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DOI: 10.1002/eji.201344062

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TGF-β-producing regulatory B cells induce regulatory T cells and promote transplantation tolerance Kang Mi Lee1 , Ryan T Stott1 , Gaoping Zhao1,2 , Julie SooHoo1 , Wei Xiong1,2 , Moh Moh Lian1 , Lindsey Fitzgerald1 , Shuai Shi1 , Elsie Akrawi1 , Ji Lei1 , Shaoping Deng1,2 , Heidi Yeh1 , James F Markmann1 and James I Kim1 1

Transplantation Unit, Department of Surgery, Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA 2 Department of Surgery, Sichuan Provincial People’s Hospital and Sichuan Academy of Medical Sciences, Chengdu, Sichuan Province, China Regulatory B (Breg) cells have been shown to play a critical role in immune homeostasis and in autoimmunity models. We have recently demonstrated that combined antiT cell immunoglobulin domain and mucin domain-1 and anti-CD45RB antibody treatment results in tolerance to full MHC-mismatched islet allografts in mice by generating Breg cells that are necessary for tolerance. Breg cells are antigen-specific and are capable of transferring tolerance to untreated, transplanted animals. Here, we demonstrate that adoptively transferred Breg cells require the presence of regulatory T (Treg) cells to establish tolerance, and that adoptive transfer of Breg cells increases the number of Treg cells. Interaction with Breg cells in vivo induces significantly more Foxp3 expression in CD4+ CD25− T cells than with naive B cells. We also show that Breg cells express the TGF-β associated latency-associated peptide and that Breg-cell mediated graft prolongation post-adoptive transfer is abrogated by neutralization of TGF-β activity. Breg cells, like Treg cells, demonstrate preferential expression of both C-C chemokine receptor 6 and CXCR3. Collectively, these findings suggest that in this model of antibody-induced transplantation tolerance, Breg cells promote graft survival by promoting Treg-cell development, possibly via TGF-β production.

Keywords: Breg cells



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TGF-β

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Transplant

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Treg cells

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Tolerance

Additional supporting information may be found in the online version of this article at the publisher’s web-site

Introduction B cells play a central role in humoral immunity, but also promote naive T-cell differentiation into Th1/Th2 and Tmem cell subsets, function as antigen presenting cells, produce cytokines, and provide costimulatory signals [1–3]. The experimental autoimmune encephalomyelitis mouse model provided early evidence that B cells also serve a protective role in maintaining self-

Correspondence: Prof. James F Markmann e-mail: [email protected]  C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

tolerance, a property classically attributed to regulatory T (Treg) cells [4]. The absence of B cells exacerbated disease and delayed the appearance of Treg cells in the CNS [5, 6]. Adoptive transfer of IL-10-producing B cells to B-cell-deficient animals ameliorated disease severity [5]. In the transplant setting, adoptively transferred B cells protect against graft versus host disease as well as prolong islet allograft survival [7–9]. In 2007, we first reported a model of transplantation tolerance that is B-cell-dependent using cardiac allograft recipients treated with anti-CD45RB antibody [10]. We recently extended these findings to islet allograft recipients treated with both anti-CD45RB and anti-T cell immunoglobulin domain and mucin domain-1 (TIM-1) www.eji-journal.eu

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antibody [8, 10]. Using B cell-deficient recipients or depleting B cells with anti-CD20 antibody abrogates tolerance induced by anti-CD45RB with anti-TIM-1 “dual antibody” treatment. Islet allograft tolerance is conferred to naive recipients by adoptive transfer of B cells from grafted animals tolerized by dual antibody treatment [8]. Dual antibody treatment also significantly expands the recipient Treg-cell population. Ding et al. demonstrated that B-cell depletion diminishes Treg-cell induction by anti-TIM-1 antibody treatment, again suggesting an interaction between Treg and B cells [7]. In an autoimmune model, Mann et al. have demonstrated the absence of B cells results in a delay in the recruitment of Treg cells to the site of inflammation [6]. To probe this interaction further in a model of transplant tolerance, we sought to identify soluble factors produced by B cells that might explain their Treg-cell inducing activity. TGF-β promotes T-cell survival by inhibiting activation-induced cell death and blocks T-cell proliferation by inhibiting IL-2 production [11, 12]. Through its effects on T-helper (Th)-cell differentiation, TGF-β modulates T-cell activation [12]. TGF-β also promotes Treg-cell development while inhibiting Th1- and Th2-cell development [13, 14]. Based on these findings, we hypothesized that regulatory B (Breg) cells could contribute to regulatory T-cell induction by producing TGF-β.

