What are really the multiple roles of B cells in organ transplantation? In this review or point of view, we discuss the need to re-examine, integrate and discriminate B-cell multifunctionality in organ transplantation, involving both regulatory (REG) and inflammatory (INFLAMMA) activities, highlighting the emerging debate on the role of regulatory B cells (Bregs).
FACING MULTIFUNCTIONAL IMMUNOLOGIC COMPLEXITY
Our understanding of B-cell functions in organ transplantation is actually moving in the same direction as several other topics in immunology are today. That is, realizing that no single immune molecule or cell type has a fixed intrinsic functional activity, although some may, indeed, have a predominant functional profile in several pathological and physiologic contexts. We may cite several examples, such as IFN-γ, which is considered a dominant inflammatory cytokine and yet it seems to play a role in immunotolerance, as several protocols to induce immune tolerance are, in fact, ineffective in IFN-γ-deficient mice . Dendritic cells may induce both effector/proinflammatory  and tolerogenic immune responses , and regulatory T cells (Treg) and Th17 T cells may have a plastic functional phenotype and transit in both directions . These are examples of immunologic plasticity, which is most likely influenced by multiple factors such as the microenvironment and its metabolic, immunologic and tissue-specific factors, and also by the neuroimmune cross-talking capacity to sense cues of homeostatic disturbance in a particular physio/pathologic context .
Similar examples may be pointed in transplantation, such as T-cell alloreactivity, which despite being critical for triggering allograft rejection , may also induce immunoregulation, as reported by several groups [6–9] including ours . It seems that the scientific community is now more focussed on investigating the REG and INFLAMMA components/mechanisms of any given physiologic and pathological immune phenomenon, understanding that the functional immunologic outcome is dependent on how inflammation is fine-tuned. It depends on the REG/INFLAMMA balance, locally and systemically.
During the past decades, the focus of B-cell research in clinical transplant immunology has been mostly directed to avoid the production of donor-specific alloantibodies (DSAs). However, B cells are more than mere antibody factories. B cells have all the apparatus to present antigen to T cells , they express high levels of class II MHC and costimulatory molecules, they secrete various cytokines [12–16], can prime T cells and initiate or regulate an effector immune response. Like T cells, B cells also have multiple functional activities.
Indeed, the importance of B cells in cellular immune responses has been highlighted, ever since experiments using B-cell-deficient mice exhibited significant impact on the immune system's function. It was shown that B cells are, in fact, crucial for CD4+ T-cell expansion, cytokine secretion , for the establishment of T-cell memory  and, more recently, that they are also present in the thymus and may have a role in T-cell ontogeny . Additionally, the observation that mice devoid of B cells have a worse progression of experimental autoimmune encephalomyelitis [20–22] indicates their important role in regulating pathological autoimmunity.
REVISITING THE PRIMARY ROLE OF B CELLS IN TRANSPLANTATION: ANTIBODIES
The inflammatory role of B cells in organ transplantation has been consistently associated with anti-HLA alloantibodies and DSAs, but also with antibodies of other specificities [23,24]. Nevertheless, despite consistent data on the deleterious role of DSAs [25–27], especially for de-novo antibodies detected after transplantation [28–30], the debate regarding alloantibodies not associated to poor graft outcome remains [31–33]. This raises the discussion about the need for a finer view of alloantibody function, because not all alloantibodies seem to be deleterious, even DSAs. Accordingly, it was recently reported that the detection of multiple IgG subclasses was higher in liver chronic rejection and that recipients with IgG3 DSAs presented higher risk of graft loss, whilst those presenting normal graft function had mostly IgG1 DSAs . The mechanisms underlying these observations await elucidation.
