Regulatory and Effector B cells: A New Path Toward Biomarkers and Therapeutic Targets to Improve Transplant Outcomes?

Introduction:

In addition to antibody secretion, B cells shape the immune response through antigen presentation, co-stimulation, and cytokine production^1–3. In this regard, distinctly polarized B cell subsets expressing either pro- or anti- inflammatory cytokines respectively, can promote or inhibit adaptive and innate immunity^2–5. For example, regulatory B cells (Bregs), inhibit autoimmune diseases and transplant rejection, and promote tumor growth^1–4,6,7. Although their suppressive function is mainly attributed to the expression of the immunomodulatory cytokine- IL-10, Bregs also use other suppressive cytokines and mechanisms, including IL-35, Fas ligand, PD-L1, TGF-β, and granzyme B^8–17. While initially identified in mice, evidence now suggests that Bregs also play a significant role in human disease and transplantation^8–17. In contrast, effector B cells (Beff) expressing pro-inflammatory cytokines such as IL-6, IL-17, TNFα and IFNγ, can profoundly augment anti-microbial responses, autoimmunity, and transplant rejection^{2–5,18–20}. While the central focus of this review is on Bregs and the important role they play in experimental and clinical transplantation, the net modulating effect of B cells on the alloimmune response is likely a summation of the opposing activities of both Bregs and Beff cells, and both will be addressed herein.

B cell depletion studies make a case for both Bregs and Beff Cells:

B cell depletion in humans with anti-CD20 can reduce inflammatory T cell responses and rapidly ameliorate Rheumatoid Arthritis, Diabetes Mellitus, and Multiple Sclerosis (MS), without affecting autoantibody levels^18,21–23, suggesting a pro-inflammatory role^{2–4,24–27}. On the other hand, B cell depletion can also promote inflammatory T cell responses, exacerbate autoimmunity, and promote renal allograft rejection^28–30,31, suggesting a regulatory role. Murine models confirm this duality. For example, in murine models of IBD or contact hypersensitivity, B cell deficiency or depletion can worsen autoimmunity^32–35. In EAE (a murine model of MS), B cell depletion can either worsen or ameliorate disease depending on the timing. Moreover, B cell deficiency can either augment or inhibit anti-tumor responses and tumor growth^27,36–40. As detailed below, Breg/Beff cell ratios (based on cytokine expression) are decreased in MS and strongly predict outcomes in renal transplantation^41–43. These findings strongly suggest that B cells can play both a regulatory or proinflammatory effector role and the influence of B cells on immune response in a given patient is likely a summation of the opposing activity of both Bregs and Beff cells. If such cells could be independently targeted, the immune response might be augmented or inhibited, as required by the clinical setting.

The problem in defining “Bregs” and “Beff cells”: Lack of a specific phenotype

There are no phenotypic markers or transcription factors that specifically identify either Bregs or Beff cells. Rather, Bregs and Beff cells are defined by their expression of either anti- or pro- inflammatory cytokines. Thus, at present, Bregs are best characterized by the expression of their ‘signature cytokine’, IL-10. However, IL-10 is expressed at very low frequency (~1%) in the overall B cell population^33–35,44. As a result, multiple B cell phenotypes that are enriched for IL-10+ B cells have been used as a “surrogate” for Bregs to elucidate their biology and function. For example, in mice, CD1d+ B cells in the gut were initially shown to transfer IL-10-dependent inhibition of intestinal inflammation³³. Subsequent studies found that a number of other subsets, including CD1d^hi CD5⁺ (“B10”) and CD21^hiCD23^hiCD24^hi (T2-MZP) B cells were enriched for IL-10 expression and again transferred IL-10-dependent amelioration of murine EAE and SLE^35,45. Importantly, while enriched, IL-10+ B cells comprise a minority of cells (e.g. 15%) within each of these B cell subsets. Furthermore, the IL-10+ B cells in each of these small phenotypic subsets, comprise only 10–20% of all IL-10+ B cells in secondary lymphoid organs (SLO). Even though the frequency of IL-10+ expression is low in the remaining 80–90% of B cells, these make up the large majority of IL-10+ B cells. However, the frequency of IL-10+ cells in these B cell subsets is too low to demonstrate activity in B cell transfer models. Another challenge that impedes better understanding of the role of Bregs cells in health and disease, is the IL-10 secretion itself. Due to low levels of expression, IL-10 is usually only observed after in vitro stimulation of B cells^33–35,44. This limits the ability to perform meaningful transfer experiments with freshly isolated IL-10+ cells, examine their function or perform transcriptional analysis with an aim to uncover a unifying marker or understand their downstream function, without prior stimulation.