Results Breg-cell-mediated Treg-cell expansion is necessary for tolerance induction We have previously demonstrated that dual Ab treatment (antiCD45RB plus anti-TIM-1 antibodies) of islet transplant recipients significantly expands the Treg-cell population, and Treg-cell depletion with anti-CD25 antibody (PC61) abrogates this Breg cell dependent transplant tolerance [8]. These findings could result from the antibodies directly inducing Treg cells or from the Breg cells inducing Treg cells. We therefore examined whether Breg cells alone induce Treg cells using an adoptive transfer model. B cells purified from islet allograft recipients treated with antiCD45RB plus anti-TIM-1 exhibit regulatory activity starting at day 14 post-transplant and beyond; we refer to B cells from such treated recipients as Breg cells. Breg cells, purified total B cells, from long-term survivors were adoptively transferred to B celldeficient (μMT−/− B6) recipients grafted with BALB/c islet allografts on the same day. Long-term graft survivors (LTS) are WT C57BL/6 recipients of BALB/c islet allografts, which have survived >100 days following dual anti-CD45RB / anti-TIM-1 antibody treatment. Recipients of adoptively transferred B cells from LTS did not receive any additional treatment after B cell transfer. Adoptive transfer of Breg cells from LTS mice confers indefinite graft survival (>100 days) to grafted μMT−/− B6 recipients, while transfer of naive B cells yields no prolongation (Fig. 1A, p < 0.05). Furthermore, there was a statistically significant increase in the absolute number of Treg cells in the recipient spleens after Breg-cell adoptive transfer, even in the absence of  C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Figure 1. Treg cells are necessary for graft survival prolongation by adoptive transfer of Breg cells. Total B cells are enriched from C57BL/6 recipients whose islet allografts have survived longer than 100 days (LTS, long-term survivors) after anti-CD45RB/anti-TIM-1 antibody treatment. These LTS B cells are adoptively transferred to grafted B cell-deficient μMT−/− B6 recipients; grafted recipients do not receive any additional anti-CD45RB / anti-TIM-1 antibody treatment. Naive B cells are from unmanipulated C57BL/6. (A) μMT−/− B6 recipients and μMT−/− B6 recipients receiving naive B cells rapidly reject islet allografts, while in contrast, most μMT−/− B6 recipients receiving LTS B cells maintain graft function long-term. Treg-cell depletion of μMT−/− B6 recipients plus LTS B cells results in rapid rejection of islet allograft (**p < 0.01). (B) μMT−/− B6 recipients were grafted and/or received adoptive transfer of enriched B cells. 14 days after adoptive transfer into μMT−/− B6 recipients, spleens are examined for CD4 and Foxp3 expression. Islet allograft alone, LTS B cell transfer alone, or islet allograft with transferred naive B cells does not significantly increase the number of Foxp3+ T cells. Islet allograft plus transferred LTS B cells significantly increases the absolute number of Foxp3+ T cells. Data are shown as mean + SEM of n = 3 samples per group and are pooled from three independent experiments. *p < 0.05, **p < 0.01. Right, CD4+ T cells are gated, and percentage of Foxp3+ T cells of the CD4+ T-cell population are plotted on the graph.

antibody treatment (Fig. 1B). Absolute number of splenocytes was significantly increased in grafted recipients receiving adoptive transfer of LTS B cells. Adoptive transfer of B cells or graft alone did not result in significant increase in spleen cell number (Supporting Information Fig. 1). This suggests that, in the presence of antigen, Breg cells are able to modulate an increase in Treg cells. These data suggest that Breg cells may promote tolerance indirectly through the induction of Treg cells. We hypothesized that www.eji-journal.eu