Autoantibodies also comprise the immunobiology of organ transplantation. This is in accordance with the observations that transplantation-induced allostimulation modifies autoreactivity both at the cellular  and antibody levels [35–37]. The detection of autoantibodies directed to a variety of molecules such as alpha-tubulin [38▪,39], vimentin [40,41], or angiontensin receptor  in kidney transplantation, nucleolin in kidney and heart transplantation [43▪], as well as type V collagen in lung transplantation  has been associated to rejection and poor graft outcome. Additionally, antibody-mediated chronic kidney rejection was associated with the detection of a broad array of autoantibodies, suggesting a B-cell dysregulation . Some of these autoantibodies exhibit deleterious effects on the graft by various mechanisms, such as activation of the angiotensin II receptor signalling pathway  and inhibition or induction of endothelial cell apoptosis [43▪]. On the other hand, autoantibodies also comprise the physiologic repertoire [46,47] and may play a beneficial role in keeping the biologic REG/INFLAMMA balance [48,49].
THE IMPACT OF ORGAN TRANSPLANTATION ON THE B-CELL COMPARTMENT
Beyond inducing antibody production, allotransplantation induces several impacts on the B-cell compartment; many associated with an unfavourable graft outcome. One example is the uncontrolled mobilization and activation of B cells [50,51], associated with increased risk of DSAs development and graft loss, suggesting that overactivation of B cells may drive an inflammatory outcome.
Another interesting emerging issue regarding the role of B cells in transplantation is the formation of the so-called tertiary lymphoid tissue (TLT) within the graft [52▪▪], mostly in chronic rejection [53,54▪▪], and their putative role in keeping long-term alloantibody production and continued graft damage. Most investigators highlight an inflammatory role for B cells in this context, probably because this intragraft lymphoid organization is mainly observed during rejection . Recently, in a murine model of skin transplantation, the authors claim that B-cell tolerance is actually broken in the intragraft TLT, as they detected more autoantibodies within the graft than in the blood [54▪▪]. These are interesting data, but it is not clear whether these antibodies were in fact produced within the graft or if they are in fact deleterious. Additionally, we cannot exclude that Bregs can also comprise this intragraft TLT and play a beneficial role in the graft outcome.
B cells have been underscored as important players in transplant immune responses, as recently reviewed [55–59]. For example, B cells participate in the control of graft versus host disease, in which animals without circulating B cells develop a more severe disease . In experimental models, mice displaying MHC-II-deficient B cells exhibit increased cardiac allograft survival compared with wildtype mice , whilst no difference was observed in skin allograft . Moreover, it was recently shown that different B-cell populations can exhibit different functions in transplantation, also depending on the intensity of the allogeneic response [62▪▪]. Animals partially depleted of B cells, using an anti-CD20 antibody, presented accelerated skin allograft rejection, across minor antigen disparities, suggesting a role for B cells in controlling inflammation [62▪▪]. In contrast, in a model of chronic renal allograft rejection, a broader depletion of various B-cell subpopulations, using an anti-CD19 antibody, actually increased allograft survival [62▪▪]. These data indicate a dual role for B cells in transplantation, exhibiting both regulatory and inflammatory functional activities.
EMERGING ROLE OF BREGS IN ORGAN TRANSPLANTATION
The capacity of B cells to regulate effector immune responses has been much attributed to Bregs . Recently, Breg phenotypes and their working mechanisms have been much debated [11,63,64]. Nevertheless, it has been consistently observed that Bregs play an important role in controlling effector immune responses and autoimmune diseases [22,65–67], mostly by IL-10 secretion and direct contact with CD4+ T cells [65,66]. In humans, a Breg subpopulation, called B10 cells, has been characterized by the CD19+CD24HICD27+ phenotype and an IL-10 dependent capacity to suppress monocyte proinflammatory cytokines [68▪▪]. Another Breg subpopulation displaying the CD19+CD24HICD38HI phenotype exerts immunoregulation also mediated by IL-10, requiring CD80/CD86 costimulation and the activation of the CD40/STAT3 signalling pathway [69▪▪].