In summary, current Breg phenotypes actually represent the subsets most enriched for IL-10 expression in any given disease model, and this may be influenced by the type of stimulation used to elicit IL-10 expression. Importantly, these individual subsets are not specific, nor are they necessarily representative of most IL-10+ Bregs. While there are a growing number of examples where Bregs use mechanisms other than IL-10 to suppress immune responses, such Bregs are poorly understood and their relation to IL-10+ Bregs is unknown.

More recently, TIM-1⁺ and CD9⁺ B cells were found to be more inclusive markers for Bregs^44,46. While still not specific (IL-10+ B cells comprising only ~15–20% of TIM-1⁺ or CD9⁺ populations), each of these subsets encompass ~75–85% of all IL-10⁺ B cells^44,46. Notably, TIM-1 and CD9 have functional roles, as they have been shown to positively or negatively regulate IL-10 expression respectively (see below)^44,46–49. Both TIM-1+ and CD9+ populations contain IL-10⁺ B cells belonging to each of the different canonical B cell subsets, including transitional 1 and 2 (T1, T2), marginal zone (MZ), MZ precursors (MZP), follicular (FO), and plasma cells (PC)^44,46. The frequency of IL-10 expression varies significantly as does the size of each subset^44,46,50,51. Our recent findings examining unstimulated B cells isolated from IL-10 reporter mice, show that PCs, FO, and MZ B cells each contain 25–30% of IL-10+ B cells⁵². It will be important to determine whether IL-10+ B cells belonging to different subsets exhibit distinct functions.

B cells can also express various pro-inflammatory cytokines. While various phenotypic markers have been used to identify IL-10+ Bregs, far less is known about the phenotypic identity of pro-inflammatory Beff cells, hampering our ability to understand Beff biology. Harris et al first demonstrated that B cells, dubbed “Be1”, could be polarized in vitro to express IFNγ. Since then, “innate-like B cells” have been shown to express various pro-inflammatory cytokines that contribute to rapid host responses to microbial infections. Examples include IL-2 (Polygyrus; Th2 response), IL-17 (T. cruzii; reduced parasitemia), and IFNγ (Listeria; monocyte activation and Salmonella, Th1 responses)^5,53,54. Of these, a small subset of CD11a^HiFcɣRIII^Hi B cells, which rapidly and transiently expresses IFNγ in response to TLR ligands, remains the only identified innate-like subset of Be1/Beff cells^5,53,54.

In addition to innate-like Beff cells described above, pro-inflammatory B cells are also active in more protracted autoimmune and tumor settings^41,42. For example, loss of B cell IL-6 reduces Th1 and Th17 responses, reducing the severity of EAE^4,20,55. Additionally, loss of B cell IFNγ reduces Th1 responses, resulting in increased Tregs and resistance to proteoglycan-induced arthritis^4,20,55. The phenotype of these Beff cells was previously unknown. In this regard, we recently showed that another TIM-family member, TIM-4, is a broad marker for Be1 cells that are enriched for IFNγ and low in IL-10 expression (and encompass the innate-like CD11a^HiFcɣRIII^Hi subset). TIM-4⁺ B cells promote Th1 polarization while reducing Tregs and regulatory cytokines such as IL-10. As such, they enhance allograft rejection and reduce tumor growth and metastasis in an IFNγ-dependent manner⁴⁷. B cells also play a requisite role in chronic rejection of murine cardiac allografts⁵⁶. While the phenotype and exact mechanism are unknown, this role is not antibody-mediated, and is driven by both antigen presentation and maintenance of splenic lymphoid architecture required for productive immunity.