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Figure 2. Breg cells induce Foxp3+ T cells in vivo. Naive Foxp3− GFP− CD4+ T cells are FACS sorted and adoptively transferred to B6.RAG recipients bearing islet allografts along with either Breg cells or naive B cells. Starting purity of T cells is over 99% CD4+ Foxp3− GFP− (Supporting Information Fig. 2). Breg cells, total B cells purified from a recipient day 14 post islet transplant plus administration of both antibodies, were enriched by magnetic sorting. On day 14 post-adoptive transfer, CD4+ T cells are examined for Foxp3 expression by flow cytometry. Representative flow is shown on left. Transfer without B cells or cotransfer with Breg cells results in 1.86-fold increase in Foxp3+ T cells over co-transfer with naive B cells (right). Data are shown as mean + SEM of n = 6 naive B cell recipients, n = 9 regulatory B cell recipients, n = 4 no B cells. Flow are representative of one out of three independent experiments. p = 0.055; two-way ANOVA.

the adoptive transfer of LTS Breg cells would not prolong graft survival in Treg-cell-depleted recipients. To test this possibility directly, we depleted CD25+ Treg cells by pretreatment of recipients with anti-CD25 antibody prior to Breg transfer. Anti-CD25 depletes existing Treg cells at the time of depletion as well as cells that upregulate CD25 upon activation. Treg depletion completely abrogated Breg-mediated graft survival prolongation suggesting that Breg cells may suppress alloreactivity indirectly through Treg cells (Fig. 1A).

TGF-β-producing Breg cells induce Foxp3 expression in CD4+ CD25− T cells Breg cells could increase Treg-cell numbers by expanding existing Treg populations or converting naive Foxp3− T cells into Foxp3+ Treg cells. To distinguish between these alternatives, naive CD4+ Foxp3− GFP− T cells were FACS sorted (Supporting Information Fig. 2) and adoptively cotransferred with Breg cells or with naive B cells to islet allograft-bearing and otherwise untreated RAG recipients. Fourteen days after adoptive transfer, examination of the spleen for Foxp3 induction revealed significant upregulation in CD4+ T cells cotransferred with Breg cells compared  C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

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with naive B cells (1.86-fold with Breg-cell transfer versus naive B cell transfer, p = 0.055, Fig. 2). Since the starting population of adoptively transferred cells was Foxp3− , we conclude that the Treg cells are induced Treg cells, or iTreg cells. In the absence of any B cell transfer, there was no significant upregulation of Foxp3 (Fig. 2, right). Both draining and nondraining lymph nodes were also examined but did not demonstrate significant Foxp3 induction. We next determined whether these iTreg cells were functional using a standard in vitro suppression assay. CD4+ CD25+ induced Treg cells from mice receiving a cotransfer of Breg cells and CD4+ Foxp3− T cells were purified on day 14. CD4+ T cells from CD45.1 congenic B6 mice were CFSE labeled and used as responders. All wells were normalized for cell number. Treg cells were cultured with CFSE-labeled responder T cells at a 1:1 ratio. We observed that CD4+ CD25+ iTreg cells suppressed T-cell proliferation as well as natural Treg cells purified from a naive C57BL/6 (Supporting Information Fig. 3). We conclude that Treg cells induced in vivo upon cotransfer with Breg cells are functional suppressor T cells. Based on their ability to induce Treg cells in this model, we hypothesized that Breg cells contribute to Treg expansion by secreting TGF-β. TGF-β is critical for the induction of Treg cells [13, 15], and some studies show that TGF-β may have an important role in Breg-cell control of autoimmunity [16–18]. We examined B cells for expression of latency associated peptide (LAP), the C-terminal pro-region of TGF-β; bound to TGF-β, it serves as a marker for its expression and has been widely used in other systems for this purpose [19, 20]. We found that mice undergoing dual antibody treatment with islet transplantation had a significant increase in LAP+ B cells (Fig. 3A and B) on day 14 posttransplant compared with naive mice (p < 0.0001), supporting the hypothesis that dual antibody treatment induces TGF-β producing Breg cells. Dual antibody treatment alone, without transplant, significantly elevates the percentage of LAP+ B cells, while transplant alone, without dual antibody treatment, does not (Fig. 3A and B). We believe that in the absence of the inflammation surrounding the transplant procedure, antibody treatment alone is able to induce the highest levels of TGF-β. We have previously demonstrated that this dual antibody treatment generates TIM-1+ Breg cells. We examined B cells from naive and transplanted animals for LAP and TIM-1 expression (Fig. 3C, left). A significantly higher percentage of B cells from grafted, antibody-treated animals co-express TIM-1 and LAP versus naive animals (Fig. 3C, right). There is no significant increase in TIM-1+ LAP+ B cells in grafted recipients not receiving antibody treatment (data not shown). We next examined levels of IL-10 expression on TIM-1+ LAP+ B cells versus TIM-1− LAP− B cells. IL-10 is a hallmark cytokine of Breg cells. IL-10 is upregulated in B cells upon transplantation and dual antibody treatment (Fig. 3E). Upon examination of Breg cells, IL-10 expression was significantly elevated on TIM-1+ LAP+ B cells compared with TIM-1− LAP− B cells (Fig. 3D). The IL-10 expression MFI of TIM-1+ LAP− T cells lower than that of TIM-1+ LAP+ T cells (data not shown). These data demonstrate that TIM-1+ LAP+ IL-10+ www.eji-journal.eu

