the antigen-specific T cell response and prevents colitis. Subsequent work showed that high IL-10 and TGF-β expression plays a significant role in the immunosuppressive effect of Tr1 cells [10].

The major difference between Tr1 cells and other immunoregulatory cells is the existence of Foxp3 expression. Tr1 cells exert their immunologic effects by directly inhibiting the T cell response due to their expression of IL-10 and TGF-β, but not Foxp3. Tr1 cells exert a greater suppressive effect on T cell proliferation than Foxp3⁺ Treg cells, which lose their suppressive function in the presence of severe inflammation. Barrat et al. [22] reported that Tr1 cells could be generated in vitro and in vivo by priming naive T cells with antigen in the presence of IL-10 using dexamethasone and vitamin D3. These Tr1 cells are Foxp3^– regulatory CD4⁺ T cells that produce IL-10 and have nonredundant roles in the control of inflammation [39]. Tr1 cells express costimulatory molecules, which include CD49b and LAG3. We have confirmed that Tr1 cells generated using dexamethasone and vitamin D3 are satisfied with this characteristic of Tr1 cell-specific (Tr1 cells were detected as a relative highly dual expression of LAG-3 and CD49b as well as high expression of IL-10 in Fig. 1A, B). Even though under the inflammatory condition, Tr1 cells showed the constant characteristics, which highly increased IL-10 level and stably increased Foxp3⁺CTLA4⁺ and Foxp3⁺ICOS⁺ cells in contrast to Treg cells (Fig. 1C–E). Although the expression of costimulatory molecules alone is not sufficient to explain the immunomodulatory effects, our results suggest that Tr1 cells are more stable and functional and have less plasticity than Treg cells in the presence of severe inflammation.

Tr1 cells generally inhibit T cell responses through the Tr1 cell–dendritic cell–effector T cell pathway. The expression of costimulatory or inhibitory molecules, such as CTLA-4, PD-1, and ICOS, in Tr1 cells results in immunomodulatory effects similar to those of Foxp3⁺ Treg cells [15]. Although Tr1 cells do not constitutively express Foxp3 [40], they could exhibit transient upregulation of Foxp3 activation at the nonexceeding expression levels of naive Foxp3⁺ Tregs [3,15,41]. In this study, Foxp3 expression was higher in the adoptive Tr1 cells transfer group than the GVHD group (6.94% vs. 4.85%, Fig. 6A, upper panel), and these Foxp3⁺CD4⁺ cells were absolutely originated from the adoptive-transferred Tr1 cells (13.2% vs. 0% in Tr1 cell therapy group vs. GVHD, respectively, Fig. 6A, lower panel), due to the presence of transferred Tr1 cells (Fig. 6B). The costimulatory molecules of CTLA-1 and ICOS dependent manner were expressed from adoptive transfer of Tr1 cells without PD-1 expression; the expressed 10.2% of CTLA-4 and 5.29% of ICOS were originated entirely from adoptive-transferred Tr1 cells according to eGFP analysis, in contrast to 0.04% and 0% of CTLA-4 and ICOS, respectively, in the GVHD group (Fig. 6C, D). And eGFP-PD-1 expression did not differ from each other (Fig. 6E). Therefore, adopted Tr1 cells exert an immunoregulatory effect by upregulating ICOS and CTLA-4 expressions, and a portion may differentiate into Foxp3⁺CD4 cells under inflammatory conditions, such as acute GVHD.

Foxp3⁺ Treg cells are involved in initiating immune responses and inducing tolerance. However, accumulating evidence suggests that unstable Foxp3 expression of memory CD4⁺Foxp3⁺ T cells reduces their suppressive effect and proliferative capacity under severe inflammatory conditions [4]. Therefore, immunomodulatory cell therapy may be effective against GVHD. Clinical studies have shown that Tr1 cells are associated with long-term persistent chimerism after allo-BMT in patients with immunologic dysfunction [42,43]. Naturally occurring Foxp3⁺ Treg cells, which are present from birth, are immediately effective, particularly against self-reactivity, whereas Tr1 cells are induced in the periphery and are involved in regulation later in life. Also, Foxp3⁺ Treg and Tr1 cells have different effects on their targets. Foxp3⁺ Treg cells exert systemic immunoregulatory effects and contribute to homeostasis, whereas Tr1 cells regulate local immune microenvironments [37,44]. Bacchetta et al. demonstrated that severely combined immunodeficiency patients who underwent transplantation of human leukocyte antigen (HLA)-mismatched hematopoietic stem cells have circulating host-reactive T cell clones that generate high amounts of IL-10 in the absence of IL-4. Their appearance correlates with the absence of GVHD, as well as immunosuppression-free long-term graft tolerance [42]. Furthermore, higher frequency of IL-10-producing Tr1 cells in thalassaemic patients with a prolonged state of mixed chimerism after successful BMT has been reported [43]. Increased IL-10 production in peripheral blood before HSCT has been associated with a low incidence of GVHD and reduced number of transplant-related deaths [45,46]. The Infusion of T-Regulatory Cells in Kidney Transplant Recipients (ONE study) is based on these findings.

