Regulatory B cells in Solid Organ Transplantation: From Immune Monitoring to Immunotherapy

Abstract

Regulatory B cells (Bregs) modulate the immune response in diverse disease settings including transplantation. Despite the lack of a specific phenotypic marker or transcription factor, their significance in transplantation is underscored by their ability to prolong experimental allograft survival, the possibility for their clinical use as immune monitoring tools, and the exciting prospect for them to form the basis for cell therapy. IL-10 expression remains the most widely used marker for Bregs. Several Breg subsets with distinct phenotypes that express this ‘signature Breg cytokine’ have been described in mice and humans. Although T cell immunoglobulin and mucin family-1 (TIM-1) is the most inclusive and functional marker that accounts for murine Bregs with disparate mechanisms of action, the significance of TIM-1 as a marker for Bregs in humans still needs to be explored. While the primary focus of this review is the role of Bregs in clinical transplantation, the net modulatory effect of B cells on the immune response and clinical outcomes is the result of the balancing functions of both Bregs and effector B (Beff) cells. Supporting this notion, B cell IL-10/TNFα ratio is shown to predict immunological reactivity and clinical outcomes in kidney and liver transplantation. Assessment of Breg:Beff balance using their IL-10/TNFα ratio may identify patients that require more immunosuppression and provide mechanistic insights into potential therapies. In summary, current advances in our understanding of murine and human Bregs will pave way for future definitive clinical studies aiming to test them for immune monitoring and as therapeutic targets.

INTRODUCTION

B cells differentiate into plasma cells that secrete antibodies, a defining feature of the humoral immune response. In addition to their ability to secrete antibodies, B cells also profoundly influence alloimmune as well as autoimmune, tumor, and anti-microbial immunity through their antibody independent functions such as antigen presentation, T cell co-stimulation/co-inhibition and cytokine secretion. In fact, B cells modulate both innate and adaptive immune responses either through the elaboration of either proinflammatory cytokines such as TNFα, GM-CSF, IFNγ, IL-12, IL-17 and IL-6 or through anti-inflammatory cytokines such as IL-10, IL-35 and granzyme B (GZB)^1–17. B cells can also be classified into subsets based on their cytokine milieu in a similar fashion to helper T cell subsets. Regulatory B cells (Bregs) are one such functional B cell subset that was defined by Mizoguchi and colleagues based on the secretion of their ‘signature cytokine’- IL-10¹¹. These IL-10 expressing cells contribute to the maintenance of immunological equilibrium^18–21. Whether Bregs represent a distinct B cell lineage with a common progenitor or whether they represent plasticity within B cell sub-populations that can adopt regulatory function upon receiving appropriate immunological signals still remains elusive. Nonetheless, they have been extensively studied in a number of disease settings including transplantation.

Two observational studies from either side of the Atlantic that described a Breg centric ‘B cell signature’ re-kindled the enthusiasm for the assessment of Bregs in experimental and clinical transplantation^22,23. While this review focuses primarily on the role of Bregs in transplantation, the net modulatory effect of B cells on immune response is best reflected by the balance of both Bregs and those B cells that secrete pro-inflammatory cytokines and termed as B effector (Beff) cells. In this overview, we will first describe the phenotype of murine Bregs and their significance in experimental transplant models. We will then highlight the plasticity that underpins human Breg phenotypes and provide an overview of how the assessment of Bregs or Breg:Beff balance could be used as potential monitoring tools in clinical transplantation. We will finally review how newer pharmacological agents could enhance Bregs and explore the possibility of a Breg based cell therapy product that can be used to promote tolerance or to treat rejection in the recipients of solid organ transplants.

Murine regulatory B cells:

B cells with immunoregulatory function were first described in the 1970s in studies examining delayed type hypersensitivity reaction in guinea pigs, although the mechanisms behind their suppressive ability was not investigated²⁴. Next, B cells were shown to play a significant role in protection against the autoimmune response in murine models of experimental autoimmune encephalomyelitis (EAE)²⁵. This protective ability of B cells was subsequently shown to be dependent on IL-10 secretion². IL-10 secreting B cells were also shown to confer protection against disease in mouse models of arthritis and chronic intestinal inflammation^8,11. While lacking an inclusive marker or a transcription factor, these earlier studies established that B cells suppress inflammation through the elaboration of IL-10. Since IL-10+ B cells could not be identified without prior ex vivo stimulation and intracellular cytokine staining after cell membrane fixation 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, CD1d+(MZ), CD21hiCD23hiCD24hi (T2-MZP), and CD1dhi CD5+(“B10”) subsets are all enriched for IL-10 expression and can transfer IL-10-dependent amelioration of murine colitis, EAE, and SLE^10,11,26. Although Bregs are still primarily identified by the secretion of their ‘hallmark cytokine’- IL-10, a variety of other regulatory mechanisms including PD-L1, FasL, TIGIT, GZB, transforming growth factor-β (TGFβ), and IL-35 have been described^7,27–31. Whilst several phenotypic subsets of B cells with disparate suppressive mechanisms have so far been identified, how these cells are related to each other, and to IL-10 expressing Bregs remains unclear.

