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Background: Immunologic rejection is a major barrier to successful long-term outcomes in clinical transplantation. The importance of B lymphocytes-and their secretory products, alloantibodies-in the pathogenesis of allograft rejection is accepted. Furthermore, it is now clear that the dominant regulator of peripheral B-cell homeostasis and tolerance is the B-lymphocyte stimulator (BLyS), also referred to as the B-cell activating factor (BAFF). Recently, a novel class of clinical immunotherapeutic agents specific for BLyS, and its family of cytokines, has emerged for the treatment of B-cell-mediated diseases. In this study, we demonstrate the potential utility of BLyS-directed immunotherapy in preventing allograft rejection using a murine islet transplantation model., Methods: A transient period of mature peripheral B-cell depletion was induced by means of in vivo BLyS neutralization using a murine analog of the monoclonal antibody, Benlysta. Subsequently, fully major histocompatibility complex-mismatched islets were transplanted into naïve diabetic mice followed by a short course of rapamycin., Results: After BLyS neutralization, indefinite islet allograft survival was achieved. Induction therapy with rapamycin was necessary, but not sufficient, for the achievement of this long-term graft survival. The tolerant state was associated with (1) abrogation of the donor-specific antibody response, (2) transient preponderance of immature/transitional B cells in all lymphoid organs, (3) impaired CD4 T-cell activation during the period of B-cell depletion, and (4) presence of a "regulatory" cytokine milieu., Conclusions: In vivo BLyS neutralization effectively induces humoral tolerance and promotes long-term islet allograft survival in mice. Therefore, B-lymphocyte-directed immunotherapy targeting the homeostatic regulator, BLyS, may be effective in promoting transplantation tolerance.

Transplantation tolerance remains an elusive goal as B-cell-initiated chronic humoral rejection evades current immunosuppression. B-cell-directed therapy is thus emerging as a key component in achieving transplantation tolerance and long-term graft survival. Here, we propose strategies of B-cell repertoire remodeling to achieve humoral unresponsiveness to donor antigens with implementation of fundamental B-cell immunobiology and use of newly developed B-cell-directed agents.

T lymphocytes are the primary targets of immunotherapy in clinical transplantation. However, B lymphocytes are detrimental to graft survival by virtue of their capacity to present antigen to T cells via the indirect pathway of allorecognition and the generation of donor specific alloantibody. Furthermore, the long-term survival of organ allografts remains challenged by chronic rejection, a process in which activated B cells have been found to play a significant role. Therefore, the achievement of transplantation tolerance will likely require induction of both T and B cell tolerance to alloantigens. Moreover, human and animal investigations have shown that subsets of B cells, Transitional and Regulatory, are inherently tolerogenic. Developing therapeutic strategies that exploit these populations may be key to achieving transplantation tolerance. In this review we describe the current evidence for the essential role of B cells in transplant tolerance and discuss emerging B cell directed strategies to achieve allograft tolerance., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Current strategies for immunotherapy after transplantation are primarily T-lymphocyte directed and effectively abrogate acute rejection. However, the reality of chronic allograft rejection attests to the fact that transplantation tolerance remains an elusive goal. Donor-specific antibodies are considered the primary cause of chronic rejection. When naive, alloreactive B-cells encounter alloantigen and are activated, a resilient "sensitized" state, characterized by the presence of high-affinity antibody, is established. Here, we will delineate findings that support transient B-lymphocyte depletion therapy at the time of transplantation to preempt sensitization by eliminating alloreactive specificities from the recipient B-cell pool (ie, "repertoire remodeling"). Recent advances in our understanding of B-lymphocyte homeostasis provide novel targets for immunomodulation in transplantation. Specifically, the tumor necrosis factor-related cytokine BLyS is the dominant survival factor for "tolerance-susceptible" transitional and "preimmune" mature follicular B-cells. The transitional phenotype is the intermediate through which all newly formed B-cells pass before maturing into the follicular subset, which is responsible for mounting an alloantigen-specific antibody response. Systemic BLyS levels dictate the stringency of negative selection during peripheral B-cell repertoire development. Thus, targeting BLyS will likely provide an opportunity for repertoire-directed therapy to eliminate alloreactive B-cell specificities in transplant recipients, a requirement for the achievement of humoral tolerance and prevention of chronic rejection. In this review, the fundamentals of preimmune B-cell selection, homeostasis, and activation will be described. Furthermore, new and current B-lymphocyte-directed therapy for antibody-mediated rejection and the highly sensitized state will be discussed. Overall, our objective is to propose a rational approach for induction of humoral transplantation tolerance by remodeling the primary B-cell repertoire of the allograft recipient., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Acute allograft rejection requires the activation of alloreactive CD4 T cells. Despite the capacity of B cells to act as potent APCs capable of activating CD4 T cells in vivo, their role in the progression of acute allograft rejection was unclear. To determine the contribution of B cell APC function in alloimmunity, we engineered mice with a targeted deficiency of MHC class II-mediated Ag presentation confined to the B cell compartment. Cardiac allograft survival was markedly prolonged in these mice as compared to control counterparts (median survival time, >70 vs 9.5 days). Mechanistically, deficient B cell-mediated Ag presentation disrupted both alloantibody production and the progression of CD4 T cell activation following heart transplantation. These findings demonstrate that indirect alloantigen presentation by recipients' B cells plays an important role in the efficient progression of acute vascularized allograft rejection.