IL-10 and TGF-β are thought to be involved in the mechanism of Tr1 cell-mediated immune suppression and promotion of immune tolerance [15,21,38]. IL-10 and TGF-β directly inhibit the T cell response by suppressing IL-2 and IFN-γ production and T cell proliferation and indirectly act on antigen-presenting cells by downregulating the production of costimulatory molecules, HLA class II, and proinflammatory cytokines [3,47]. Previously, we reported the enhanced immunoregulation of MSCs by Tr1 cells in a collagen-induced arthritis model with synergistic induction of indoleamine 2,3-dioxygenase through the signal transducer and activator of transcription 1 pathway [21]. Thus, we focused on the immunomodulatory effects of IL-10-producing Tr1 cells in a model of GVHD, as IL-10 is a soluble factor vital to control inflammation, mitigating T cell response and preserving immunologic tolerance post-transplantation [48]. Our results show that transferred Tr1 cells exert immunomodulatory effects in GVHD by upregulating CTLA-4 and ICOS and by differentiating into Foxp3⁺ Treg cells.

In summary, adoptive transfer of Tr1 cells improved the survival outcomes and clinical manifestations of acute GVHD after allo-BMT. Tr1 cell-based therapy resulted in downregulation of Th1/Th17 responses and upregulation of Th2/Th1 responses in a manner dependent on CTLA-4 and ICOS; moreover, some of the transferred Tr1 cells differentiated into Foxp3⁺ Treg cells. In addition, the transferred Tr1 cells were stable, nonplastic, and enhanced immune tolerance. These findings may facilitate the development of novel therapeutic interventions for GVHD after allo-BMT.

Footnotes

Acknowledgments

This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIP) (no. NRF-2016R1A2B4007282). Also, this research was supported by the grant about Chief Executive Officer (CEO)-Fostering Project from Research Fund of the Seoul St. Mary's Hospital, The Catholic University of Korea.

Author Disclosure Statement

No competing financial interests exist.

References

von Herrath

and Harrison

. (2003). Antigen-induced regulatory T cells in autoimmunity. Nat Rev Immunol, 3:223–232.

Lim

, Park

, Im

, Kim

, Jeon

, Kim

, Cho

and Cho

. (2014). Combination cell therapy using mesenchymal stem cells and regulatory T-cells provides a synergistic immunomodulatory effect associated with reciprocal regulation of TH1/TH2 and th17/treg cells in a murine acute graft-versus-host disease model. Cell Transplant, 23:703–714.

Gregori

, Passerini

and Roncarolo

. (2015). Clinical outlook for type-1 and FOXP3(+) T regulatory cell-based therapy. Front Immunol, 6:593.

Passerini

, Rossi Mel

, Sartirana

, Fousteri

, Bondanza

, Naldini

, Roncarolo

and Bacchetta

. (2013). CD4(+) T cells from IPEX patients convert into functional and stable regulatory T cells by FOXP3 gene transfer. Sci Transl Med, 5:215ra174.

Bennett

, Christie

, Ramsdell

, Brunkow

, Ferguson

, Whitesell

, Kelly

, Saulsbury

, Chance

and Ochs

. (2001). The immune dysregulation, polyendocrinopathy, enteropathy, X-linked syndrome (IPEX) is caused by mutations of FOXP3. Nat Genet, 27:20–21.

Roncarolo

and Battaglia

. (2007). Regulatory T-cell immunotherapy for tolerance to self antigens and alloantigens in humans. Nat Rev Immunol, 7:585–598.

