Elsevier

Seminars in Immunology

Volume 24, Issue 2, April 2012, Pages 109-114
Seminars in Immunology

Review
Primary B cell repertoire remodeling to achieve humoral transplantation tolerance

https://doi.org/10.1016/j.smim.2011.08.016Get rights and content

Abstract

The current mainstay of immunotherapy in clinical transplantation is T lymphocyte directed. However, it has long been appreciated that the emergence of an alloimmune response mounted by the B lymphocyte compartment and detectable as donor-specific antibodies is a critical challenge to long-term graft survival. Thus, achieving robust transplantation tolerance will require induction of tolerance in both the T- and B-cell compartments. Here we propose that the natural developmental propensity of the B-lymphocyte compartment acquisition of tolerance to self-antigens can be recapitulated to achieve humoral transplantation tolerance. It is our contention B-lymphocyte directed induction immunotherapy would be an important component of emerging strategies for induction of Transplantation tolerance.

Highlights

► T- and B-lymphocyte acceptance of alloantigen is critical for transplantation tolerance. ► Recent studies suggest purging alloreactive B cells at the time of transplantation is effective. ► Following B cell depletion, newly emerging Transitional B cells may impart allograft tolerance. ► Stringent selection, under BLyS-limiting conditions, may promote tolerance.

Introduction

Solid-organ transplantation offers a significant survival advantage over other modalities and is the treatment of choice for patients with end-organ failure [1]. Calcineurin inhibitors and other T-cell targeted therapies have ushered in an era of excellent one-year graft survival rates in kidney, liver, and heart transplantation (91%, 82%, and 87%, respectively). However, the long-term survival of organ allografts remains challenged by chronic rejection, demonstrating that transplantation tolerance remains an elusive objective.

Donor specific alloantibodies (DSA) are emerging as important mediators of chronic allograft rejection. There is now solid epidemiological evidence that DSA compromise long-term graft survival [2], [3]. Even DSA specific for non-HLA alloantigens are associated with worse outcomes [4]. DSA can be acquired by prior organ transplantation, blood transfusion, pregnancy or generated de novo after transplantation.

Understanding the biology underlying the generation of an alloantibody response and the mechanistic basis of acquired self-tolerance are critical to innovating rational strategies for achievement of humoral transplantation tolerance. Currently, the mainstay of clinical immunotherapy in transplantation is T-cell directed and effectively prevents acute T-cell mediated allograft rejection, but is insufficient to achieve a tolerant B cell compartment as evident by the generation of DSA. Therefore, achievement of transplantation tolerance will likely require induction of tolerance in both the T- and B-cell compartments.

In this review, we will summarize B cell compartment development, its inherent propensity for acquisition of tolerance to self-antigens, its survival factors, and our proposed approach to B-cell directed therapy for transplantation. We will argue that the next generation clinical trials should continue current mainstay T-cell directed induction, but also include B-cell directed therapies. B-cell depletion should be rigorously monitored and the compartment should be allowed to reconstitute under conditions promoting stringent selection. We refer to this approach as “repertoire remodeling” [5]. This strategy aims to promote the negative selection of alloreactive B cell clones from the primary repertoire upon reconstitution. Overall, the resulting “remodeled” B cell compartment should remain unresponsive to the newly transplanted alloantigens. Recent experimental and preclinical studies have supported this preemptive approach to reduce the likelihood of chronic humoral rejection [6].

