Regulatory B cells: TIM‐1, transplant tolerance, and rejection

Abstract Regulatory B cells (Bregs) ameliorate autoimmune disease and prevent allograft rejection. Conversely, they hinder effective clearance of pathogens and malignancies. Breg activity is mainly attributed to IL‐10 expression, but also utilizes additional regulatory mechanisms such as TGF‐β, FasL, IL‐35, and TIGIT. Although Bregs are present in various subsets defined by phenotypic markers (including canonical B cell subsets), our understanding of Bregs has been limited by the lack of a broadly inclusive and specific phenotypic or transcriptional marker. TIM‐1, a broad marker for Bregs first identified in transplant models, plays a major role in Breg maintenance and induction. Here, we expand on the role of TIM‐1+ Bregs in immune tolerance and propose TIM‐1 as a unifying marker for Bregs that utilize various inhibitory mechanisms in addition to IL‐10. Further, this review provides an in‐depth assessment of our understanding of Bregs in transplantation as elucidated in murine models and clinical studies. These studies highlight the major contribution of Bregs in preventing allograft rejection, and their ability to serve as highly predictive biomarkers for clinical transplant outcomes.


| INTRODUC TI ON
B lineage cells uniquely produce antibodies -the sine qua non of humoral immunity. However, B (lineage) cells also play non-antibody-mediated roles that significantly impact the immune response through antigen presentation, T cell co-stimulation/co-inhibition, and cytokine secretion. B cells produce an array of pro-and anti-inflammatory cytokines (including TNFα, IFNγ, IL-4, IL-6, IL-10, IL-12, IL-17, and IL-35) that can profoundly influence both innate and adaptive immune responses. [1][2][3][4][5][6][7][8][9][10][11][12] Regulatory B cells (Bregs) expressing anti-inflammatory cytokines such as IL-10, potently downregulate the immune response, ameliorating autoimmune disease and allograft rejection, while limiting anti-tumor and infectious immunity. 2,3,7,9,10,13,14 In contrast, effector B cells (Beffs), which express pro-inflammatory cytokines, have the opposite effect. [15][16][17][18][19][20][21] While this review focuses on Bregs, the net modulatory effect of B cells on the immune response likely results from the balance of the opposing activities of both Bregs and Beffs. A better understanding of these subsets could lead to novel therapeutic approaches to either enhance or dampen the immune system. Despite significant advances, a number of aspects of Breg biology remain poorly understood. These include: the lack of a specific phenotypic or transcriptional marker; poor insight into their development (stochastic vs distinct lineage); effector function in vivo; and the relationship between various Breg subsets identified in the literature. For example, Bregs are still primarily identified by their expression of IL-10, their best studied suppressive cytokine, leading to a number of different phenotypic "subsets" whose function and lineal relationship to one another are unclear.
In the first part of this review, we provide a detailed assessment of these salient issues in murine models and suggest that TIM-1, first identified in transplantation, may represent a functional marker that helps unify Bregs with different phenotypes and mechanisms of action. Since most Bregs have phenotypes resembling those of B cells belonging to various canonical B cell subsets, we address the potential localization and in vivo intercellular interactions of Bregs with other immune cells.
Murine transplantation represents a good model for dissecting the immune response, immunoregulation, and tolerogenic mechanisms, since it parallels the human clinical setting where otherwise naïve animals receive a potent immune stimulus in the form of an allograft.
In the second portion of this review, we shift the focus to Bregs in clinical transplantation. Similar to murine models, we discuss attempts to identify human Bregs despite lack of a specific marker. However, the clinical setting raises issues not generally considered in animal models.
Thus, we not only review the evidence for a role for Bregs in transplantation, but also whether Breg numbers or activity can predict allograft outcomes. This would allow physicians to proactively monitor and individualize immunosuppressive therapy, for example, by identifying transplant recipients at low risk for rejection whose immunosuppression can be safely reduced or pre-emptively increasing immunosuppression in high-risk recipients to reduce future rejection episodes and premature allograft loss. Further, we address the effects of currently used immunosuppressive regimens on Bregs with the prospect of therapeutic manipulation of Bregs to promote immunological tolerance.