Human Bregs and Beffs:

Based on the studies of Bregs in mice, various groups set out to identify subsets of human B cells enriched for IL-10 expression that might be defective in autoimmune settings. While no specific phenotype was discovered, Blair et al. found that IL-10+ B cells were enriched amongst the CD24^hiCD38^hi TrB subset. Moreover, TrB cells from SLE patients were defective in IL-10 expression when stimulated through the CD40 pathway, compared to normal subjects¹⁷. Subsequently, Iwata et al. showed that human CD24^hiCD27⁺ memory B cells expressed the most IL-10, but no defects in IL-10 expression were seen in a variety of autoimmune subjects, including SLE patients⁸‘⁵⁷. Since then, various additional phenotypes have been reported to enrich for IL-10+ human B cells including, TNFR2⁺, CD25^hiCD71^hiCD73⁻, CD27⁺CD43⁺CD11b⁺, and TIM1⁺ B cells, and CD27^intCD38^hi plasmablasts, and each of these subsets has been shown to suppress pro-inflammatory T cell responses in vitro^{8,17,50,58–61}. Of note, there is a significant overlap between some of these subsets. For example, TNFR2+ and TIM1+ B cells are both enriched in IgM+ memory B cells and TrBs^59,60.

As in mice, only a small proportion of B cells within each of these human subpopulations actually express IL-10, and IL-10 is expressed by multiple B cell subsets. Indeed, we found that several major B cell subsets (e.g. TrB, Memory, and naïve) all express IL-10 at relatively high frequencies (10–15%)¹¹. Importantly, we found that B cells within these same subsets also co-express inflammatory cytokines like TNFα, and that the ratio of IL-10/TNFα correlated best with in vitro regulatory activity. Thus, TrBs (high IL-10/TNFα ratio) were able to suppress T cell inflammatory cytokine expression in vitro, whereas neither memory nor naïve B cells (low IL-10/TNFα ratios) could not. However, both naïve B cells and memory B cells became suppressive in vitro when TNFα was neutralized, and conversely, TrBs lost their suppressive activity when IL-10 was neutralized. Finally, while IL-10 alone was unchanged, the TrB IL-10/TNFα ratio fell with acute renal allograft rejection. These data highlight the limitations of current makers, demonstrate the importance of measuring cytokines rather than just phenotype, and importantly, suggest that Bregs and Beff cells might both contribute to outcomes. These findings are supported by studies in MS patients. B cells from patients with MS expressed lower IL-10 with high TNFα expression. Importantly, the ratio of B cell IL-10/TNFα expression correlated with disease relapses. However, the phenotype of the cells that express either IL-10 or TNFα was not examined^18,62.

In summary, none of the current phenotypic markers for human Breg populations are specific. They identify subpopulations enriched for IL-10 but are not necessarily representative of the majority of IL-10+ B cells that might also exhibit potent Breg activity. Further, even more poorly defined Beff cells are present in the same canonical B cell subsets and may counteract Breg activity and influence the outcomes observed^44,47. Thus, we believe that the balance of IL-10/TNFα, expressed particularly by immature TrBs, is a better read-out of Breg (or Breg/Beff) activity than either B cell subsets or IL-10 alone.

Bregs and clinical transplantation: Is there a Breg signature of operational tolerance?

Evidence accumulating over the last two decades strongly supports a potent immunomodulatory role for B cells in clinical transplantation. Bregs have been extensively studied in the context of operational kidney transplant tolerance (defined as patients with stable renal function despite withdrawal of immunosuppression). Several studies have shown an increase in TrBs and/or IL-10+ B cells in peripheral blood of operationally tolerant renal transplant patients when compared to patients with stable function on immunosuppression, or those with chronic rejection^9,63–65. A similar B cell “signature” was also noted in patients rendered tolerant via induction of mixed chimerism, and this phenotype remained stable over time⁶⁶. It must be emphasized that these studies failed to detect differences in TrBs or their IL-10 expression, between tolerant patients and healthy subjects, and subsequent studies have shown that the differences seen in tolerant vs. stable transplant patients might be due to immunosuppression itself^67,68. Moreover, since these studies were not prospective, it is unknown whether these changes in TrBs could predict tolerance in patients prior to immunosuppression withdrawal.