Generation of the B Cell Receptor (BCR) repertoire via a pseudo-random process of somatic combinatorial rearrangement of immunoglobulin genes in individual B-cell clones guarantees the generation of impressive diversity. Of course, repertoire diversity generated in this fashion is a double-edged word; on the one hand protecting against novel foreign pathogenic variants, and on the other, generating potentially deadly self-reactive clones. Nonetheless, this process transpires continually in the bone marrow as millions of new B cells are generated daily throughout the organism's life (Fig. 1). The defining moment in bone marrow B cell development is the successful expression of a productively rearranged B cell receptor (BCR), which is created in the stochastic process of VDJ recombination. By some estimates, this stochastic process produces up to 50% high affinity self-reactive clones. As such, central and peripheral mechanisms have evolved to deal with this large frequency of auto-reactive B cells. In the bone marrow, immature B cells expressing a self-reactive BCR are induced to either “edit” their receptor or undergo apoptosis, a process termed clonal deletion [7], [8]. Receptor editing is a predominant mechanism in the development of central tolerance [9]. Indeed, persistence of RAG1 and RAG2 expression in auto-reactive B cells gives these cells a second chance to rearrange new light chains, thereby eliminating their self reactivity and permitting entry into the peripheral B cell pool [10], [11]. Overall, the central bone marrow checkpoint permits fewer than 10% of immature B cells formed in the bone marrow to exit into the periphery as Transitional (TR) B cells [12], [13] (Fig. 1).

TR B cells are newly emerging B cells from the bone marrow that have successfully rearranged their BCR, survived the central tolerance checkpoint and have entered the periphery. The TR compartment will inevitably contain autoreactive clones having evaded the central checkpoint for a variety of reasons [14], [15], [16]. Therefore, a peripheral tolerance checkpoint has also evolved as a final barrier to maturation of potentially pathogenic autoreactive B cells. This peripheral checkpoint is far more elastic compared to the “all-or-none” central tolerance mechanisms in the bone marrow [17], [18]. This elasticity is regulated by the homeostatic demands of the host (i.e., on the perceived systemic need for B cell numbers) (Fig. 1). Specifically, it has been established that follicular entry of TR B cells and their maturation into the follicular phenotype relies on the B Lymphocyte Stimulator (BLyS) cytokine [19]. Indeed, BLyS is critical for the survival of mature Follicular (FO)B cells. An integrated signaling cross-talk between the BCR and BLyS-receptor starts at the TR B cell stage and persists in the resting follicular niche [20]. Autoreactive cells are excluded from the follicular niche due to their specificity based on an inability to compete efficiently for the limited BLyS [16], [21], [22], [23], [24], [25]. On the one hand, an abundance of BLyS can easily over-ride follicular exclusion of autoreactive B cells at the peripheral checkpoint [26]; on the other hand, limiting ambient BLyS increases competition for follicular entry and at an extreme blocks follicular entry entirely [19]. Thus, the stringency of peripheral B cell tolerance at the TR  FO checkpoint is tightly regulated by BLyS. Ultimately, in healthy individuals, in addition to effective T-cell tolerance mechanisms, exclusion of self-reactive B cells from the follicular compartment during development eliminates the probability of a germinal center (GC) reaction forming against self antigens.

B cell activation is a multistep process that leads to the formation of antibody secreting cells (ASCs) and plasma cells (PCs). The first step in B cell activation depends on the interaction between the B cell receptor and its cognate antigen. This event typically occurs in secondary lymphoid organs. Naive B cells continuously circulate through secondary lymphoid organs, including the spleen and peripheral lymph nodes, thereby increasing their likelihood of encountering cognate antigen (Ag). Following Ag recognition, B cells are activated and migrate to the T–B cell border of the lymphoid follicle, where they present Ag and engage in cognate interaction with Ag-specific T cells to form a GC. In this specialized environment Ag-specific B cells undergo expansion, class-switching, and somatic hyper mutation to affinity mature and eventually differentiate into ASCs, memory cells, or PCs. A complete discussion of the mechanisms for the generation and maintenance of the alloantibody response is beyond the scope of this review and has been reviewed recently [27].

B cells are by far the largest population of antigen presenting cells found in vivo, and although antigen-presenting dendritic cells are sufficient to activate CD4+ T cells, B cells are particularly effective antigen-presenting cells [28]. Therefore, B cell depletion therapy at the time of transplantation will eliminate this function and likely alter graft outcomes (Fig. 2). It has been shown that when B cells are deficient in antigen-presenting function due to restricted absence of MHC class II expression, the tempo of acute rejection is significantly attenuated [29]. In confirmatory experiments it was also noted that CD8 and CD4 memory was impaired when the antigen-presenting function of B cells was absent [30]. Furthermore, B cells have been shown to help alloreactive T cells to differentiate into memory T cells [31]. Therefore, the strategy of depleting B cells at the time of transplantation will directly impair T-cell alloimmune responses, thereby promoting allograft survival.