Additional Breg phenotypes have been reported in the transplantation models, such as B cells bearing high expression of the inhibitory Fc-gamma receptor IIB, in cardiac allograft tolerance [70▪▪], or TIM-1 (T cell Ig domain and mucin domain), in islet allograft transplantation [71▪]. In the latter, the stimulation with a low-affinity anti-TIM-1 antibody prolonged allograft survival in an IL-4/IL-10 dependent manner [71▪]. In both models, adoptively transferred total B cells or TIM1+ Bregs prolonged allograft survival in relation to untreated animals, suggesting the capacity to induce infectious tolerance [72,73]. Thus, B cells also play a regulatory role in the context of transplantation.
In line with this idea, observations on operational tolerance (stable graft function after immunosuppression withdrawal for more than 1 year [35,74–77]) have brought additional elements supporting the B-cell regulatory function in allotransplantation. Taken that operational tolerance individuals have managed to control graft inflammation, the operational tolerance differential immunologic profile may help identify relevant immunoregulatory checkpoints in human transplantation tolerance.
On the basis of the data from several research groups, there is a current belief that B cells play a relevant role in operational tolerance. Several immunologic features have been proposed for a B-cell gene signature in operational tolerance kidney transplant recipients in the peripheral blood mononuclear cells: higher transcript expression of immunoglobulin light chains (IGKV4-1, IGLLA and IGKV1D-13) , CD20 , genes related to B-cell proliferation and cell cycle ; higher numbers of circulating total, naive and transitional B cells [35,78,79]; increased IL-10 expression in transitional B cells  (phenotypically similar to human Bregs [69▪▪]) and B cell increased expression of the inhibitory Fc-gamma receptor IIB .
However, there are some yet unclear and contradictory interpretations on the B-cell data in operational tolerance. For example, no immunoregulatory function has been reported for the IGKV4-1, IGLLA and IGKV1D-13 genes . Additionally, there is a contradictory interpretation regarding the increase in circulating B cells in operational tolerance [35,78,79]. As operational tolerance individuals have B-cell numbers similar to healthy individuals, but higher than recipients with chronic rejection, we interpret that rather than having increased mobilization of B cells, operational tolerance individuals seem to preserve a healthy profile of the B-cell compartment [35,78–80▪].
We have emphasized this distinct interpretation regarding the B-cell compartment in operational tolerance in a recent work [80▪]. We confirmed that operational tolerance individuals exhibit preservation in the numbers of circulating B cells, particularly the CD19+CD24HICD38HIBregs, in contrast to individuals with chronic rejection. Further comprising the operational tolerance B-cell profile, operational tolerance's Bregs display a conserved capacity to activate the CD40/STAT3 signalling pathway (pathway important for IL-10 production, as described by Blair et al.[69▪▪]) and preserved B-cell repertoire diversity [80▪].
In addition to the above-mentioned operational tolerance B-cell profile, microRNAs (miRNAs) also appear to integrate this complex network. The emerging role of miRNAs in immunoregulation is quite unexplored in organ transplantation. MicroRNAs are noncoding sequences of RNA bearing important roles in gene-expression regulation . It was recently observed that operational tolerance's circulating B cells have preserved expression of the miR-142-3p miRNA, whilst individuals with stable graft function under conventional immunosuppression and those with chronic rejection have decreased expression [82▪▪]. Taken that this specific miRNA is involved in the regulation of several genes associated with the proposed operational tolerance B-cell signature [35,74,78], this miRNA and possibly others are likely to integrate the immunotolerance network in human organ transplantation.
Recent data from our group and others support the view that the differential expression of some important immune molecules, including miRNAs, together with the preservation of the B-cell compartment and the upregulation of some specific pathways may be relevant immunologic checkpoints in achieving/maintaining this homeostatic state. Understanding how operational tolerance individuals manage to keep a healthy B-cell profile and what operating mechanisms disturb the REG/INFLAMMA B-cell compartment balance, in chronic rejection, may help the design of novel therapeutic strategies to favour allograft tolerance.
