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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
Eur J Immunol. Author manuscript; available in PMC 2015 June 1.
Published in final edited form as:
PMCID: PMC4048633
NIHMSID: NIHMS584942

TGF-β-producing regulatory B cells induce regulatory T cells and promote transplantation tolerance

Summary

Regulatory B cells (Bregs) have been shown to play a critical role in immune homeostasis and in autoimmunity models. We have recently demonstrated that combined anti-TIM-1 and anti-CD45RB antibody treatment results in tolerance to full MHC-mismatched islet allografts in mice by generating Bregs that are necessary for tolerance. Bregs are antigen-specific and are capable of transferring tolerance to untreated, transplanted animals. Here we demonstrate that adoptively transferred Bregs require the presence of Tregs to establish tolerance, and that adoptive transfer of Bregs increases the number of Tregs. Interaction with Bregs in vivo induces significantly more Foxp3 expression in CD4+CD25− T cells than with naive B cells. We also show that Bregs express the TGF-β associated latency-associated peptide (LAP) and that Breg-mediated graft prolongation post-adoptive transfer is abrogated by neutralization of TGF-β activity. Regulatory B cells, like regulatory T cells, demonstrate preferential expression of both CCR6 and CXCR3. Collectively, these findings suggest that in this model of antibody-induced transplantation tolerance, Bregs promote graft survival by promoting Treg development, possibly via TGF-β production.

Keywords: regulatory T cells, TGF-β, regulatory B cells, tolerance, transplant

Introduction

B cells play a central role in humoral immunity, but also promote naive T cell differentiation into Th1/Th2 and Tmem subsets, function as antigen presenting cells, produce cytokines, and provide costimulatory signals [13]. The experimental autoimmune encephalomyelitis mouse model provided early evidence that B cells also serve a protective role in maintaining self-tolerance, a property classically attributed to regulatory T cells [4]. The absence of B cells exacerbated disease and delayed the appearance of regulatory T cells in the central nervous system [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 [79].

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-TIM-1 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 regulatory T cell population. Ding et al. demonstrated that B cell depletion diminishes regulatory T cell induction by anti-TIM-1 antibody treatment, again suggesting an interaction between regulatory T 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 regulatory T 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 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 differentiation, TGF-β modulates T cell activation [12]. TGF-β also promotes Treg development while inhibiting Th1 and Th2 development [13, 14]. Based on these findings we hypothesized that Bregs could contribute to regulatory T cell induction by producing TGF-β.

Results

Breg-mediated Treg expansion is necessary for tolerance induction

We have previously demonstrated that dual Ab treatment (anti-CD45RB plus anti-TIM-1 antibodies) of islet transplant recipients significantly expands the Treg population, and Treg depletion with anti-CD25 antibody (PC61) abrogates this Breg-dependent transplant tolerance [8]. These findings could result from the antibodies directly inducing Tregs or from the Bregs inducing Tregs. We therefore examined whether Bregs alone induce Tregs using an adoptive transfer model. B cells purified from islet allograft recipients treated with anti-CD45RB plus anti-TIM-1 exhibit regulatory activity starting at day 14 post-transplant and beyond; we refer B cells from such treated recipients as Bregs. Bregs, purified total B cells, from long-term survivors were adoptively transferred to B cell-deficient (µMT−/−B6) recipients grafted with BALB/c islet allografts on the same day. Long-term graft survivors (LTS) are wild-type 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 Bregs from LTS mice confers indefinite graft survival (>100 days) to grafted µMT−/−B6 recipients, while transfer of naive B cells yields no prolongation (Figure 1A, p<0.05). Furthermore, there was a statistically significant increase in the absolute number of Tregs in the recipient spleens after Breg adoptive transfer, even in the absence of antibody treatment (Figure 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 (supplemental figure 1). This suggests that, in the presence of antigen, Bregs are able to modulate an increase in Tregs.

Figure 1
Tregs are necessary for graft survival prolongation by adoptive transfer of Bregs

These data suggest that regulatory B cells may promote tolerance indirectly through the induction of regulatory T cells. We hypothesized that the adoptive transfer of LTS Bregs would not prolong graft survival in Treg-depleted recipients. To test this possibility directly, we depleted CD25+ Tregs by pre-treatment of recipients with anti-CD25 antibody prior to Breg transfer. Anti-CD25 depletes existing Tregs 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 Bregs may suppress alloreactivity indirectly through Tregs (Figure 1A).

TGF-β-producing Bregs induce Foxp3 expression in CD4+CD25− T cells

Bregs could increase Treg numbers by expanding existing Treg populations or converting naive Foxp3− T cells into Foxp3+ Tregs. To distinguish between these alternatives, naive CD4+Foxp3−GFP- T cells were FACS sorted (supplemental figure 2) and adoptively co-transferred with Bregs 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 co-transferred with regulatory B cells compared with naive B cells (1.86-fold with Breg transfer versus naive B cell transfer, p=0.055, Figure 2). Since the starting population of adoptively transferred cells were Foxp3−, we conclude that the Tregs are induced Tregs, or iTregs. In the absence of any B cell transfer, there was no significant upregulation of Foxp3 (Figure 2, right). Both draining and non-draining lymph nodes were also examined but did not demonstrate significant Foxp3 induction.