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Figure 3. TIM-1+ B cells are enriched for LAP+ IL-10+ B cells. C57BL/6 recipients received BALB/c islet allograft and are treated with standard course of anti-CD45RB and anti-TIM-1 antibodies. At 14 days posttransplant, mice are sacrificed and B cells are purified from spleen and cultured overnight either with or without LPS, PMA, and ionomycin. Stimulated cells are shown. Cells are stained for CD19, IL-10, and LAP expression. (A) B cells from grafted recipients treated with anti-TIM-1/anti-CD45RB antibodies produce significantly elevated levels of LAP compared with naive or grafted alone animals (p < 0.001 versus naive animals, and p < 0.001 versus grafted only animals; naive 3.9% ± 0.5, graft alone 4.8% ± 0.3, graft plus antibody treatment 7.5 ± 0.1). (B) Antibody treatment alone but not islet transplant alone is sufficient to increase LAP expression on B cells (p < 0.01, naive versus antibodies alone; ns, naive versus graft alone). Recipients are examined 2 weeks after antibody treatment or transplant. LAP+ B cells were analyzed for TIM-1 and IL-10 c-expression. (C) A significantly higher percentage of B cells from grafted and dual antibody-treated recipients were TIM-1+ LAP+ compared with naive animals (5.6% ± 1.1 versus 1.4% ± 0.08, respectively). (D) IL-10 expression is higher in LAP+ TIM-1+ Breg cells versus LAP−TIM-1− B cells (MFI is 697.7 ± 24.7 versus 325.7 ± 26, respectively; p < 0.01). Dotted lines are B220+ CD19+ LAP− TIM-1− gated cells, while thick lines are LAP+ TIM-1+ cells. (E) B cells upregulate IL-10 expression after islet allograft and dual antibody treatment. Lymphocytes are examined two weeks after transplant. (A-D) Data are shown as mean ± SEM of n = 3–7 animals per group and (A–E) are representative of one out of two independent experiments.

Breg cells are generated upon transplantation and dual antibody treatment. We examined whether Breg and Treg cells express similar chemokine receptors. C-C chemokine receptor 6 (CCR6) and CXCR3 have been found to be critical for Treg function, and we examined Breg cells for expression of these molecules [21–23]. We found, as others have reported, that among CD4+ T cells, CCR6 and CXCR3 were preferentially expressed on Foxp3+ cells compared with Foxp3− cells (13.56% ± 1.7% versus 1.7% ± 0.7%, respectively, for CCR6, and 25.9% ± 4.3% versus 13.6% ± 4.3% for CXCR3) (Fig. 4A and B). Dual antibody treatment significantly increased the percentage of CCR6+ cells in both Foxp3+ Treg cells and Foxp3− T cells, while dual antibody treatment significantly increased the percentage of CXCR3+ cells in only Foxp3+ T cells and not in Foxp3− T cells. Interestingly, compared with non-Breg cells (gated as TIM-1− B cells), a significantly higher percentage of Breg cells expressed  C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