Much of what is known about the basics of developmental B cell tolerance was deciphered from transgenic systems over the past three decades [32]. Utilizing these known principals, Parsons et al. [33] demonstrated that it is possible to “tolerize” a recipient B cell compartment to cardiac allografts in the absence of immuno suppression. Reminiscent of experiments by the Chong laboratory [34], it was demonstrated that alloreactive B cells could be deleted upon de novo emergence in the presence of transplanted alloantigen [33].

In the clinical setting, B cell tolerance has also been demonstrated in the developing immune system of infants. ABO-incompatible heart transplantation during infancy has been shown to result in development of B cell tolerance to donor blood group A and B antigens [35]. Tolerance in this setting has been found to occur by elimination of donor-reactive B lymphocytes and may be dependent upon persistence of some degree of antigen expression in the setting of B cell development. Further study has shown that generation of de novo anti-HLA antibodies following cardiac transplantation is clearly age-dependent [36]. These findings suggest that intentional exposure to non-self group antigens to the developing B cell compartment can lead to tolerance in the clinical setting. These studies have led our group to propose that remodeling of the primary B cell repertoire at the time of organ transplantation may be required for establishment of robust humoral tolerance [37].

A potential advantage of B cell depletion therapy at the time of transplantation is that the emergence of the reconstituting B cell pool occurs in the presence of alloantigen provided by the transplanted organ. Whether this process recapitulates the ontogeny of B cell development and humoral tolerance is intriguing [37]. The experimental evidence in support of this view and mentioned above is reported by: (1) Parsons et al. demonstrating deletion of alloreactive B cells upon their de novo emergence in the presence of a transplanted cardiac allograft [33], (2) Li et al. [34] who observed clonal deletion of primary B cells in recipients of cardiac allografts after cost imulation blockade, and (3) Fan et al. [35] who observed donor-specific B cell tolerance after ABO-incompatible infant heart transplant.

Therefore, these studies suggest, it is reasonable to hypothesize that B cell targeted therapy would deplete the mature B cells and allow tolerance-promoting TR B cells to reconstitute the peripheral B cell compartment in the presence of allograft and subsequent clonal deletion of alloreactive clones.

An additional consideration in this strategy is that it is known that after B cell depletion therapy, BLyS levels rise and could allow the re-emergence of alloreactive clones [38]. Furthermore, it is also known that the susceptibility of transitional B cells to negative selection is under homeostatic control by the B cell survival factor BLyS [39] (Fig. 1). Therefore, transient depletion of the mature B cell repertoire at the time of transplantation followed by reconstitution under BLyS-limiting conditions is predicted to increase the stringency of alloreactive clonal negative selection, and thus induce a tolerant B cell compartment.

Furthermore, it has been shown that BLyS directed immunotherapy increases the ratio of transitional to follicular B cells [40], which has been identified as an important biomarker of a tolerance susceptible immune system [40], discussed below (see Transitional B Cell Signatures in Transplantation Tolerance). BLyS directed immunotherapy at the time of transplantation, using Benlysta and its murine analog 10F4,which is an antibody that binds BlyS and sequesters it, is currently under investigation. In recipients of cardiac allograft in whom the B cell compartment was concomitantly depleted, allowing for the re-emergence of the B cell repertoire in the presence of the allograft and a BLyS deficient environment (achieved with anti-BLyS therapy), there was abrupt rejection of the cardiac allograft by the alloreactive T cells. However there was an absence of DSA, suggesting the elimination of alloreactive B cell clones, while the alloantibody response to a third party donor was preserved (Vivek et al., unpublished).