Figure 2
Regulatory B cells induce Foxp3+ T cells in vivo

We next determined whether these iTregs were functional using a standard in vitro suppression assay. CD4+CD25+ induced Tregs from mice receiving a co-transfer of Bregs 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. Regulatory T cells were cultured with CFSE-labeled responder T cells at a 1:1 ratio. We observed that CD4+CD25+ iTregs suppressed T cell proliferation as well as natural Tregs purified from a naive C57BL/6 (supplemental Figure 3). We conclude that Tregs induced in vivo upon co-transfer with Bregs are functional suppressor T cells.

Based on their ability to induce regulatory T cells in this model, we hypothesized that Bregs contribute to Treg expansion by secreting TGF-β. TGF-β is critical for the induction of Tregs [13, 15], and some studies show that TGF-β may have an important role in Breg control of autoimmunity [1618]. 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 {Andersson, 2008 #1149; Nakamura, 2004 #2100}. We found that mice undergoing dual antibody treatment with islet transplantation had a significant increase in LAP+ B cells (Figures 3A & B) on day 14 post-transplant compared to naive mice (p<0.0001), supporting the hypothesis that dual antibody treatment induces TGF-β producing Bregs. Dual antibody treatment alone, without transplant, significantly elevates the percentage of LAP+ B cells, while transplant alone, without dual antibody treatment, does not (Figures 3A & 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.

Figure 3Figure 3
TIM-1+ B cells are enriched for LAP+IL-10+ B cells

We have previously demonstrated that this dual antibody treatment generates TIM-1+ regulatory B cells. We examined B cells from naive and transplanted animals for LAP and TIM-1 expression (Figure 3C, left). A significantly higher percentage of B cells from grafted, antibody-treated animals co-express TIM-1 and LAP versus naive animals (Figure 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 regulatory B cells. IL-10 is upregulated in B cells upon transplantation and dual antibody treatment (Figure 3E). Upon examination of regulatory B cells, IL-10 expression was significantly elevated on TIM-1+LAP+ B cells compared to TIM-1−LAP− B cells (Figure 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+ regulatory B cells are generated upon transplantation and dual antibody treatment.

We examined whether regulatory B and T cells express similar chemokine receptors. CCR6 and CXCR3 have been found to be critical for Treg function, and we examined Bregs for expression of these molecules [1921]. We found, as others have reported, that among CD4+ T cells, CCR6 and CXCR3 were preferentially expressed on Foxp3+ cells compared to 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) (Figures 4A & B). Dual antibody treatment significantly increased the percentage of CCR6+ cells in both Foxp3+ Tregs 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 to non-regulatory B cells (gated as TIM-1− B), a significantly higher percentage of Bregs expressed CCR6 and CXCR3 (Figures 4A & 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 mean fluorescence intensity (MFI), CCR6 expression on TIM-1+ Bregs 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 function, may be necessary for Breg function.

Figure 4
TIM-1+ regulatory B cells exhibit higher expression of CCR6 and CXCR3 than TIM-1− B cells, in both naive and transplanted animals

Breg-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 (Figure 5A). Furthermore, recipients receiving LTS B cells plus anti-TGF-β antibody promptly rejected alloislets, when they would ordinarily accept them indefinitely (Figure 5B; MST 13 days for LTS B cells with anti-TGF-β antibody versus MST >100 days for LTS B cells alone). Anti-TGF-β antibody alone does not deplete regulatory T cells but does diminish the increase in Tregs induced by dual antibody treatment (Figure 5C).

Figure 5
Blocking TGF-β activity abrogates Breg activity

Discussion

We have identified a population of regulatory B cells that induce tolerance indirectly through Treg induction, possibly via production of TGF-β, the potent pleiotropic cytokine necessary for Treg induction and function [22]. Although attempts by our group at more definitive localization of the source of Treg 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 regulatory B cell population increases the number of Tregs in the recipient and neutralizing anti-TGF-β antibody blocks Treg induction by Bregs. To further elucidate this issue, we are currently generating B cell-specific TGF-β-deficient mice using the cre-lox system. One could also use transgenic mice expressing a dominant-negative TGF-β type II receptor which is unable to respond to TGF-β signals. By adoptive transfer of regulatory B cells to the dnTGFBRII mouse, one could determine whether Treg 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 Treg induction by Bregs in a transplant setting, other groups have reported TGF-β-producing B cells that induce Tregs in vitro or in autoimmune models. Shah et al. demonstrated that resting B cells are able to expand Tregs in vitro, but this capacity decreases upon activation of B cells, which they speculate is the result of decreasing TGF-β3 [23]. In contrast, we observe in vivo that activated regulatory B cells promote increased Treg conversion compared to 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 co-culture with regulatory B cells could induce Foxp3 expression upon activation by anti-CD3 and anti-CD28 in a TGF-β-dependent manner [24]. More recently, the same group has demonstrated co-localization of Bregs and Tregs histologically [25], consistent with our findings that Bregs and Tregs both express CCR6 and CXCR3.