Bregs in murine models of transplantation:

Allograft acceptance without maintenance immunosuppression (IS) is the holy grail for the field of transplantation. As such, experimental transplantation focused on identifying tolerogenic pathways and mechanisms that could also lead to the development of newer therapeutic targets. Indirect evidence for the role of Bregs in transplantation emanated from one such study that examined murine cardiac transplant tolerance utilizing anti-CD45RB antibody³². Anti-CD45RB treatment failed to induce tolerance when allogeneic hearts were transplanted in B cell deficient hosts. In the absence of B cells, tolerance did not develop, and in addition the immunosuppressive effect of anti-CD45RB antibody was lost, directly implicating host B cells in the induction of anti-CD45RB mediated tolerance. Furthermore, B cells stimulated ex vivo through TLR4 and TLR9 receptors not only inhibited ex vivo T cell proliferation but also suppressed B cell proliferation and prolonged islet allograft survival^33,34. These TLR-stimulated Bregs were enriched for IL-10 as well as other immunomodulatory molecules such as TIM-1, LAP and PDL-1.

B cells were also identified as a major target for the tolerogenic anti-T cell immunoglobulin and mucin family-1 (TIM-1) monoclonal antibody that prolongs murine islet allograft survival and ameliorates EAE by decreasing pro-inflammatory Th17 and Th1 responses, while augmenting Th2 cells and Foxp3+ Tregs¹. In transplanted mice 10–15% of the splenic B cells expressed TIM-1, in contrast to T cells where TIM-1 expression was rare. Adoptive transfer of TIM-1+, but not TIM-1-, B cells from alloimmunized hosts promoted islet allograft tolerance in an alloantigen specific manner. TIM-1+ B cells demonstrated a 20–25-fold enrichment for IL-10 expression when compared to TIM-1- B cells. TIM-1+ B cells accounted for ~75% of all IL-10+ splenic B cells and anti-TIM1 treatment resulted in a further 4-fold increase in IL-10+ B cells, suggesting that TIM-1 signals play a functional role in IL-10+ Breg induction. Moreover, anti-TIM-1 failed to prolong islet allograft survival in tamoxifen-inducible B cell–specific IL-10 knockout mice, suggesting that anti-TIM-1 mediated prolongation of islet allograft survival is dependent on B cell IL-10³⁵. TIM-1 is a phosphatidyl serine receptor (PtdS) on B cells and promotes IL-10 secretion by interacting with PtdS on the surface of apoptotic cells³⁶. Mice with a mutant form of TIM-1 with a deletion of the mucin domain (TIM-1Δmucin), exhibited defects in both basal and induced IL-10+ Bregs^37–39. Transcriptional assessment of TIM-1+ and TIM-1- B cells from wild type mice and from TIM-1Δmucin mice revealed that TIM-1 signaling not only regulated IL-10, but a host of other inhibitory cytokines and co-inhibitory molecules including, Ebi3, GITRL, Fgl2, CTLA-4, Lag3, and TIGIT³¹. Thus, TIM-1 regulates an array of potentially inhibitory molecules and may link different “types” of Bregs that utilize distinct mechanisms¹⁹. Taken together, murine transplant models highlight the role of Bregs in the induction of tolerance and raise a distinct possibility that these cells could form the basis for cell therapy in clinical transplantation.

Human Breg phenotype:

In line with the observations in mice, there exists no specific human Breg marker. Like in mice, human Bregs are characterized based on their expression of IL-10^18,40. Since IL-10 expressing resting human B cells are rare, a period of 24 to 96 hours of ex vivo stimulation is typically used to demonstrate B cell IL-10¹⁹. Blair and colleagues were the first to describe the immunomodulatory properties of IL-10 expressing CD24hi CD38hi transitional B cells (TrBs)⁴¹. They found that TrBs were enriched for IL-10 after 48-hour ex vivo stimulation with CD40L expressing CHO cells. Further, TrBs inhibited IFNγ, TNFα and IL-17 expression and increased FoxP3 expression of anti-CD3-treated autologous CD4+ T cells in healthy donors^41,42. In contrast, naïve and memory B cell subsets had no discernable effect on T cell cytokine expression. They go on to show that TrB modulation of T cell cytokines is dependent on both IL-10 and co-stimulatory signaling through CD80 and CD86. Compared to healthy donors, patients with either SLE or rheumatoid arthritis not only had reduced numbers of TrBs in their peripheral blood but also the TrBs lacked ex vivo Breg activity^41,42. Moreover, they demonstrated that agonistic CD40L stimulation leads to phosphorylation of STAT3, a process that was defective in SLE patients. Contrasting these observations, Iwata and colleagues showed that CD24hi CD27+ memory B cells expressed most IL-10⁶. However, they found no defect in the memory B cell IL-10 expression in patients with various autoimmune disorders including SLE. In addition, a subset of memory B cells defined as CD24hiCD27+CD39hi B cells were shown to express more IL-10, GZB and PD-L1 when compared to TrBs⁴³. Further, they were also shown to be enriched for functional Breg markers such as TIGIT and suppress autologous T cell proliferation in an IL-10, PD-L1 and GZB dependent fashion⁴³. In addition to inhibiting T cells, TIGIT+ Memory B cells also downregulated expression of co-stimulatory molecules and pro-inflammatory cytokines by immature monocyte-derived dendritic cells (MDDCs) ex vivo which in turn were more efficient in suppressing CD4+ T cells. Thus, each of the canonical B cell subsets have IL-10 expressing B cells. But IL-10 expressing cells constitute a small minority. Apart from the memory B cells and TrBs described above, studies have reported that several distinct B cell subsets such as CD5+CD1dhi B cells, CD9+ B cells, CD11b+ cells, CD38+CD1d+IgM+CD147+ B cells, CD25hiCD71hiCD73lo B cells, CD39+CD73+ B cells and CD27intCD38hi plasmablasts also suppress T cell proliferation and pro-inflammatory cytokine expression ex vivo^7,40,44–50. In addition to IL-10, human Breg activity has also been attributed to various immunomodulatory molecules such as GZB, indoleamine 2,3-dioxygenaze (IDO), adenosine, and transforming growth factor β. Finally, the concept that IL-10+ Bregs in various subsets can be suppressive averts the concern that a minor subset, or TrBs themselves that are short-lived and either differentiate into mature naïve B cells or undergo cell death, would be sufficient to have a major suppressive impact on immune responses.

TIM-1 is an inclusive and functional Breg marker in mice. Several studies now highlight TIM-1 as a marker for human Bregs. Aravena and colleagues reported that ~5% of human peripheral blood B cells and 15%−35% of the TrBs express TIM-1⁵¹. TIM-1 expression was shown to be increased 1.5-fold in all the canonical B cell subsets after 48hrs of ex vivo B cell receptor (BCR) activation by anti-human IgG and IgM antibodies⁵¹. Reportedly, ~40% of all TrBs express both TIM-1 and IL-10, and TIM1+ but not TIM1- B cells suppress pro-inflammatory autologous T cell cytokines. Compared to healthy controls, patients with systemic sclerosis not only had less TIM-1+ B cells but they were also functionally defective in an ex vivo Breg assay. TIM-1 as a human Breg marker was further corroborated in subsequent studies^52,53.In addition, as observed in mice, anti-TIM-1 was shown to increase B cell IL-10⁵². Taken together, TIM-1 is a useful human Breg marker and future studies will probably address its significance in clinical transplantation.

The same caveats that apply to the study of mouse Bregs also apply to human Bregs. IL-10 expressing cells constitute a small minority of the purported Breg subsets. In addition, IL-10 secretion typically requires ex vivo stimulation with non-physiological concentrations of various stimulation cocktails that include anti-CD40, CPG-ODN-2006, and/or anti-human Ig, for time periods ranging from 24hrs to 5 days. It is indeed very hard to extrapolate which ex vivo conditions best reflect what occurs in vivo. Critically, ex vivo stimulation of B cells alters their phenotype and function. Also, the suppressive assays used to establish the regulatory capacity of a given B cell subset commonly utilize B cells at supra-physiological concentrations (1:1 to 1:10 concentrations) compared to the respective T cell populations. In summary, ex vivo stimulation for extended periods of time and the current Breg functional assays limit our ability to understand the biological significance of these cells in health or in a specific human disease setting.

IL-10/TNFα ratio and Breg:Beff cell balance.

Given the contrasting evidence that identified different canonical B cell subsets as Bregs, we compared IL-10 expression in peripheral blood B cells from healthy donors and found that all major B cell subsets including transitional, naïve, and memory B cells have comparable IL-10 expression. However, B cells from the same subsets also co-express pro-inflammatory cytokines such as TNFα⁵⁴. These findings emphasize that both Bregs and Beff cells co-exist in all B cell subsets. When we examined the ratio of cells that expressed IL-10 to those that expressed TNFα, we found that TrBs, especially the most immature T1 TrBs had the highest ratio of IL-10/TNFα expressing cells compared to either the naïve or memory B cells^54,55. Moreover, the ability of B cells to selectively suppress autologous CD4+ T cell pro-inflammatory cytokines in an ex vivo functional assay correlated with their IL-10/TNFα ratio. Thus, TrBs, which have the highest IL-10/TNFα ratio had the strongest B regulatory capacity. In fact, adding neutralizing anti-IL-10 mAb to the Breg functional assay nullified TrB Breg activity, while blocking TNFR1 uncovered the Breg activity of both naïve and memory B cells. Taken together, the relative expression of IL-10 and TNFα within individual B cell subsets correlates with their Breg:Beff balance and thus with their ability to modulate T cell responses ex vivo.