Brunstein

, Miller

, Cao

, McKenna

, Hippen

, Curtsinger

, Defor

, Levine

, June

, et al. (2011). Infusion of ex vivo expanded T regulatory cells in adults transplanted with umbilical cord blood: safety profile and detection kinetics. Blood, 117:1061–1070.

Lee

, Lim

, Im

, Kim

, Nam

, Jeon

and Cho

. (2015). Adoptive transfer of treg cells combined with mesenchymal stem cells facilitates repopulation of endogenous Treg cells in a murine acute GVHD model. PLoS One, 10:e0138846.

Trzonkowski

, Bieniaszewska

, Juścińska

, Dobyszuk

, Krzystyniak

, Marek

, Myśliwska

and Hellmann

. (2009). First-in-man clinical results of the treatment of patients with graft versus host disease with human ex vivo expanded CD4+CD25+CD127- T regulatory cells. Clin Immunol, 133:22–26.

10.

Groux

, O'Garra

, Bigler

, Rouleau

, Antonenko

, de Vries

and Roncarolo

. (1997). A CD4+ T-cell subset inhibits antigen-specific T-cell responses and prevents colitis. Nature, 389:737–742.

11.

Gagliani

, Magnani

, Huber

, Gianolini

, Pala

, Licona-Limon

, Guo

, Herbert

, Bulfone

, et al. (2013). Coexpression of CD49b and LAG-3 identifies human and mouse T regulatory type 1 cells. Nat Med, 19:739–746.

12.

Bacchetta

, Sartirana

, Levings

, Bordignon

, Narula

and Roncarolo

. (2002). Growth and expansion of human T regulatory type 1 cells are independent from TCR activation but require exogenous cytokines. Eur J Immunol, 32:2237–2245.

13.

Akdis

, Verhagen

, Taylor

, Karamloo

, Karagiannidis

, Crameri

, Thunberg

, Deniz

, Valenta

, et al. (2004). Immune responses in healthy and allergic individuals are characterized by a fine balance between allergen-specific T regulatory 1 and T helper 2 cells. J Exp Med, 199:1567–1575.

14.

Häringer

, Lozza

, Steckel

and Geginat

. (2009). Identification and characterization of IL-10/IFN-gamma-producing effector-like T cells with regulatory function in human blood. J Exp Med, 206:1009–1017.

15.

Gregori

, Goudy

and Roncarolo

. (2012). The cellular and molecular mechanisms of immuno-suppression by human type 1 regulatory T cells. Front Immunol, 3:30.

16.

Gagliani

, Jofra

, Stabilini

, Valle

, Atkinson

, Roncarolo

and Battaglia

. (2010). Antigen-specific dependence of Tr1-cell therapy in preclinical models of islet transplant. Diabetes, 59:433–439.

17.

Allen

, Zhang

, Moodie

, Clemens

, Smith

, Gregoire

, Bell

, Muscat

and Gustafson

. (2006). Halofenate is a selective peroxisome proliferator-activated receptor gamma modulator with antidiabetic activity. Diabetes, 55:2523–2533.

18.

Tang

, Bluestone

and Kang

. (2012). CD4(+)Foxp3(+) regulatory T cell therapy in transplantation. J Mol Cell Biol, 4:11–21.

19.

Bacchetta

, Lucarelli

, Sartirana

, Gregori

, Lupo Stanghellini

, Miqueu

, Tomiuk

, Hernandez-Fuentes

, Gianolini

, et al. (2014). Immunological outcome in haploidentical-HSC transplanted patients treated with IL-10-anergized donor T cells. Front Immunol, 5:16.

20.

Asiedu

, Goodwin

, Balgansuren

, Jenkins

, Le Bas-Bernardet

, Jargal

, Neville

Jr. and Thomas

. (2005). Elevated T regulatory cells in long-term stable transplant tolerance in rhesus macaques induced by anti-CD3 immunotoxin and deoxyspergualin. J Immunol, 175:8060–8068.

21.

Lim

, Im

, Lee

, Kim

, Nam

, Jeon

and Cho

. (2016). Enhanced immunoregulation of mesenchymal stem cells by IL-10-producing type 1 regulatory T cells in collagen-induced arthritis. Sci Rep, 6:26851.