In vivo, the absence of B cells results in a lower percentage of Foxp3+ regulatory T cells compared to wild-type animals [26]. Shah and Qiao report that naive B cells have the capacity to expand regulatory T cells. Thus it is not unexpected that adoptive transfer of either naive B cells or regulatory B cells are able to induce Foxp3 expression to some level in conventional T cells. We hypothesize that higher levels of TGF-β expression in regulatory B 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 (Figure 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 function, our results demonstrate that TGF-β may be equally important in the function of Bregs in transplantation. IL-10 production by Bregs also suppresses T cell-mediated immune responses [3, 5, 7, 18, 27], altering Th1/Th2 proportions, and decreasing pro-inflammatory cytokine production. Carter et al. demonstrated that in the absence of Bregs or when Bregs are IL-10-deficient, there is a significant drop in Foxp3+ Tregs and an increase in Th1/Th17 cells. We will examine whether IL-10-deficient Bregs are deficient in TGF-β production. Breg production of IL-10 maintains the balance between Tregs and Th1/Th17 cells and may explain the increase in Foxp3 upon adoptive transfer of naive B cells (Figure 2) [28]. However, in their model, Bregs are able to suppress autoimmunity in the absence of Tregs, while Tregs 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 Th2 (IL-4 and IL-10) cytokine production and increased Th1 (IFN-γ) cytokine production. However, TGF-β blocks Th1 differentiation by decreasing IL-12Rβ2 and Tbet expression [12, 29, 30], 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-1Δmucin mutant mice, deficient in the mucin domain of TIM-1, demonstrate that the TIM-1 protein plays a critical role in Breg function, while the TIM-1-deficient and full-length TIM-1 transgenic overexpression animals demonstrate no significant phenotype [31, 32]. TIM-1Δmucin mutant animals initially appear normal but gradually exhibit a Breg IL-10 production defect at around 10 months of age. Consistent with the proposed role of Bregs, the absence of TIM-1Δmucin 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 [33]. This complex has been reported on Foxp3+ Tregs, 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 contact-dependent manner.

While Bregs have been demonstrated in other models involving inflammation [3, 27, 28], it remains unclear whether regulatory B cells exist in normal mice. Furthermore, we continue to compare TIM-1+ B cell population with other reported regulatory B 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

Wild-type 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 hundred islets were transplanted into the renal sub-capsular space of diabetic recipients. A functioning graft was defined as a non-fasting blood glucose level < 200 mg/dl and rejection was diagnosed at blood glucose of > 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 [34] as previously described.

Immunotherapy and adoptive transfer

Recipient mice received 100 µg anti-mouse CD45RB (Bio × 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 × 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 × cell) on days 0, 2, 4, 6, and 8 post-transplant. Anti-CD20 (5D2 from Genentech) treatment is a day-8 iv dose at 10 mg/kg, then 5 mg/kg ip 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%. 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 iv injection on day 0. On day -14, B6.RAG animals are grafted with BALB/c islets under the kidney capsule. To generate Bregs, 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 Bregs 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. 340k CFSE-labeled T cell responders were cultured with either an additional 340k unlabeled CD45.1 congenic CD4+ T cells, 340k unlabeled natural Tregs, or 340k unlabeled induced Tregs. Wells are stimulated with 0.7uL anti-CD3 / anti-CD28 beads (Invitrogen). Regulatory T cells are by magnetic column (Miltenyi Regulatory T cell kit). On day four, cells were analyzed by flow cytometry for proliferation.

Flow cytometry

Single-cell suspensions were recovered from spleens and lymph nodes by passing through a 70 µm nylon mesh. Erythrocytes 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% FBS in 96-well plates with the following fluorochrome-tagged 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.).

Cell stimulation

Single cell suspensions were stimulated in Complete Medium (RPMI 1640 containing 10% fetal bovine serum (HyClone FetalClone III, Thermo Scientific), 50 M 2-mercaptoethanol (ACROS Organics), 1mM sodium pyruvate, 1× MEM eagle non essential amino acids, 2mM L-glutamine, 100IU mL−1 Penicillin, and 100 g mL−1 Streptomycin, all from MP Biomedicals) with PMA (50 ng mL−1, Sigma), ionomycin (1 g mL−1, Sigma), monensin (GolgiStop; 4 g mL−1, BD), and either with or without LPS (10 g mL−1 Escherichia coli serotype 0111: B4, Sigma) in 6 well tissue culture treated plates for 5 hours 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.

Supplementary Material

01

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.

Abbreviations

Breg
regulatory B cell
Treg
regulatory T cell
LAP
latency-associated peptide
B6
C57BL/6
LTS
long-term graft survivors.

Footnotes

Conflict of interest

The authors of this manuscript have no conflicts of interest to disclose as described by the European Journal of Immunology.

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