| The problem in defining Breg subsets and phenotype without a specific marker
There are no specific markers that definitively identify Bregs. As a result, many studies have utilized IL-10 expression as a surrogate marker since IL-10 was the first mechanism of Breg activity described and remains dominant in many models. 11,27 However, defining Bregs by their expression of IL-10 alone is also a "narrow" definition since it may ignore Bregs that utilize other mechanisms of action. The spleen is the largest reservoir for Bregs and is the focus of most murine Breg studies. 28 IL-10+ Bregs are rare, comprising approximately 1% of all B cells in naïve spleen. 11,28 However, they can expand up to 3%-5% of the total splenic B cell population after antigen challenge. 13  in varying frequency. 20,[29][30][31][32][33][34] However, the exact role and relative activity of these different Breg "subsets" remains completely unclear.
Until recently, B cell IL-10 expression was only identified by intracellular staining after in vitro mitogenic stimulation. 11 Since IL-10+ B cells could not be identified without prior in vitro stimulation and permeabilization, most studies assessing Breg function depended on adoptive transfer of freshly isolated B cell subsets found to be relatively enriched for IL-10 expression. For example, the CD1d + (MZ), CD21 hi CD23 hi CD24 hi (T2-MZP), and the unusual CD1d hi CD5 + ("B10") subsets are all enriched for IL-10 expression and can transfer IL-10-dependent amelioration of murine colitis, EAE, and SLE. 11,12,35 However, this approach confuses the frequency of Bregs within transferred populations, with the actual activity of IL-10+ B cells within a given subset. Although enriched, IL-10+ B cells still comprise a minority of cells (eg, 10%-15%) within each of these subsets.
Moreover, the IL-10+ B cells in each of these subsets comprise only 10%-20% of the total number of IL-10+ B cells in secondary lymphoid organs (SLO). Thus, most IL-10+ B cells (potential Bregs) are not included in studies of these individual subsets, and most transferred B cells are in fact, not Bregs. As a specific example, ~15% of CD5 + CD1d hi B cells express IL-10. However, this subset makes up only ~2% of splenic B cells and therefore encompasses only ~20% of all IL-10+ B cells. 2,13 While the majority of the IL-10+ B cells are contained within the remaining 98% of splenic B cells, their frequency is too low (~1%) to demonstrate Breg activity upon adoptive transfer.
We systematically addressed both the relative frequency of IL-10+ B cells within each B cell subset and identified the relative contribution of each subset to total B cell IL-10. 29 We found that IL-10 expression, albeit at lower levels, can consistently be identified without in vitro stimulation both by intracellular staining, and Not surprisingly, the frequency of IL-10 expression increased in every subset after the commonly used approach of in vitro simulation with LPS, PMA, and ionomycin (LPIM) for 5 hours (Table 1). 29 In most B cell subsets, the frequency of IL-10 expression increased 3to 5-fold; however, the rank order of IL-10 expression amongst the subsets was unchanged. 29   cells are regulatory and are antigen-specific. Such antigen-specificity has been corroborated in autoimmune models like EAE and CIA. 42,43 This also suggests that BCR-signaling is required to activate and/or expand relevant Breg clones. This notion is supported by the finding that TIM-1 + B cell expansion in response to anti-TIM-1 requires immunization. 2

| TIM-1 contributes to Breg induction and suppressor function
TIM-1 may be distinct amongst phenotypic markers, in that it plays an important functional role in Breg expansion and suppressor function.
As noted above, TIM-1 ligation with anti-TIM-1 (RMT1-10) induces Breg expansion in vivo. 2,36 Moreover, knock-in mice that express a mutant form of TIM-1 with a deletion of the mucin domain (TIM-1 Δmucin ), exhibit defects in both basal and induced IL-10+ Bregs. 44,45 Specifically, anti-TIM-1 binds to mutant TIM-1 but fails to induce IL-10+ Bregs. Gray and colleagues previously showed that apoptotic cells induce IL-10+ Bregs. 46 Phosphatidyl serine (PtdS), exposed on the surface of apoptotic cells is a natural ligand for TIM-1. PtdS binds to WT TIM-1 + B cells and induces IL-10 expression, but does not bind to, or induce IL-10, in WT TIM-1 -B cells or TIM-1 + B cells from TIM-1 Δmucin mice. 44,47 Thus, TIM-1 is the major PtdS receptor on B cells and promotes IL-10 expression ( Figure 1). Further, these data indicate that TIM-1 Δmucin is a loss-of-function mutant. As a consequence, these mice exhibit progressive spontaneous splenomegaly with age and more severe SLE when crossed onto a MRL-Fas lpr background. 45 Even young TIM-1 Δmucin mice exhibit more aggressive alloimmune responses. For example, TIM-1 Δmucin mice acutely reject single Class II MHC mismatched BM12 cardiac allografts that survive > 100 days in WT recipients. 44 Acute rejection is ameliorated by transfer of WT TIM-1 + but not TIM-1 -B cells. Thus, TIM-1 is not only a marker for IL-10+ Bregs but plays a key role in their development and induction.
This further suggests that Bregs may normally be maintained and expanded by sensing levels of apoptotic cells through TIM-1.