CCR6 and CXCR3 (Fig. 4A and B). In contrast to CD4+ T cells, antibody treatment did not significantly affect CCR6 or CXCR3 expression on either B-cell population. Also, as determined by MFI, CCR6 expression on TIM-1+ Breg cells was significantly higher than TIM-1− B cells (765.9 ± 51 versus 477 ± 44.2, respectively; data not shown), as was CXCR3 expression (162 ± 22.5 versus 49 ± 3.2, data not shown). Thus, these chemokine receptors critical for Treg-cell function, may be necessary for Breg-cell function.

Breg-cell-mediated tolerance is dependent on TGF-β To demonstrate the functional relevance of TGF-β expression, we neutralized TGF-β activity with anti-TGF-β antibody. The addition of anti-TGF-β (every other day for 8 days starting on day 0) to dual antibody treatment effectively impeded tolerance induction and resulted in prompt rejection (Fig. 5A). Furthermore, recipients receiving LTS B cells plus anti-TGF-β antibody promptly

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Figure 4. TIM-1+ Breg cells exhibit higher expression of CCR6 and CXCR3 than TIM-1− B cells, in both naive and transplanted animals. C57BL/6 recipients received BALB/c islet allograft and are treated with standard course of anti-CD45RB and anti-TIM-1 antibodies. On day 14, splenocytes from naive animals and from islet grafted / dual antibody-treated recipients were stained for CD4, B220, Foxp3, CCR6, CXCR3, and TIM-1. As labeled on the x-axis, cells were gated on CD4+ Foxp3+ or Foxp3− T cells, or B220+ TIM-1+ or TIM-1− B cells. (A) 14.9% ± 5.3% of TIM-1+ Breg cells expressed CCR6 while 0.9% ± 0.4% TIM-1− B cells were CCR6+ (p < 0.01). The percentage of CCR6 expression increased on CD4+ T cells, both Foxp3– and Foxp3− , upon treatment with anti-CD45RB and anti-TIM-1 (p < 0.01). The percentage of CCR6 expression did not change significantly in B cells. (B) 17.3% ± 2.0% of TIM-1+ Breg cells expressed CXCR3 while 1.1% ± 0.2% TIM-1− B cells were CXCR3+ (p < 0.01). The percentage of CXCR3 expression increased on CD4+ Foxp3+ Treg cells upon treatment with anti-CD45RB and anti-TIM-1, but not in any other population examined. (A,B) Data are shown as mean + SEM of n = 3 to 4 animals per group and are representative of one out of two independent experiments.

rejected alloislets, when they would ordinarily accept them indefinitely (Fig. 5B; MST 13 days for LTS B cells with anti-TGF-β antibody versus MST >100 days for LTS B cells alone). Anti-TGFβ antibody alone does not deplete Treg cells but does diminish the increase in Treg cells induced by dual antibody treatment (Fig. 5C).

Discussion We have identified a population of Breg cells that induce tolerance indirectly through Treg-cell induction, possibly via pro-

duction of TGF-β, the potent pleiotropic cytokine necessary for Treg-cell induction and function [24]. Although attempts by our group at more definitive localization of the source of Treg-cell promoting TGF-β production to B cells by adoptive transfer of TGF-β−/− B cells to B-cell-deficient hosts have been thwarted by the failure of homozygous deficient fetuses on a B6 background to survive to birth, adoptive transfer of our Breg cell population increases the number of Treg cells in the recipient and neutralizing anti-TGF-β antibody blocks Treg-cell induction by Breg cells. To further elucidate this issue, we are currently generating B-cellspecific TGF-β-deficient mice using the cre-lox system. One could also use transgenic mice expressing a dominant-negative TGF-β