Next, using high-dimensional computational flow cytometry, Glass and colleagues confirmed our initial observations that Breg activity is better quantified by the relative expression of IL-10 and pro-inflammatory cytokines like TNFα, and that TrBs have the highest IL-10/TNFα ratio⁵⁶. Consistent with the notion that Bregs lack a lineage specific marker, they highlight that B cell IL-10 production ex vivo is a kinetically defined B cell function rather than a lineage-defining one⁵⁶. In addition, Bigot and colleagues have also shown that TrBs enrich for IL-10, while proinflammatory cytokines such as IL-1α, IL-1β and tumor necrosis factor β were under-represented in this subset⁵⁷. Moreover, markers associated with regulatory function and IL-10 expression such as ICOS-L, CD9, CD5 and GARP were significantly enriched in the TrBs⁵⁷. The notion that Breg:Beff balance contributes to disease pathology and outcomes is supported by studies of patients with multiple sclerosis (MS)^58,59. When compared to healthy donors, B cell IL-10 expression in patients with MS is markedly reduced, while expression of pro-inflammatory cytokines such as TNFα, lymphotoxin, and GM-CSF is increased. Further, B cells polarized to a pro-inflammatory cytokine profile fail to suppress T cell proliferation ex vivo. Importantly, favorable clinical response following B cell depletion with Rituximab is associated with normalization of IL-10/TNFα expression in the reconstituting B cells and restoration of their ex vivo suppressive function. In summary, these data point to the possibility that Breg:Beff balance could also contribute to allograft outcomes in clinical transplantation.

Bregs and clinical transplantation

Two randomized controlled clinical trials evaluating the effect of pre-transplant B cell depletion using Rituximab in kidney and heart transplant patients respectively, conducted almost a decade apart, highlighted the possible immunomodulatory role and relevance of Bregs in clinical transplantation. In the first study, B cell depletion in HLA-compatible kidney transplant recipients led to a striking increase in acute rejection (AR, 83%) within the first three months post-transplantation⁶⁰. In the second study, heart transplant recipients who received Rituximab experienced significantly increased cardiac allograft vasculopathy (CAV) at one-year post-transplantation⁶¹. In contrast to patients with MS, where B cells have lower IL-10/TNFα ratio, standard immunological risk transplant recipients lack active alloimmunity, and likely have relatively higher IL-10/TNFα expressing B cells (i.e., more Bregs and less Beff cells). While B cell depletion in MS results in the disruption of the predominance of Beff cells, it possibly results in the relative depletion of Bregs in the early post-transplant period in transplant patients and contributes to enhanced alloimmune graft injury. Thus, data from these two B cell depletion clinical trials in transplantation underscore the role of Bregs and Beff cells in transplantation.

Bregs and tolerance:

Solid organ transplant recipients need lifelong IS to maintain their allograft function. Around a third of liver transplant patients could be weaned off IS and still maintain long-term function. In contrast, only a handful of kidney transplant patients world-wide maintained stable transplant function despite discontinuation of their IS. These rare patients have been the focus of several studies that aimed to gain more insights into their clinical tolerant state^{22,23,62–64}. Tolerant kidney transplant patients exhibited a ‘B cell signature’ with enrichment of genes involved in B cell activation and differentiation compared to stable recipients on IS²³. Furthermore, analysis of peripheral blood mononuclear cells by flow cytometry highlighted the expansion of total B cells as well as TrBs and their IL-10 expression in these clinically tolerant patients. Comparison of tolerant transplant patients who remained off maintenance IS to transplant patients on chronic IS makes maintenance IS a potential confounding variable in these studies. Subsequently, several studies reported that transplant patients maintained on immunosuppressive agents such as calcineurin inhibitors (CNI), prednisone or azathioprine had a dose-dependent reduction in B cells and TrBs in their peripheral blood^65,66. Nonetheless, in a longitudinal study, Newell and colleagues have demonstrated that patients rendered tolerant through the induction of transient mixed chimerism, and those weaned to minimal IS, showed similar increases in genes such as IGKV1D-13 that were a part of the original B cell tolerant signature⁶⁷. Although B cells from tolerant liver allograft recipients express more IL-10 and less pro-inflammatory cytokines like TNFα, the B cell centric gene signatures observed in kidney transplant patients were not noted in tolerant liver allograft recipients^56,68. Therefore, an increase in Bregs that has been reported in the studies of spontaneously tolerant kidney transplant patients is confounded by the effects of IS and is not broadly applicable to transplant recipients of other organs.