TrBs from operationally tolerant patients have been shown to be more suppressive of autologous T cell responses than those from stable patients still on immunosupression⁶⁴. In this regard, Nova-Lamperti et al. showed that B cells from tolerant patients exhibited high CD40 expression, low CD86 expression, and low Erk phosphorylation upon BCR engagement, when compared to both stable patients on immunosuppression and healthy volunteers. All three mechanisms were related to increased B cell IL-10^64,65. Another study showed that B cells that secrete granzyme B in vitro were specifically increased in operationally tolerant patients when compared to both stable patients on maintenance immunosuppression and healthy volunteers¹². These cells exhibited a plasma cell phenotype (CD138^hi) and inhibited T cell IL-21 secretion, while other T cell proinflammatory cytokines including TNFα and IFNγ remained unaffected¹². Despite the excitement over these potentially important findings, it should be noted that the ‘B cell signature’ of tolerance with predominance of Bregs, is seen in only kidney transplantation. Specifically, it is not found in other organs such as liver, where patients prospectively undergo immunosuppression withdrawal^9,63–65.

Bregs and clinical transplantation: Can Bregs predict transplant outcomes?

In the vastly more common non-tolerance setting, Bregs appear to play an important role inhibiting rejection and promoting good outcomes. As previously mentioned, B cell depletion in the peri-transplant period can markedly increase the rate of acute renal allograft rejection, and in cardiac allograft recipients, it may be associated with an increased rate of cardiac transplant vasculopathy. This suggests that Bregs, inadvertently depleted by anti-CD20, may play a critical protective role in the peri-transplant period in transplant patients with “standard immunological risk”^31,69. In contrast, rituximab (anti-CD20) has been added successfully to plasmapheresis and IVIG in peri-transplant desensitization protocols in HLA-incompatible transplantation⁷⁰. Interestingly, B cell repopulation after rituximab treatment of such high-risk patients, is characterized by an increase in allo-specific TrBs, a decrease in allo-specific memory B cells, and no increase in acute rejection. This suggests that allospecific TrBs could contribute to improved outcomes in HLA incompatible transplant recipients⁷¹. This scenario is reminiscent of MS, which is responsive to B cell depletion therapy. While MS patients exhibit high TNFα and low IL-10 in their peripheral B cells, this aberrant cytokine ratio is corrected in newly emerging B cells after Rituximab therapy. Taken together, these studies suggest that the immunological status of a given patient may reflect the relative proportion of Bregs/Beffs – and therefore affect the relative depletion of these two subpopulations after B cell depletion therapy. In renal transplant patients of “standard risk”, there may be a relatively a higher proportion of Bregs than Beffs, and B cell depletion jeopardizes engraftment of the kidney transplant. In autoimmune or allosensitized patients, Beffs may predominate, and B cell depletion will likely have a salutary effect on engraftment/autoimmunity.

In several small single center studies, a higher number of TrBs in peripheral blood was independently associated with protection from rejection and positively correlated with eGFR and superior graft survival in kidney transplantation^72–75. For example, we showed that a lower number of TrBs two years after renal transplantation was associated with higher rates of rejection and DSA, and a lower eGFR at 2 years, along with a significant decline in eGFR from 6 months to 2 years.⁷⁵. Subsequently, Shabir et al. showed that higher TrB frequency in the 1st year was associated with protection from acute rejection over a 5-year follow-up period. In contrast, a low TrB frequency was associated with significantly increased risk of rejection⁷³. Finally, in a small prospective study, patients with acute renal allograft rejection were subsequently found to have an increase in CD86+ B cells and plasmablasts and a significant reduction in TrBs and granzyme B+ B cells at one year⁷⁶.