Figure 5. Blocking TGF-β activity abrogates Breg activity. (A) C57BL/6 recipients received a BALB/c islet allograft plus anti-TIM-1 / anti-CD45RB treatment. Additional treatment of grafted recipients with anti-TGF-β antibody abrogates graft survival. p < 0.0001 versus dual treatment. (B) μMT−/− B6 recipients received a BALB/c islet allograft plus adoptive transfer of tolerant LTS B cells without anti-CD45RB/anti-TIM-1 treatment. p < 0.0001 versus LTS B cell transfer. (C) C57BL/6 mice were injected with antibodies as described on the x-axis. Splenocytes were examined on day 14 after the start of injection. Coinjection with anti-TGF-β antibody blocks Foxp3 upregulation mediated by anti-CD45RB antibody alone or in combination with anti-TIM-1. Data are shown as mean ± SEM of n = 3 to 10 animals per group and are representative of one out of two independent experiments.

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type II receptor which is unable to respond to TGF-β signals. By adoptive transfer of Breg cells to the dnTGFBRII mouse, one could determine whether Treg-cell induction is critical to graft survival. Using a bead-based assay to measure cytokines, we attempted to measure secreted TGF-β1 in vitro by stimulated B cells, however, measurements gave unreliable results. While this is the first demonstration of TGF-β-dependent Tregcell induction by Breg cells in a transplant setting, other groups have reported TGF-β-producing B cells that induce Treg cells in vitro or in autoimmune models. Shah et al. demonstrated that resting B cells are able to expand Treg cells in vitro, but this capacity decreases upon activation of B cells, which they speculate is the result of decreasing TGF-β3 [25]. In contrast, we observe in vivo that activated Breg cells promote increased Treg-cell conversion compared with resting B cells. Interestingly, they demonstrate that TGF-β1 is increased in B cells upon activation, which is consistent with our findings. Starting with sorted Foxp3− T cells in an allergic airway disease model, Singh et al. demonstrated in vitro that coculture with Breg cells could induce Foxp3 expression upon activation by anti-CD3 and anti-CD28 in a TGF-β-dependent manner [26]. More recently, the same group has demonstrated colocalization of Breg cells and Treg cells histologically [27], consistent with our findings that Breg cells and Treg cells both express CCR6 and CXCR3. In vivo, the absence of B cells results in a lower percentage of Foxp3+ Treg cells compared with WT animals [28]. Shah and Qiao report that naive B cells have the capacity to expand Treg cells. Thus, it is not unexpected that adoptive transfer of either naive B cells or Breg cells is able to induce Foxp3 expression to some level in conventional T cells. We hypothesize that higher levels of TGF-β expression in Breg cells versus naive B cells results in higher induction of Foxp3. However, despite their ability to induce some level of Foxp3 expression, adoptive transfer of naive B cells to transplant recipients does not prolong graft survival (Fig. 1A). Naive B cells lack sufficient antigen-specificity and IL-10 production to regulate the alloimmune response [8]. While IL-10 expression is considered central to the mechanism of Breg-cell function, our results demonstrate that TGF-β may be equally important in the function of Breg cells in transplantation. IL-10 production by Breg cells also suppresses T-cell-mediated immune responses [3, 5, 7, 18, 29], altering Th1/Th2 cell proportions, and decreasing pro-inflammatory cytokine production. Carter et al. demonstrated that in the absence of Breg cells or when Breg cells are IL-10-deficient, there is a significant drop in Foxp3+ Treg cells and an increase in Th1/Th17 cells. We will examine whether IL-10-deficient Breg cells are deficient in TGF-β production. Breg-cell production of IL-10 maintains the balance between Treg cells and Th1/Th17 cells and may explain the increase in Foxp3 upon adoptive transfer of naive B cells (Fig. 2) [30]. However, in their model, Breg cells are able to suppress autoimmunity in the absence of Treg cells, while Treg cells are necessary in our transplant model. Ding et al. demonstrated that in the absence of B cells or with B cells lacking IL-4 responsiveness, T cells exhibit decreased Th2cell (IL-4 and IL-10) cytokine production and increased Th1-cell  C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