Bregs and transplant rejection:

In clinical transplantation, a much more common scenario than tolerance is allograft rejection. In fact, several representative B cell specific genes (for example, MS4A1, TCL1A, CD79B, TOAG-1) that constituted various clinical tolerance signatures have been shown to be downregulated in patients with renal allograft rejection^69,70. As highlighted earlier, B cell depletion markedly increases kidney allograft rejection raising the possibility that Bregs might prevent rejection and enumerating them could be an effective immune monitoring strategy after clinical transplantation. Although lacking data on cytokine secretion or immunomodulation ability, several reports have shown that transitional B cells are reduced in number in transplant patients with allograft rejection. First, we examined B cells and their subsets in the peripheral blood of patients who either received Alemtuzumab or Basiliximab induction after renal transplantation. At two years post-transplantation, when patients were divided into tertiles based on the absolute number of TrBs in their peripheral blood, those within the lowest tertile had the worst renal function and the highest incidence of donor-specific antibodies (DSA) at the time of the assessment⁷¹. In general agreement with these observations, Shabir and colleagues showed that the frequency of peripheral blood TrBs, when assessed two weeks post-transplantation, inversely correlated with acute renal transplant rejection over the ensuing 4 years⁷². While no patients with a TrB frequency of ≥ 3% had rejection, 50% of patients with a TrB frequency of < 1% had rejection. In a multivariable logistic regression model, the association between TrB frequency and rejection was independent of all potential clinical confounders. These findings were further corroborated by several other small single center studies in kidney and lung transplantation^73,74. In fact, TrBs have been shown to be enriched for CD9 expressing B cells, and their frequency was shown to correlate with the diagnosis of bronchiolitis obliterans in lung allograft recipients⁷⁵.

Shiu and colleagues have further reiterated the role of Bregs in kidney transplant patients with or without antibody mediated rejection (AMR)^76–78. In an IFNγ ELISPOT assay, they observed that CD4+ T cells from patients with no evidence of alloimmune injury do not respond to donor HLA peptides unless their B cells were depleted. Depleted B cells from these patients were polarized to more IL-10 production. Similarly, approximately half of the patients with AMR did not demonstrate CD4+ T cell anti-donor IFNγ response unless either CD25+ Tregs or CD19+ B cells (that secrete IL-10) were depleted. Importantly such AMR patients with a suppressed anti-donor T cell IFNγ response ex vivo demonstrated clinical stabilization following IS optimization. Of note, clinical stabilization was associated with a relative increase in T1 TrBs compared to T2 TrBs. In addition to TrBs, several other B cell subsets shown to be enriched for cells expressing immunoregulatory molecules such as IL-10, GZB and TIGIT also correlate with clinical transplant outcomes. For example, CD25+ B cells were shown to correlate with higher regulatory T cell numbers as well as better renal function assessed by glomerular filtration rate in kidney transplant recipients⁷⁹. Next, TIGIT+ Memory B cells were shown to be reduced in kidney and liver allograft recipients with DSA compared to those without DSA⁴³. Similarly, stem cell transplant recipients with graft versus host disease (GVHD) and liver transplant recipients that experienced acute rejection, demonstrated a decline in the number of CD24hiCD27+ IL-10+ B cells^80,81. Table 1 highlights the significance of various B cell subsets that have been shown to be regulatory ex vivo in clinical transplantation.

Table 1:

Human B cell populations enriched for putative Bregs and their relevance to clinical transplantation.

Human B cell subpopulation	Breg mechanism	Relevance to transplantation
Transitional B cells
CD24hiCD38hi B cells	I. IL-10 secretion   II. Contact dependent mechanisms utilizing CD80/CD86 III. IL-10/TNFα ratio	I. Increased in number as well as increased IL-10 secretion in tolerant renal transplant recipients when compared to those on standard immunosuppression23,67.   II. Reduced in number in patients with acute renal and lung transplant rejection72–74.   III. Reduced IL-10/TNFα ratio in patients with late renal transplant rejection54.   IV. Modulate allospecific T cell cytokine expression in renal transplant recipients with ABMR76,96.    V. A low ratio of T1/T2 transitional B cells predicts premature renal transplant failure55.   VI. A low IL-10/TNFα ratio in T1- TrBs has the potential to be a biomarker for renal transplant rejection and clinical course82.   VII. A low IL-10/TNFα ratio in T1- TrBs risk stratifies renal transplant recipients with early borderline rejection83. VIII. Reduced in number in lung allograft recipients with bronchiolitis obliterans75.
Memory B cells
CD24hiCD27+ B cells	IL-10 secretion	I. Reduced IL-10 expressing CD24hiCD27+ B cells in liver transplant rejection when compared to stable patients81. II. HLA-G enhances IL-10 secretion by these cells ex vivo97.
CD24hiCD27+CD39hi B cells	IL-10, Granzyme B, PD-L1, TIGIT	I. These cells inhibit T cell pro-inflammatory cytokines ex vivo in a granzyme B and TIGIT dependent fashion43. II. Reduced in liver transplant patients with DSA43.
CD73-CD25+CD71+ B cells	TIM-1, Granzyme B, IL-10	Expanded cells prolong skin allograft survival in a humanized mouse and could form the basis for cell therapy53.
Plasma cells
CD5+CD27+CD138+ B cells	Granzyme B secretion	Higher number in tolerant renal transplant recipients when compared to those on standard IS28.
CD307bhiCD258hiCD72hiCD21loPD-1hi B cells	Granzyme B secretion	Cells could be expanded with αBCR, CpG, CD40L, IL-21, IL-2 and could form the basis for cell therapy95.
CD9+ B cells	IL-10 secretion	Enriched in TrBs and reduced in lung allograft recipients with bronchiolitis obliterans75.