While the above studies may be informative, cytokine expression by B cells/subsets was not examined. This limits our ability to understand the underlying changes in Bregs/Beff that occur in various clinical settings that could improve accuracy, understanding and ultimately treatment strategies. In this regard, we examined 47 patients 2–20 years post-transplant who had for-cause renal transplant biopsies¹¹. Patients with graft dysfunction who experienced rejection exhibited a reduction in TrB number and frequency compared to patients with graft dysfunction without rejection, a comparable group of stable patients, or healthy controls. However, the TrB IL-10:TNFα ratio was specifically decreased and showed the strongest association in patients with graft dysfunction and rejection. Moreover, the TrB IL-10:TNFα ratio was highly correlated with in vitro Breg function. Indeed, TrBs from rejecting patients specifically lost their in vitro regulatory activity. Importantly, at the time of the late for-cause biopsy the TrB IL-10:TNFα ratio (but not IL-10 alone) could strongly predict the presence of rejection (ROC AUC, 0.82, p<0.0001)¹¹. Moreover, amongst patients exhibiting rejection, patients with a higher TrB IL-10/TNFα ratio exhibited significantly better allograft survival over the following three years. Taken together, these findings again implicate the balance between Bregs and Beffs as a possible driving force for adverse transplant outcomes. Of note, most patients in this study suffered from chronic antibody-mediated rejection (CAMR). These findings are generally supported by Nouel et al, who showed that B cells from patients with CAMR exhibit a decreased number of TrBs and lose suppressive capacity in vitro compared to B cells from healthy volunteers or those with stable allograft function⁷⁷. However, we believe that the TrB cytokine ratio adds valuable information related to diagnosis, prognosis, and Breg/Beff function in the setting of allograft rejection versus quiescence.

The above studies were extended by showing that the ratio of T1/T2 transitional B cells closely reflects the changes in the TrB IL-10:TNFα ratio and might serve as a simpler marker for Breg/Beff activity. Importantly, a low T1/T2 ratio in stable patients 2-year post-transplant was independently associated with and strongly predicted graft outcomes over a 5-year follow-up period (ROC AUC >0.8, p<0.0005), whereas clinical parameters including DGF, Creatinine and DSA were not predictive (ROC AUC range 0.56–0.66)⁷⁸. Based on promising results in patients with late rejection and stable function at 2 years, we are now prospectively examining the TrB cytokine ratio in the early post-transplant period as a predictive biomarker for subsequent rejection and outcomes.

Bregs, especially TrBs, have also been studied in stem cell transplant settings. For example, TrBs constitute a majority of B cells in the cord blood and are enriched for IL-10+ B cells that suppress in vitro T cell proliferation and pro-inflammatory cytokine expression⁷⁹. Importantly, in patients who received cord blood transplants, development of GVHD was associated with a sharp decline in IL-10+ B cells amongst the reconstituting B cells, and B cells from such patients lose their ability to suppress allogeneic T cells in vitro⁸⁰. Similarly, putative Breg subsets (TrB and IgM+ memory B cells) were also reduced in number and lost in vitro Breg activity in stem cell transplant recipients with chronic GVHD⁷⁹. Taken together, these studies suggest that Bregs, or the balance of Bregs/Beffs, may help establish an important “immunological set-point”. Moreover, they might potentially serve as strong biomarkers that can aid clinical decisions in transplantation.

Effects of therapeutic agents on Bregs:

Given the potentially important role of Bregs in modulating alloresponses, promoting a state of allograft tolerance, and preventing allograft rejection, it is important to consider the impact of drugs routinely used to treat transplant patients on Breg number, function, and their ability to promote allograft survival. A variety of immunosuppressive agents targeting T cells, also inhibit or deplete B cells and potentially affect Breg number. For example, Alemtuzumab (anti-CD52) profoundly depletes peripheral B cells along with T cells. B cell reconstitution following Alemtuzumab is predominantly comprised of immature TrBs and is associated with a prolonged suppression of memory B cells. Interestingly, a reduced number of TrBs in the reconstituting B cell pool after Alemtuzumab induction, was associated with increased rejection and DSA detection along with poor allograft function^75,81–83. In comparison, Thymoglobulin leads to B cell depletion, though its effect on memory B cells may be less pronounced^83–85. In contrast, Basiliximab (anti-CD25) induction does not deplete peripheral B cells, but may result in a phenotype dominated by memory cells⁸⁶. Rituximab (anti-CD20), which has been used both as an induction agent especially in HLA incompatible transplant recipients and to treat Antibody-Mediated Rejection (ABMR), results in a significant long-lasting B cell depletion, particularly of naïve B cells and possibly memory B cells⁸⁷. As noted above, wholesale B cell depletion with Rituximab may have variable effects on autoimmune and transplant patients – perhaps depending on their immunological status and relative Breg/Beff ratio. Finally, glucocorticoids, used both in induction and maintenance regimens, may affect B cell function by promoting apoptosis within the specific B cell subsets^67,88. This reduces B cell numbers, particularly within the TrB subset. Interestingly, despite the routine use of glucocorticoids for the treatment of acute allograft rejection, early ex vivo studies suggest that in therapeutic dose ranges, they actually enhance antibody production^89,90.