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(IFN-γ) cytokine production. However, TGF-β blocks Th1 differentiation by decreasing IL-12Rβ2 and Tbet expression [12, 31, 32], and we propose that this alteration in Th1/Th2 profile may also be the result of the absence of TGF-β production which suppresses Th1 cytokine production. TIM-1mucin mutant mice, deficient in the mucin domain of TIM-1, demonstrate that the TIM-1 protein plays a critical role in Breg-cell function, while the TIM-1-deficient and full-length TIM-1 transgenic overexpression animals demonstrate no significant phenotype [33, 34]. TIM-1mucin mutant animals initially appear normal but gradually exhibit a Breg-cell IL-10 production defect at around 10 months of age. Consistent with the proposed role of Breg cells, the absence of TIM-1mucin and diminished IL-10 production results in autoimmune disease and hyperactivated T cells. In addition to secreted TGF-β, the membrane-bound LAP/ TGF-β complex exerts suppressive function as well [35]. This complex has been reported on Foxp3+ Treg cells, conventional CD4+ T cells, and CD8+ T cells, and regulatory function has been ascribed to all of them. It is hypothesized that the surface complex can stimulate TGF-β signaling in the target cell in a contactdependent manner. While Breg cells have been demonstrated in other models involving inflammation [3, 29, 30], it remains unclear whether Breg cells exist in normal mice. Furthermore, we continue to compare TIM-1+ B-cell population with other reported Breg-cell populations and subsets, to understand cytokine production by B cells and B-cell function in vivo. Lineage relationships between TIM-1+ B cells, B10, B1a, marginal zone, and other transitional B cells are likely. However, very few overlapping characteristics have been identified other than IL-10.

Materials and methods Mice WT C57BL/6 (B6, H-2b ), B cell deficient C57BL/6 (μMT−/− B6, H-2b ), and BALB/c (H-2d ) mice were purchased from the Jackson Laboratory. Foxp3− GFP (C57BL/6 background) were provided by Mohamed Oukka [15]. All mice were housed under specific pathogen-free conditions in the animal facility of Massachusetts General Hospital. All protocols detailed below were performed following the principles of laboratory animal care and approved by the Institutional Committee for Research Animal Care.

Transplantation Diabetes was induced in C57BL/6 mice with streptozotocin (200 mg/kg i.p.; Sigma-Aldrich) and was defined as blood glucose levels > 300 mg/dL for at least 2 consecutive days. Islets were isolated by collagenase digestion (liberaseRI, Roche) and then separated by discontinuous Euroficoll gradients (densities: 1.11; 1.096; 1.066) from the pancreatic digest. Four to five www.eji-journal.eu

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hundred islets were transplanted into the renal subcapsular space of diabetic recipients. A functioning graft was defined as a nonfasting blood glucose level 200 mg/dL for at least 2 consecutive days. Mice were monitored at least twice per week by measuring blood glucose until the mice were sacrificed. Nephrectomy was performed to rule out recovery of native islet function in mice that remained normoglycemic after 100 days. Skin grafts were transplanted to mice according to the technique of Billingham and Medawar [36] as previously described.

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were lysed with ammonium chloride buffer and collected cells were washed and counted using a hemocytometer. One million cells were suspended in PBS containing 0.1% azide and 2% fetal bovine serum in 96-well plates with the following fluorochrometagged antibodies CD3, CD4, CD19, B220, Foxp3, IL-10, CXCR3, and CCR6. Antibodies were purchased from eBioscience. Anti-LAP Ab is purchased Biolegend. Intracellular Foxp3 and IL-10 in lymphocytes were measured using a fixation/permeabilization staining kit (eBioscience). All samples were run on an Accuri flow cytometer (Accuri cytometers Inc.) or LSRII (Becton Dickinson) and analyzed using Flow Jo analysis software (Tree Star Inc.).