Transitional B cell IL-10/TNFa ratio and renal transplant outcomes:

Based on the notion that Breg:Beff balance within B cells and subsets as determined by their IL-10/TNFα cytokine polarization, modulates immune responses, and contributes to clinical outcomes after transplantation, we asked if the cytokine ratio would correlate with adverse transplant outcomes. When we assessed B cell subset IL-10/TNFα ratio in 41 kidney transplant patients who underwent for-cause allograft biopsies late in the course of their transplantation, we found that the ratio of IL-10/TNFα expressing cells within the TrBs was significantly lower in patients experiencing rejection on their biopsies when compared to those who had allograft dysfunction and no rejection or to those with stable allograft function⁵⁴. Neither the absolute number of TrBs nor their IL-10 expression alone could differentiate these patient subgroups. In an ex vivo Breg functional assay, TrBs from patients with rejection failed to suppress autologous T cell pro-inflammatory cytokine expression confirming the balance-tilt towards a more pro-inflammatory TrB profile in these patients. In patients with late allograft rejection, TrB IL-10/TNFα ratio below the median was associated with 50% allograft loss in the ensuing 3 years, while no allografts were lost in patients with a TrB IL-10/TNFα ratio above the median. Thus, TrB cytokine ratio appeared to risk stratify patients at higher risk for poor subsequent outcomes following an episode of late rejection⁵⁴. Moreover, within the TrBs, the most immature T1 TrBs had the highest ratio of IL-10/TNFα expressing cells compared to the relatively mature T2 TrBs. As the ratio of T1/T2 TrBs paralleled their IL-10/TNFα expression, we asked if the ratio of T1/T2 TrBs in the peripheral blood of clinically stable kidney transplant recipients two years post-transplantation could effectively risk stratify them for subsequent allograft functional decline. A T1/T2 ratio lower than the median was found to be associated with graft loss or graft dysfunction⁵⁵. Together, these data suggested that assessment of TrB cytokines early in the course of transplantation could serve as an effective risk stratification tool and be a valuable biomarker to predict rejection and subsequent clinical course.

Next, we prospectively examined B cell subsets and their IL-10/TNFα cytokine expression profiles in 244 consecutive kidney transplant patients that received Thymoglobulin induction and steroid free maintenance IS with tacrolimus and mycophenolate⁸². Of all the B cell parameters, T1 TrB IL-10/TNFα ratio best discriminated patients with and without AR within the first post-transplant year. A low T1B IL-10/TNFα ratio, at 3 months post-transplantation (a time-point when B cell repopulation after Thymoglobulin induced depletion is rather complete), strongly predicted any AR in the first post-transplant year (ROC AUC 0.89). Importantly, in patients with no AR at the time of the biomarker assessment, the T1B cytokine ratio predicted subsequent AR with an average lead time of around 8 months providing a valuable time window for potential intervention. In fact, patients categorized as high-risk (based on the T1B cytokine ratio cut-off of 1.3), had ~12-fold increase in incidence of subsequent late AR and a markedly high level of persistence of rejection despite standard of care treatment. These findings were validated in an external cohort of patients receiving induction IS with Basiliximab. The T1B IL-10/TNFα ratio strongly predicted AR irrespective of donor type, HLA matching, delayed graft function, detection of DSA, induction IS, CNI adherence, and whether or not surveillance biopsies were performed. Furthermore, the T1B cytokine ratio was not affected by opportunistic viral infections. The equally good performance of the T1B cytokine ratio across patient sub-groups stratified by these key confounding variables suggests generalizability. High-risk patients defined by the T1B cytokine ratio had ~2-fold increased incidence of interstitial fibrosis and tubular atrophy (IFTA) at 1 year, decreased 5-year GFR, and a ~5-fold increase in 5-year- graft loss. Thus, T1B IL-10/TNFα ratio predicts early and persistent inflammation that translates into premature fibrosis and increased risk for allograft loss.