Almost all commonly used maintenance immunosuppressive agents in transplantation influence B cells and may directly or indirectly affect Bregs. For example, Cyclosporine A reduces immature B cells (TrBs) with less effect on memory or mature B cells⁷². Mycophenolic Acid (MPA) has been shown to cause a dose dependent reduction in B cell IL-10 and decrease expression of CD80 and CD86^91,92. Further, MTOR inhibitors are associated with a reduction in TrB numbers and an increase in CD27+ memory B cells⁹³. Finally, Belatacept, a selective T cell co-stimulation blocker, may promote a more regulatory B cell phenotype with increased IL-10+ B cells, higher TrB cell frequency, and reduced B cell differentiation into plasmablasts, when compared to CNI therapy^94,95. A recent study has demonstrated that addition of Belumimab (anti-BLyS, a B cell survival factor) to Tacrolimus, mycophenolate and prednisolone in Basiliximab-induced renal transplant patients led to a significant increase in the ratio of B cell IL-10/IL-6 in the first three months post-transplant. However, the clinical implications of this finding remain to be studied⁹⁶. As mentioned above, a recent analysis of operationally tolerant kidney transplant recipients reported that TrB number is decreased with the use of either prednisolone or azathioprine and conversely, withdrawal of steroids was associated with a significant increase in their number⁶⁷. The fact that almost all immunosuppressive agents routinely used in the clinical management of transplant patients influence B cells, and specifically Bregs, has potentially important diagnostic and therapeutic implications. More studies are needed to understand which combinations might promote, rather than inhibit, IL-10+ B cells. Moreover, studies examining B cell subsets, including Bregs, as prognostic or diagnostic markers in transplantation, must carefully consider the effects of these agents on B cell phenotype and the potential for the resultant bias.

Strategies to expand Bregs in humans

Given evidence for their beneficial role in murine and human transplantation, expansion of Bregs and inhibition of Beffs, could be an important pro-tolerogenic strategy. Murine studies suggest that specific expansion of Bregs or inhibition of Beffs may be possible. In this regard, TIM-1 and CD9, both broad markers for IL-10+ Bregs, have been shown to have functional roles in mice. For example, TIM-1, positively regulates IL-10 expression by B cells and treatment of mice with an anti-TIM-1 antibody (RMT1–10) results in 2–4 fold expansion of IL-10+ Bregs, which are essential for prolonged allograft survival^{44,47–49,97}. Moreover, a loss-of-function TIM-1 mutation decreases both basal Bregs and promotes allograft rejection^48,49. This suggests that binding of apoptotic cells to TIM-1, a phosphatidylserine receptor, may be important for maintaining basal Breg levels. Moreover, apoptotic cells increase IL-10+ Bregs in vitro and in vivo through TIM-1 binding, and can inhibit collagen-induced arthritis^47,48,98. TIM-1 is enriched on human IL-10+ B cells, suggesting a therapeutic potential.

Other factors negatively regulate inhibit Breg expansion. For example, CD9-deficient mice have almost double the number of IL-10+ B cells, indicating an inhibitory effect of CD9 on Breg expansion⁴⁶. Similarly, CD22 negatively regulates BCR signaling and inhibits IL-10 expression⁹⁹. Finally, Breg frequency is reduced in the presence of inflammatory cytokines such as IFNγ and TNF-α^11,47. Though the mechanisms are unclear, this suggests a reciprocal relationship between Bregs and Beff. In this regard, anti-TIM-4 is a potent tolerogenic agent in murine allograft models⁴⁷. Its tolerogenic activity is wholly dependent on the presence of TIM-4+ B cells [Qing Be1 paper]. Anti-TIM-4 inhibits IFNγ expression by TIM-4+ Beff cells and this leads to a reciprocal increase in IL-10 expression by TIM-1+ Bregs, though the mechanisms are unclear.