22.

Barrat

, Cua

, Boonstra

, Richards

, Crain

, Savelkoul

, de Waal-Malefyt

, Coffman

, Hawrylowicz

and O'Garra

. (2002). In vitro generation of interleukin 10-producing regulatory CD4(+) T cells is induced by immunosuppressive drugs and inhibited by T helper type 1 (Th1)- and Th2-inducing cytokines. J Exp Med, 195:603–616.

23.

, Park

, Kim

, Lim

, Park

, Lee

, Nam

, Lee

, Cho

and Cho

. (2014). Induction of mixed chimerism using combinatory cell-based immune modulation with mesenchymal stem cells and regulatory T cells for solid-organ transplant tolerance. Stem Cells Dev, 23:2364–2376.

24.

Shi

, Adachi

, Cui

, Li

, Lian

, Zhang

, Yanai

, Shima

, Imai

and Ikehara

. (2011). Combination of intra-bone marrow-bone marrow transplantation and subcutaneous donor splenocyte injection diminishes risk of graft-versus-host disease and enhances survival rate. Stem Cells Dev, 20:759–768.

25.

White

and Wraith

. (2016). Tr1-like T cells—an enigmatic regulatory T cell lineage. Front Immunol, 7:355.

26.

Wang

, Han

, Foks

, Huizinga

and Toes

. (2010). Neutralization of IL-4 reverses the nonresponsiveness of CD4+ T cells to regulatory T-cell induction in non-responder mouse strains. Mol Immunol, 48:137–146.

27.

Bucher

, Koch

, Vogtenhuber

, Goren

, Munger

, Panoskaltsis-Mortari

, Sivakumar

and Blazar

. (2009). IL-21 blockade reduces graft-versus-host disease mortality by supporting inducible T regulatory cell generation. Blood, 114:5375–5384.

28.

Zheng

and Flavell

. (1997). The transcription factor GATA-3 is necessary and sufficient for Th2 cytokine gene expression in CD4 T cells. Cell, 89:587–596.

29.

Lazarevic

, Glimcher

and Lord

. (2013). T-bet: a bridge between innate and adaptive immunity. Nat Rev Immunol, 13:777–789.

30.

Sawant

and Vignali

. (2014). Once a Treg, always a Treg?. Immunol Rev, 259:173–191.

31.

Osorio

, LeibundGut-Landmann

, Lochner

, Lahl

, Sparwasser

, Eberl

and Reis e Sousa

. (2008). DC activated via dectin-1 convert Treg into IL-17 producers. Eur J Immunol, 38:3274–3281.

32.

Beriou

, Costantino

, Ashley

, Yang

, Kuchroo

, Baecher-Allan

and Hafler

. (2009). IL-17-producing human peripheral regulatory T cells retain suppressive function. Blood, 113:4240–4249.

33.

Kimura

and Kishimoto

. (2010). IL-6: regulator of Treg/Th17 balance. Eur J Immunol, 40:1830–1835.

34.

, Wang

, Liu

, Kaosaard

, Semple

, Anasetti

and Yu

. (2011). Prevention of GVHD while sparing GVL effect by targeting Th1 and Th17 transcription factor T-bet and RORgammat in mice. Blood, 118:5011–5020.

35.

Taylor

, Noelle

and Blazar

. (2001). CD4(+)CD25(+) immune regulatory cells are required for induction of tolerance to alloantigen via costimulatory blockade. J Exp Med, 193:1311–1318.

36.

Edinger

, Hoffmann

, Ermann

, Drago

, Fathman

, Strober

and Negrin

. (2003). CD4+CD25+ regulatory T cells preserve graft-versus-tumor activity while inhibiting graft-versus-host disease after bone marrow transplantation. Nat Med, 9:1144–1150.

37.

Zeng

, Zhang

, Jin

and Chen

. (2015). Type 1 regulatory T cells: a new mechanism of peripheral immune tolerance. Cell Mol Immunol, 12:566–571.

38.

Roncarolo

, Gregori

, Battaglia

, Bacchetta

, Fleischhauer

and Levings

. (2006). Interleukin-10-secreting type 1 regulatory T cells in rodents and humans. Immunol Rev, 212:28–50.

39.