THE REG/INFLAMMA BALANCE: SHAPING THE B-CELL REPERTOIRE TOWARDS TRANSPLANTATION TOLERANCE
Considering the primary idea that B cells were essentially proinflammatory players in clinical transplantation, several therapeutic strategies have been put forth to control B-cell activity. One strategy is the use of the chimeric anti-CD20 antibody (rituximab) to deplete B cells in patients with DSAs [83–85]. The CD20 molecule is a B-cell marker expressed during B-cell ontogeny – from pre-B to memory B cells (though a lower expression) – but not expressed by pro-B cells or by plasma cells [58,86]. Overall, the real benefits of the anti-CD20 antibody therapy in human transplantation remain controversial . It shows limited effect in the desensitization of patients with DSAs, maybe because it does not affect plasma cells. Indeed, rituximab inhibits naive B-cell proliferation, in vitro, but has little effect on memory B cells [87▪▪], which could sustain DSA production and deleterious effect on graft outcome.
Recently, rituximab was used as induction therapy, in a double-blinded prospective clinical trial, to prevent the activation and generation of antibody-producing cells . Although well tolerated, the use of rituximab plus corticosteroids, as induction therapy, showed no real benefit in a 6-month follow-up . A more striking result comes from the aborted clinical trial comparing the use of rituximab and daclizumab (an anti-CD25 monoclonal antibody), again as induction therapy in kidney transplantation. The rituximab-treated group showed higher prevalence of acute cellular rejection (83 versus 14%) in the first 3 months after transplantation . The use of corticosteroids may have contributed to preventing cell-mediated rejection in the first trial. However, these latter data emphasize the B-cell role in downregulating graft inflammation and indicate that the indiscriminate deletion of B cells in transplantation may be deleterious.
There seems to be a general understanding today that developing strategies to help reshape recipients’ B-cell repertoire may be valid to control alloantibody-producing cells and to induce Bregs, favouring transplantation tolerance. But what are really efficient strategies to favour the B-cell regulatory activity (Fig. 1)? No direct answer is possible yet. However, a broader REG/INFLAMMA view on both basic research and clinical studies may bring relevant information and help the transplantation community to rethink and develop novel strategies.
In the search for new strategies to solve the problem of circulating alloantibodies, the use of the proteasome inhibitor, bortezomib, has recently been tested [90,91,92▪]. The proteasome inhibition in plasma cells induces the activation of the unfolded protein response, leading to apoptosis [93,94]. The use of bortezomib together with plasmapheresis has been shown to dramatically reduce the circulating plasma cell numbers and alloantibodies in renal transplantation [91,95,96▪]. However, the incomplete elimination of alloantibodies has been attributed to remaining circulating memory B cells that escape bortezomib's action.
Long after the use in several animal models , the humanized version of the anti-CD52 antibody, alemtuzumab, has been attempted as a broader immunoregulatory therapeutic strategy, as CD52 is expressed on T and B cells, monocytes and dendritic cells [98,99]. As CD52 is expressed from the pro-B to the short-lived plasma cell stages , alemtuzumab provides a high spectrum depletion of circulating B cells and, potentially, a broad reconstruction of the B-cell repertoire. The benefits of alemtuzumab as induction therapy dates from 14 years ago, when it was reported to induce a state ‘near’ to tolerance in kidney transplantation, allowing significant immunosuppressant reduction [100,101]. One of the largest prospective trials to evaluate alemtuzumab's efficacy showed a marked reduction of biopsy-confirmed acute rejection in the first 3 years after transplantation, compared to anti-CD25 antibody (basiliximab), whilst no difference was found with rabbit antithymocyte globulin [102▪▪].
It is worth revisiting these data in the light of today's new knowledge on immunoregulatory mechanisms. Alemtuzumab's beneficial effect can be reinterpreted based on two recent works showing that the induction therapy, in transplanted individuals, promoted a reshaping of the B-cell repertoire, decreasing the numbers of memory B cells and increasing naive B cells and CD19+CD24HICD38HI Bregs [103▪▪,104▪▪]. Thus, alemtuzumab seems to induce a more tolerogenic B-cell repertoire in the first few years after treatment. However, it remains important to evaluate the long-term impact of alemtuzumab's therapy and which other combinatory therapies could help to induce and maintain a predominant regulatory B-cell repertoire.