In humans, a variety of agents including Interferon-β, Fingolimod, Liquinimod, Tocilizumab, and Infliximab that are used to treat autoimmune disorders, may enhance Breg numbers and/or activity^14,100–107. Additionally, other agents such as vitamin D, retinoic acid and Sotrastaurin can induce ex vivo B cell proliferation and ensuing IL-10+ Breg expansion might contribute to their therapeutic efficacy^91,108,109. Many such drugs have not been examined in the transplant arena.

Another approach to Breg-based therapy utilizes adoptive therapy of Bregs expanded ex vivo. It has been noted in mice, that various cytokines induced in inflammatory settings, including IL-1β, IL-4, IL-6, IL-21, IL-35 and IFN-α can expand Bregs in vitro or in vivo^{14,44,110–112}. Remarkably, in vitro culture using IL-4 and IL-21 along with CD40L/BAFF-expressing feeder cells was shown to expand murine Bregs up to a million-fold¹¹². Transfer of these ex vivo expanded Bregs ameliorated EAE, proving that their regulatory activity was retained. Similarly, transitional B cells from healthy human donors can be expanded with IFN-α and CpG-C ex vivo and retain their IL-10 expression, phenotype, and Breg activity¹⁴. Importantly, this same approach was unsuccessful using B cells from SLE patients. In an in vivo setting, Bregs may interact with other cell types that could provide cytokines that promote their expansion, such as macrophages (IL-1-β, IL-6), Tfh (IL-21), B cells (IL-35) and pDCs (IFN-α)^14,112.

Conclusion

Bregs and Beffs exhibit a potent ability to modulate immune response and could potentially alter the course of various autoimmune, alloimmune, and infectious processes. Accumulating evidence suggests that increased Breg frequency and intact Breg function correlates strongly with the development of a tolerant clinical state, reduced rejection episodes and improved long-term allograft survival. Therefore, strategies that specifically aim to expand Bregs or enhance their function, or conversely inhibit/deplete Beffs, represent promising therapeutic options to improve transplant outcomes. This aim is hindered by our limited understanding of Breg and Beff biology, and limited knowledge of their development, induction, and in vivo effector function. This is compounded by lack of specific markers that would allow us to identify these cell populations for monitoring, or as targets for depletion. Nonetheless, protocols that can expand B cells (in vivo or in vitro) that are highly enriched for IL-10, while expressing low levels of pro-inflammatory cytokines, have potential to reset the immune set-point and improve clinical outcomes. Finally, given that alterations within the Breg subsets may precede rejection episodes, Bregs may play a promising role as biomarkers to guide pre-emptive treatment of at-risk transplant recipients.

Key Points.

1. No specific phenotypic marker exists for Bregs or Beffs in either animal models or in humans.

2. Bregs are frequently identified by the expression of their “signature cytokine”, IL-10.

3. Relative expression of IL-10 to TNFα in immature B cells in peripheral blood is a good marker for human Breg or Breg/Beff activity.

4. Bregs may serve as a marker of human renal allograft tolerance and may be able to predict transplant outcomes.

5. Strategies to expand Bregs in vivo and ex vivo exist, suggesting that Bregs have therapeutic potential in clinical transplantation.

Synopsis.

In addition to humoral immunity, B cells shape the alloimmune response through polarized subsets exhibiting regulatory and effector function. Respectively, these Bregs and Beffs inhibit or promote immune responses through the expression of suppressive or pro-inflammatory cytokines. The summed activity of Bregs and Beffs likely dictates the influence of B cells on the alloimmune response in a given transplant patient.

Here, we review the evidence for Bregs and Beff cells in both mice and humans, discuss current limitations in their phenotypic identification, and discuss Bregs as a “signature” for clinical renal allograft tolerance and as predictive markers for allograft outcomes. Finally, we discuss the effects of therapeutic agents on Bregs and potential approaches to augment their numbers as a therapeutic tool.

In summary, Breg and Beff cells play an important role modulating the immune response and may provide both a diagnostic tool and therapeutic opportunity to improve long-term transplant outcomes.