Mascanfroni

, Takenaka

, Yeste

, Patel

, Wu

, Kenison

, Siddiqui

, Basso

, Otterbein

, et al. (2015). Metabolic control of type 1 regulatory T cell differentiation by AHR and HIF1-alpha. Nat Med, 21:638–646.

40.

Vieira

, Christensen

, Minaee

, O'Neill

, Barrat

, Boonstra

, Barthlott

, Stockinger

, Wraith

and O'Garra

. (2004). IL-10-secreting regulatory T cells do not express Foxp3 but have comparable regulatory function to naturally occurring CD4+CD25+ regulatory T cells. J Immunol, 172:5986–5993.

41.

Brun

, Neveu

, Pers

, Fabre

, Quatannens

, Bastian

, Clerget-Chossat

, Jorgensen

and Foussat

. (2011). Isolation of functional autologous collagen-II specific IL-10 producing Tr1 cell clones from rheumatoid arthritis blood. Int Immunopharmacol, 11:1074–1078.

42.

Bacchetta

, Bigler

, Touraine

, Parkman

, Tovo

, Abrams

, de Waal Malefyt

, de Vries

and Roncarolo

. (1994). High levels of interleukin 10 production in vivo are associated with tolerance in SCID patients transplanted with HLA mismatched hematopoietic stem cells. J Exp Med, 179:493–502.

43.

Serafini

, Andreani

, Testi

, Battarra

, Bontadini

, Biral

, Fleischhauer

, Marktel

, Lucarelli

, Roncarolo

and Bacchetta

. (2009). Type 1 regulatory T cells are associated with persistent split erythroid/lymphoid chimerism after allogeneic hematopoietic stem cell transplantation for thalassemia. Haematologica, 94:1415–1426.

44.

Barthlott

, Kassiotis

and Stockinger

. (2003). T cell regulation as a side effect of homeostasis and competition. J Exp Med, 197:451–460.

45.

Baker

, Roncarolo

, Peters

, Bigler

, DeFor

and Blazar

. (1999). High spontaneous IL-10 production in unrelated bone marrow transplant recipients is associated with fewer transplant-related complications and early deaths. Bone Marrow Transplant, 23:1123–1129.

46.

Holler

, Roncarolo

, Hintermeier-Knabe

, Eissner

, Ertl

, Schulz

, Knabe

, Kolb

, Andreesen

and Wilmanns

. (2000). Prognostic significance of increased IL-10 production in patients prior to allogeneic bone marrow transplantation. Bone Marrow Transplant, 25:237–241.

47.

Roncarolo

, Gregori

, Bacchetta

and Battaglia

. (2014). Tr. 1 cells and the counter-regulation of immunity: natural mechanisms and therapeutic applications. Curr Top Microbiol Immunol, 380:39–68.

48.

Roncarolo

, Battaglia

and Gregori

. (2003). The role of interleukin 10 in the control of autoimmunity. J Autoimmun, 20:269–272.

Enhancement of Graft-Versus-Host Disease Control Efficacy by Adoptive Transfer of Type 1 Regulatory T Cells in Bone Marrow Transplant Model

Abstract

Introduction

Materials and Methods

Mice

Tr1 and Foxp3+ Treg cell generation in vitro

Bone marrow transplantation and GVHD induction and scoring

Cultured Tr1 cells were transferred during BM transplantation

Clinico-histopathology of GVHD

Flow cytometric analysis

Real-time reverse transcription–polymerase chain reaction

Quantitation of cytokine levels by enzyme-linked immunosorbent assay

Statistical analyses

Results

Immunophenotypes of culture-expanded Tr1 cells in vitro

Tr1 cells could maintain stable status under the inflammatory condition

Adoptive transfer of Tr1 cells ameliorates clinical manifestations of GVHD

Adoptive transfer of Tr1 cells modulates acute GVHD-related Th1 and Th2 responses

The Treg/Th17 ratio is increased by adoptive transfer of Tr1 cells

Some adoptive-transferred Tr1 cells differentiated into CD4+Foxp3+ Treg cells after BMT

Discussion

Footnotes

Acknowledgments

Author Disclosure Statement

References

Tr1 and Foxp3⁺ Treg cell generation in vitro

Some adoptive-transferred Tr1 cells differentiated into CD4⁺Foxp3⁺ Treg cells after BMT