| TIM-1 is a broad regulator of Breg activity and identifies Bregs as major regulators of immune homeostasis
While

| Breg in vivo mechanisms of action, localization, and intercellular interactions
In both allograft and other models, Bregs skew Th differentia-

T cells and increased
IFNγ expression and proliferation by CD8 + T cells. 29 Bregs have also been shown to modulate the function of innate immune cells including monocytes, dendritic cells (DCs), neutrophils, and NK T cells.
Bregs predominantly inhibit innate cell secretion of inflammatory mediators such IL-12, IFNγ, TNFα, and nitric oxide that contribute to priming and skewing of the adaptive immune response. 5 suggest that Bregs act through direct cognate interactions, at least with CD4 + T cells. 2,3,9,11,35,42 However, Tr B cell maturation in the absence of conventional (MHCII and CD40-dependent) T:B interactions may not be entirely normal. 50,51 Further, Bregs could indirectly suppress T cell function by inhibiting antigen-presenting cell function, similar to Tregs. 52 The apparent antigen specificity of Bregs might result from a requirement for cell activation in close proximity to DCs involved in presenting antigen to responding T cells.
To directly examine the interactions between Bregs and other immune cells and identify their anatomical localization in spleen, we utilized 2-photon intravital microscopy. 29

| The IL-10/TNFα ratio as an indicator of Breg/ Beff balance
Given conflicting reports, we directly compared IL-10 expression in peripheral blood B cells from healthy individuals, and found that all major B cell subsets (eg, Tr, Naïve and Memory) express IL-10 at roughly similar frequencies (10%-15%). 61 However, we found that B cells within these same subsets co-express pro-inflammatory cytokines like TNFα, emphasizing the lack of a true Breg phenotype.

| The role of Bregs in clinical transplantation
Antibodies targeting the allograft can significantly impact allograft survival by mediating an acute form of rejection (antibody-mediated rejection) and contributing to chronic allograft rejection. 67,68 With the advent of Rituximab (humanized anti-CD20), attempts were made to reduce antibody-mediated components of rejection by preemptively depleting B cells in the peri-transplant period. Surprisingly, this led to a marked increase in acute T cell-mediated rejection (83%) in kidney transplant patients within the first three months posttransplantation. 69 In a second study, cardiac allograft recipients were randomized to receive Rituximab in an attempt to reduce cardiac allograft vasculopathy (CAV), which is thought to be antibody-mediated. 70   Upon further examination of the Tr B cells, we found that the most immature or T1 Tr B cell subset has a significantly higher IL-10/TNFα ratio than the remaining T2 subset of Tr B cells. 80 Moreover, the ratio of T1/T2 Tr B cells generally parallels the Tr B IL-10/TNFα cytokine ratio and might serve as a simpler marker of Breg/Beff activity. 80 Therefore, we examined the T1/T2 ratio in stable patients 2 years after kidney transplantation. We found that a low ratio was associated with significantly worse outcomes with decreased renal function (GFR) and 25% graft loss over the next 5 years, compared to stable GFR and no graft loss in patients with a higher ratio. Specifically, a low T1/T2 ratio

| Bregs as biomarkers for transplant tolerance and the confounding effects of immunosuppression
Outside of experimental protocols attempting to induce toler- Amongst findings that did differ from healthy controls, tolerant patients had a greater frequency of granzyme B-expressing plasma cells that suppressed T cell proliferation in vitro. 23 Taken together, an increase in Bregs appears common to studies of spontaneously tolerant kidney transplant patients, however, the confounding effects of immunosuppression limit interpretation. As a further concern, in a prospective study, liver transplant patients who were or were not successfully weaned off of immunosuppression, had no differences in total or Tr B cells. 95 Thus, the "Breg profile" seen in spontaneously tolerant kidney transplant patients does not appear to be broadly applicable.

| The effects of immunosuppressive agents on Bregs
Given evidence in both animal models and in humans that Breg/ Beff balance may play an important role modulating the immune response, strategies to expand Bregs (or inhibit Beff cells) might promote allograft survival. As discussed above, murine studies show that anti-TIM-1 specifically expands Bregs in vivo. 2  These preliminary studies suggest that some immunotherapeutic agents might actually increase Bregs. Further study will be required to determine how these, or other experimental agents, might be used to optimally expand Bregs in vivo to promote allograft survival.

| CON CLUS ION
Bregs have a profound influence on alloimmune, as well as autoimmune,

CO N FLI C T O F I NTE R E S T
None of the authors have any conflicts of interest related to the work presented in this article.