We next extended our assessment of T1B IL-10/TNFα ratio to kidney transplant patients diagnosed with borderline rejection- a diagnostic category that creates both a prognostic and therapeutic dilemma⁸³. It is left up to the clinician to try to decide, often on a case-by-case basis, whether borderline rejection does or does not represent early or mild AR that might progress without additional anti-rejection therapy. Moreover, there is no consensus on whether or how to treat borderline rejection. We found that T1B IL-10/TNFα ratio can specifically risk-stratify patients with early borderline rejection (diagnosed within the first four months post-transplantation) such that low-risk patients have excellent (92%) seven-year graft survival, comparable to those with no allograft inflammation. In contrast, 55% of high-risk patients defined by low T1B cytokine ratio lose their grafts by seven years⁸³. Therefore, T1B cytokine ratio directly addresses the clinical dilemma by identifying a sizeable (~30%) number of patients in need for augmented IS. T1B cytokine ratio is distinct from other biomarkers in transplantation in that it identifies an immunological imbalance between Bregs and Beff cells that might be amenable to therapeutic manipulation. Supporting this, we demonstrated that TNF neutralization augments both B cell and T1B IL-10/TNFα ratio and their ex vivo Breg activity. Anti-TNFα also inhibits plasma cell differentiation and suppresses complement-fixing antibody secretion. Further, it has been shown that treatment of rheumatoid arthritis patients with anti-TNFα increases IL-10+ B cells in vivo, supporting the notion that TNF-blockade may work through modulation of Breg:Beff balance⁸⁴. Although data in transplantation is limited, anti-TNFα successfully rescued small bowel allograft recipients who had rejection resistant to both steroids and T cell depletion therapy with OKT3 antibody⁸⁵. Thus, anti-TNFα could improve clinical outcomes in immunologically high-risk renal transplant recipients by restoring Breg activity. Taken together, our findings support the notion that assessment of Bregs and Beff cells not only provides a valuable insight into a patient’s immunological set-point and allograft outcomes but also points to possible therapeutic intervention.

Bregs and therapeutics in transplantation:

Solid organ transplant recipients require lifelong IS to prevent rejection of the transplanted organ. Further, both induction and maintenance IS regimens are empirically dosed, and the associated drug toxicities add to patient mortality and morbidity. Thus, there is a major need to identify newer IS strategies that will enhance allograft survival without increasing the associated toxicities. To address this, there have been major efforts to identify regulatory cells most relevant to transplantation that might be targeted or expanded ex vivo to promote organ-specific tolerance or to prevent or treat allograft rejection. Mounting evidence as discussed in the above sections suggests that Bregs play a vital role in allograft survival and could potentially be augmented by specific pharmaceutical agents or form the basis for cell therapy in clinical transplantation. Figure 1 details potential clinical applications of Bregs with disparate mechanisms of action.

Figure 1. Breg phenotypes and clinical transplantation.

Mechanisms of action and the clinical significance of putative human Breg populations that modulate T cell or innate cell cytokine responses through the elaboration of either IL-10, granzyme B (GZB), T cell Immunoglobulin and Mucin domain 1 (TIM-1) or through differential expression of both IL-10 and TNFα. DC, dendritic cell; TLR Toll like receptor, BCR, B cell receptor; TCR, T cell receptor; TIGIT, T cell immunoreceptor with Ig and ITIM domains; TNFR2, TNF receptor2; CTLA4, Cytotoxic T-lymphocyte-associated protein 4; PDL1, Programmed death ligand 1; MHC, major histocompatibility complex.

Pharmacological agents that enhance Bregs:

Immunosuppression augmentation with monoclonal antibodies or biological agents could lead to increased Breg induction and/or expansion in transplantation. For example, depletional induction with Alemtuzumab (anti-human CD52) in kidney transplant patients is associated with increased TrB numbers in the first-year post-transplantation^71,86. Such patients with increased TrBs had better renal function and less DSA. Next, kidney transplant patients treated with Belatacept, a CTLA4 fusion protein, had ~3-fold increase in TrB number and their IL-10 expression compared to patients that received CNI^57,87,88. A high serum concentration of the B-cell survival factor, BAFF, in kidney transplant recipients is associated with de novo DSA and increased AMR. In a small phase 2 randomized clinical trial comparing the addition of anti-Baff (Belimumab) or a placebo to standard of care IS, Belimumab usage was associated with a higher B cell IL-10/IL-6 ratio compared to the placebo treated group, although no differences in early clinical outcomes were observed ⁸⁹.

Cytokines like IL-6 and TNFα play an important role in B cell differentiation. IL-6 receptor blockade in patients with rheumatoid arthritis was associated with an increase in CD25+ Bregs and their TGFβ expression⁹⁰. Further, IL-6 neutralization with clazakizumab in sensitized kidney transplant patients undergoing desensitization prior to their transplantation not only resulted in a reduction in HLA antibodies but also an increase in the TrB numbers although their IL-10 expression was not tested⁹¹. Preliminary data from our group demonstrated that anti-TNF increases both B cell IL-10/TNFα ratio and their suppressive activity ex vivo, providing a rationale for testing anti-TNF to improve outcomes in transplant patients. However, in a recent CTOT supported clinical trial, a single dose of anti-TNF did not lead to improved early post-transplant outcomes, and in fact led to an alarming increase in BK viremia⁹².

Finally, as mentioned above, STAT3 phosphorylation appears to be an important step in IL-10+ Breg generation. Hypoxia-inducible factor (HIF)-1α, the oxygen-sensitive subunit of the HIF transcriptional complex, was shown to be important for B cell IL-10 secretion in a STAT3-dependent manner⁹³. HIF1α-prolyl hydroxylase enzyme inhibitors that stabilize HIF complex are now being tested for the management of renal anemia⁹⁴. Future studies of these agents in transplantation might reveal their effect on Bregs and clinical transplant outcomes. Taken together, assessment of long-term outcomes in large patient cohorts would uncover the effect of these newer IS augmentation strategies on Bregs and long-term clinical outcomes.