Nevertheless, an important side-effect of alemtuzumab therapy is the significant increase in circulating BAFF , a B-cell survival signal with a central role in the differentiation of transitional B cells to mature B cells [106,107]. BAFF-receptor knockout mice display reduced numbers of mature B cells and an accumulation of transitional B cells [106–108], whereas animals with high levels of circulating BAFF present B-cell compartment expansion and develop pathological autoimmunity [106–108]. So, changes in BAFF circulating levels may have an impact on the availability of peripheral mature B cells and on the numbers of activated memory and plasma cells. Accordingly, the effects of increased circulating BAFF could well be the cause of the increased frequency of antibody-mediated rejection in alemtuzumab-treated individuals [109▪].
So, to improve the alemtuzumab's beneficial effects in organ transplantation it may be important to control the levels of circulating BAFF following treatment, such as by using the anti-BAFF monoclonal antibody (belimumab) . It was recently reported that the combinatory therapy with anti-BAFF and rapamycin induce allograft tolerance in a model of islet transplantation, by reducing IFN-γ production, increasing IL-10 and the percentages of circulating Tregs [111▪▪]. Although it is well known that rapamycin induces Tregs , BAFF neutralization in this combinatory therapy seems to potentiate this effect. The expansion of Tregs is in concordance with another report on the expansion of CD4+FOXP3+ T cells in BAFF-transgenic mice, by a B-cell-dependent mechanism , indicating a dual functional role for BAFF in transplantation. The reshaping of the B-cell repertoire towards a predominant REG profile seems to require, on one hand, the control of alloreactive memory B cells and alloantibody-producing plasma cells, and, on the other hand, the induction/preservation of the Breg repertoire in addition to controlling BAFF circulating levels.
Bringing additional complexity to B-cell function in organ transplantation, we should remember that B cells are in constant interaction with both the graft and other immune cell types, some of which may also be relevant checkpoints in the immunoregulatory pathways to transplantation tolerance, such as Tregs and dendritic cells. Thus, quantitative and qualitative alterations in one cell population may functionally affect the others. This may be illustrated by the data showing that mice deficient for the important Treg transcription factor, FOXP3, may also present B-cell developmental defects . Moreover, we reported that operational tolerance individuals are able to keep normal numbers of Foxp3 Tregs  and Bregs [80▪], whereas chronic rejection individuals have significantly lower numbers of both. These observations suggest that the inability of chronic rejection individuals to keep the healthy profile of the regulatory T and the B cell compartments may involve a T–B cell cross-talking process. Therefore, it is possible that the beneficial or deleterious outcome of the B-cell multifunctionality is the result of various systemic and intragraft interactions with other cell types.
A significant emergent knowledge regarding B cells in organ transplantation is that B cells can also act as beneficial players. They can help to control graft inflammation and could contribute to building regulatory friendly pathways leading to operational tolerance.
Considering the B-cell regulatory function in organ transplantation, we should question the paradigm of depleting B cells as the central strategy for controlling antibody-mediated rejection. It may, indeed, be inappropriate because it may also deplete B cells displaying regulatory activity and this may have a negative impact on the graft outcome.
The scientific community faces, now, the challenge to better understand and discriminate the different B-cell subpopulation functions, specially the various Breg phenotypes, in addition to building an integrative analysis of Treg/Breg and other cell type interactions for globally evaluating the balance between the REG/INFLAMMA players in transplant immunobiology. This approach may help to develop the strategies to induce and preserve regulatory B and T cells, favouring the control of the INFLAMMA T/B cell activity that can lead to allograft rejection.
The authors thank the other participants of the Brazilian Multicenter Study on Operational Tolerance: Francine Lemos, Irene Noronha, Sandra M Monteiro, Cristina Castro, Daísa S. R. David, Florencia Maria Barbé-Tuana, Fabiana Agena. The authors also thank Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP; 07/59290-7) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq; iii/INCT, 573879-2008-7) for financially supporting our studies related to this review.
Conflicts of interest
The authors declare no conflicts of interest.
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