Immunotherapy and adoptive transfer Recipient mice received 100 μg anti-mouse CD45RB (Bio X cell) i.p. on days 0, 1, 3, 5, and 7 following transplantation. Recipient mice may also receive 500 μg anti-mouse TIM-1 (Bio X cell, RMT1–10) i.p. on day 1, and 300 μg on days 0 and 5 following transplantation. Anti-TGF-β-treated recipients were injected with 200 μg anti-TGF-β antibody (Bio X cell) on days 0, 2, 4, 6, and 8 post-transplant. Anti-CD20 (5D2 from Genentech) treatment is a day-8 i.v. dose at 10 mg/kg, then 5 mg/kg i.p. injection on day 0. Anti-CD25 (PC61) treatment is 250 μg on days 6 and 1. After 100 days of islet allograft survival by dual antibody treatment, B cells are enriched by CD90.2 magnetic bead depletion (Miltenyi, Germany). Purity of B cells is routinely over 90%. A total of 5 × 106 B cells are adoptive transferred i.v. by tail vein injection. For Breg-Treg Foxp3 induction experiment, naive CD4+ Foxp3− GFP− T cells are sorted by FACSAria (BD Biosciences), and each B6.RAG (Jackson Labs, ME) receives 4.5 × 106 by i.v. injection on day 0. On day 14, B6.RAG animals are grafted with BALB/c islets under the kidney capsule. To generate Breg cells, on day 14, C57BL/6 animals are injected i.p. with 20 × 106 irradiated BALB/c splenocytes and receive standard anti-CD45RB plus anti-TIM-1 dual antibody treatment. On day 0, B cells are magnetically sorted from splenocyte-injected mice or from naive mice. Grafted B6.RAG recipients receive GFP- T cells alone or plus either 12 × 106 Breg cells or naive B cells.

In vitro suppression assay Responder CD4+ T cells were purified from the spleen of a CD45.1 congenic and CFSE labeled. 340 k CFSE-labeled T-cell responders were cultured with either an additional 340 k unlabeled CD45.1 congenic CD4+ T cells, 340 k unlabeled natural Treg cells, or 340 k unlabeled induced Treg cells. Wells are stimulated with 0.7 μL anti-CD3 / anti-CD28 beads (Invitrogen). Treg cells are by magnetic column (Miltenyi Regulatory T-cell kit). On day 4, cells were analyzed by flow cytometry for proliferation.

Cell stimulation Single cell suspensions were stimulated in Complete Medium (RPMI 1640 containing 10% fetal bovine serum (HyClone FetalClone III, Thermo Scientific), 50 μM 2-mercaptoethanol (ACROS Organics), 1 mM sodium pyruvate, 1X MEM eagle nonessential amino acids, 2 mM L-glutamine, 100 IU/mL Penicillin, and 100 μg/mL Streptomycin, all from MP Biomedicals) with PMA (50 ng/mL, Sigma), ionomycin (1 μg/mL, Sigma), monensin (GolgiStop; 4 μg/mL, BD), and either with or without LPS (10 μg/mL Escherichia coli serotype 0111: B4, Sigma) in 6 well tissue culture treated plates for 5 h in a 37°C/5% CO2 incubator.

Statistical analysis Data were analyzed using GraphPad Prism (version 5, GraphPad Software). Graft survival between experimental groups was compared using Kaplan–Meier survival curves and Wilcoxon statistics. Foxp3− GFP experiment was analyzed using ANOVA. Other differences between experimental groups were analyzed using the Student’s t test. p values less than 0.05 were considered statistically significant.

Acknowledgments: This work was supported in part by NIH grant RO1AI057851-05 (JFM), K08-DK094965 (HY), and 5T32AI7529 (KML). We thank Alicia Johnson, PhD, for statistical analysis. We thank the MGH Flow Cytometry Core for cell sorting.

Conflict of interest: The authors declare no commercial or financial conflict of interest.

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Full correspondence: Prof. James Markmann, Transplantation Unit, Department of Surgery, Massachusetts General Hospital, Harvard Medical School 55 Fruit Street - 5 White, Boston, MA 02114 Fax: +1-617-643-4579 e-mail: [email protected]

in mammals. J. Exp. Biol. 1951. 28: 385–402.

Abbreviations: Breg: regulatory B cell · LAP: latency-associated peptide · LTS: long-term graft survivors · Th: T-helper.

 C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Received: 9/9/2013 Revised: 12/2/2014 Accepted: 27/3/2014 Accepted article online: 3/4/2014

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