Bregs for cell therapy:

In humans, TrBs have been extensively studied for their ability to modulate immune responses ex vivo and influence clinical transplant outcomes. Menon and colleagues described that IFNα secretion by plasmacytoid dendritic cells drives ~3-fold expansion of IL-10+ TrBs. They further demonstrated that the concentration of IFNα determines whether an immature B cell develops into a Breg or a plasmablast. At lower concentrations of IFNα, B cells differentiate into both plasmablasts and TrB Bregs, whereas at higher concentrations, the B cell response is channeled toward plasma cell maturation. While the expanded TrBs haven’t been tested for their utility as a cell therapy product, developing a better understanding of the feedback loop between IFNα and TrBs could potentially create opportunities for Breg based immune cell therapies. In addition, a stimulation cocktail that comprises of IL-21, anti-BCR, CpG oligodeoxynucleotide, CD40L and IL-2 has been shown to effectively expand B cells that express granzyme B and inhibit proinflammatory CD4+ T cell proliferation. However, how these expanded cells influence outcomes in either clinical or experimental transplantation remains to be studied⁹⁵.

Next, Shankar and colleagues have shown that persistent stimulation of B cells from healthy human donors with anti-human CD154 (CD40L) over a period of 14 days resulted in >900-fold expansion of IL-10 expressing B cells⁵³. Such IL-10+ B cells were also shown to be enriched for CD154 expression as well as other immunoregulatory molecules such as TIM-1, CD25, LAP and CD71. They go on to show that the expanded Bregs suppressed autologous CD4+ T cell proliferation as well as pro-inflammatory cytokine expression. In a humanized mouse model of skin transplantation, adoptive transfer of expanded Bregs but not naïve B cells prolonged skin allograft survival. Further, B cells infiltrating the skin allografts in the mice that received expanded Bregs were predominantly TIM-1+. Both ex vivo T cell responses and prolongation of allograft survival were dependent not on IL-10 but on TIM-1 and CD154. However, it is to be noted that none of these studies describe antigen specific expansion of Bregs ex vivo. While the in vivo fate of these cell subsets, especially that of the immature TrBs is not yet known, Matsumoto and colleagues have shown that immature B cells differentiate into IL-10 secreting plasmablasts ex vivo in contrast to the more mature B cells that differentiate into Antibody secreting plasmablasts with little IL-10 secreting ability⁴⁹. Irrespective of the antigen-specificity of these putative Breg populations, the promising findings highlighting prolongation of allograft survival with non-allospecific Breg expansion could pave way for future Breg cell therapy protocols in clinical transplantation.

Conclusions and future perspective:

In summary, Bregs are primarily identified by the expression of their signature cytokine IL-10, though disparate mechanisms of Breg function have been described. They play a key role in modulating immune responses and contribute to improved outcomes in both experimental and clinical transplantation. However, several aspects of Breg biology still remain poorly understood. Little is known about their development and effector function in vivo. Further, they lack a specific phenotypic marker or a transcription factor. The specific role played by Bregs identified in various B cell subsets and their relationship to each other still remains less clear. Understanding the key regulatory mechanisms that underpin Breg function, their stability and alterations in health and disease will not only allow a more accurate clinical risk stratification but could also uncover newer therapeutic targets that can enhance Bregs and improve long-term allograft survival, a major unmet need in the field. Emerging evidence, as detailed in this review provides insights into how enumerating Bregs or Breg:Beff balance by B cell subset IL-10/TNFα ratio could serve as predictive and risk-stratification tools in transplantation. Future prospective clinical studies will inform if and how clinical risk stratification by Breg based biomarkers would improve clinical transplant outcomes. Moreover, studies that assess the effect of novel anti-rejection therapies on Bregs could pave way for newer treatments that influence Bregs or Breg:Beff balance and impact long-term allograft survival. TIM-1 is the most inclusive and functional murine Breg marker. Although its biology and significance in clinical transplantation needs further exploration, promising preliminary results on TIM-1+ and granzyme B+ Breg expansion and their ability to prolong graft survival points to a tantalizing possibility that they can form the basis for future clinical trials testing Breg based cell therapy.

Abbreviations:

Bregs

Regulatory B cells

Beff

B effector cells

CHO

Chinese hamster ovary

CNI

calcineurin inhibitors

DSA

Donor specific antibody

EAE

Experimental autoimmune encephalomyelitis

GFR

glomerular filtration rate

GVHD

Graft versus host disease

GZB

Granzyme B

HLA

Human leucocyte antigen

IS

Immunosuppression

MZ

Marginal zone

MZ-P

Marginal zone precursors

PtdS

phosphatidyl serine receptor

SLE

Systemic lupus erythematosus

TGFβ

Transforming growth factor β

TLR

Toll like receptors

TIM-1

T cell immunoglobulin and mucin family-1