The above strategy to remodel the recipient immune system pertains to the pre-immune patient. In the allo-antigen experienced patient, there are many obstacles to overcome in order to achieve a tolerant B cell compartment. Once sensitized, long-lived alloantibody secreting plasma cells reside in the bone marrow where they rely on different and currently unknown survival signals than the naïve B cell pool in the periphery. Understanding these survival factors that govern plasma cell persistence will be critical to developing therapies to desensitize patients (Fig. 2). The 26S proteosome inhibitor, Bortezomib, effectively eliminates both short and long-lived PCs by activating the unfolded protein response. Reduction of alloantibody titers, but not their elimination, has been achieved with limited treatment [41]. Newer therapies certainly need to be developed to target alloantibody-secreting PCs directly. Without such therapies long-term allograft survival will continue to be challenged in the sensitized patient.

Besides remodeling the immune system to purge the system of alloreactive clones, there is emerging evidence that certain B cell subsets may have intrinsically tolerogenic properties. On the clinical front, Newell et al. [42] showed that in operationally tolerant recipients of a kidney allograft, 22 of 30 genes found to have a 2-fold increase in expression in the tolerant versus non-tolerant group (still requiring immunosuppression) were B cell specific. Many of these genes are involved in B cell activation and differentiation, including genes encoding Ig heavy, light, and joining chains and HLA. The investigators narrowed down the signature to 3 genes that predicted, with 100% accuracy, tolerant from non-tolerant recipients. The 3 genes are all expressed by TR B cells. This suggested that TR B cells are involved in the induction or maintenance of tolerance. Antigens presented by naïve B cells have been recently shown to stimulate naïve T cells toward development into regulatory T cells [43]. This may explain several, now classical observations, that transferred naïve B cells could allow for the acceptance of a skin allograft [44], [45].

Further evidence for a potentially tolerogenic role of TR B cells can be found in recent experiments using B cell depletion with rituximab. As expected, B cell depletion with rituximab has been shown to expand the TR B cell compartment when it reconstitutes [46]. Moreover, TR B cell preponderance has been associated with improved allograft survival [47]. Monkeys treated with T and B cell induction immunotherapy and rapamycin maintenance enjoyed long-term islet allograft survival when immature/TRphenotype B cells were predominant in the peripheral blood [47]. A similar mechanism is likely at work in a non-human primate model of cardiac transplantation using rituximab as an induction agent [6]. Given the capacity of BLyS neutralizing monoclonal antibodies, as well as other B cell depletion therapies, to generate a predominantly TR peripheral B cell compartment, the potential of the novel immunotherapeutic Benlysta as a tolerogenic agent in clinical transplantation cannot be under-stated [48].

Additionally, there is evidence that the TR B cell compartment may be a source of B regulatory cells (Breg). Breg are found in both the B-1 and B-2 lineage B cells and are currently defined by their ability to secrete IL-10 upon stimulation. The in vivo role of IL-10-producing B cells was first demonstrated in a murine model of experimental autoimmune encephalomyelitis, where the consequence of a deficiency of IL-10-producing B cells was determined to be the cause of disease in B cell deficient mice [49]. The involvement of Breg has been found to play a role in other autoimmune disease including ulcerative colitis, lupus and arthritis [50], [51], [52].

In humans, the TR subset has been found to secrete the highest amount of IL-10 in response to CD40 stimulation compared to other peripheral blood subsets [53]. Furthermore, in murine models, there is evidence that Breg cells increase the number of Tregs [54]. Whether those effects are mediated by IL-10 secretion, antigen processing/presentation or a combination of the two remains to be determined. Taking into consideration the TR B cell and FOXP3+ phenotype observed in tolerant transplant patients from Newell et al. as discussed previously, one could wonder if the increase in FOXP3+ observed in these patients may in fact be directly related to the subset of TR B cells that secrete IL-10 [42], [54]. Thus, moving forward, it will be important to assess the TR B cell compartment: (1) as a potential candidate Breg population and (2) as a catalyst for differentiation of Treg cells.

Section snippets

Conclusions

Transplantation tolerance will ultimately require induction of both T-cell and humoral tolerance to alloantigens. The true measure of a tolerant B cell compartment will be the sustained absence of DSA in the recipients. As is clear from the fundamental mechanisms governing the regulation of B cell mediated autoimmunity, achievement of such humoral transplantation may be possible by eliminating alloreactive specificities from the primary repertoire. Therefore, by harnessing the natural

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