Although it is well established that B-cells are required for the development of diabetes in the nonobese diabetic (NOD) mouse, the nature of their role remains unknown. Herein, we investigate the hypothesis that B-cells in this autoimmune background actively disrupt the tolerant state of those T-cells with which they interact. We demonstrate that NOD B-cells express elevated levels of crucial molecules involved in antigen presentation (including CD21/35, major histocompatibility complex class II, and CD40), alterations that invite the possibility of inappropriate T-cell activation. However, when chimeric animals are generated in which all B-cells are NOD-derived, a tolerant state is maintained. These data demonstrate that although B-cells are required for the development of autoimmunity, they are not sufficient to disrupt established tolerance. Moreover, non-B-cell antigen-presenting cells may be the critical actors in the establishment of the tolerant state; this function may be absent in NOD mice as they are characterized by deficient professional antigen-presenting cell function.

The present review highlights the two areas of research pursuit in our laboratory: (1). the regulation of the autoimmune T cell response to pancreatic islet beta cells using the nonobese diabetic (NOD) mouse model of type 1 diabetes and (2). the regulation the T cell response to alloantigens. Our work has established a central role for B lymphocytes in driving both autoimmune and allo-immmune T cell responses. Our studies indicate that: (1). B cell-deficient NOD mice are protected from autoimmune diabetes; (2). targeted disruption of cognate T/B cell collaboration via major histocompatibility complex (MHC) class II prevents both T cell-mediated islet destruction and allograft rejection; and (3). maternal transmission of islet-reactive autoantibodies potentiates the activation of diabetogenic T cells, highlighting the important role of B cells in the early targeting of islet beta cells.

Despite the impressive protection of B cell-deficient (muMT(-/-)) nonobese diabetic (NOD) mice from spontaneous diabetes, existence of mild pancreatic islet inflammation in these mice indicates that initial autoimmune targeting of beta cells has occurred. Furthermore, muMT(-/-) NOD mice are shown to harbor a latent repertoire of diabetogenic T cells, as evidenced by their susceptibility to cyclophosphamide-induced diabetes. The quiescence of this pool of islet-reactive T cells may be a consequence of impaired activation of T lymphocytes in B cell-deficient NOD mice. In this regard, in vitro anti-CD3-mediated stimulation demonstrates impaired activation of lymph node CD4 T cells in muMT(-/-) NOD mice as compared with that of wild-type counterparts, a deficiency that is correlated with an exaggerated CD4 T cell:APC ratio in lymph nodes of muMT(-/-) NOD mice. This feature points to an insufficient availability of APC costimulation on a per T cell basis, resulting in impaired CD4 T cell activation in lymph nodes of muMT(-/-) NOD mice. In accordance with these findings, an islet-reactive CD4 T cell clonotype undergoes suboptimal activation in pancreatic lymph nodes of muMT(-/-) NOD recipients. Overall, the present study indicates that B cells in the pancreatic lymph node microenvironment are critical in overcoming a checkpoint involving the provision of optimal costimulation to islet-reactive NOD CD4 T cells.

Diabetes in nonobese diabetic (NOD) mice results from the activation of I-A(g7)-restricted, islet-reactive T cells. This study delineates several characteristics of NOD CD4 T cell activation, which, independent of I-A(g7), are likely to promote a dysregulated state of peripheral T cell tolerance. NOD CD4 T cell activation was found to be resistant to antigenic stimulation via the TCR complex, using the progression of cell division as a measure. The extent of NOD CD4 T cell division was highly sensitive to changes in Ag ligand density. Moreover, even upon maximal TCR complex-mediated stimulation, NOD CD4 T cell division prematurely terminated. Maximally stimulated NOD CD4 T cells failed to achieve the threshold number of division cycles required for optimal susceptibility to activation-induced death, a critical mechanism for the regulation of peripheral T cell tolerance. Importantly, these aberrant activation characteristics were not T cell-intrinsic but resulted from reliance on B cell costimulatory function in NOD mice. Costimulation delivered by nonautoimmune strain APCs normalized NOD CD4 T cell division and the extent of activation-induced death. Thus, by disrupting the progression of CD4 T cell division, polarization of APC costimulatory function to the B cell compartment could allow the persistence and activation of diabetogenic cells in NOD mice.

B cell-deficient nonobese diabetic (NOD) mice are protected from the development of spontaneous autoimmune diabetes, suggesting a requisite role for Ag presentation by B lymphocytes for the activation of a diabetogenic T cell repertoire. This study specifically examines the importance of B cell-mediated MHC class II Ag presentation as a regulator of peripheral T cell tolerance to islet beta cells. We describe the construction of NOD mice with an I-Ag7 deficiency confined to the B cell compartment. Analysis of these mice, termed NOD BCIID, revealed the presence of functionally competent non-B cell APCs (macrophages/dendritic cells) with normal I-Ag7 expression and capable of activating Ag-reactive T cells. In addition, the secondary lymphoid organs of these mice harbored phenotypically normal CD4+ and CD8+ T cell compartments. Interestingly, whereas control NOD mice harboring I-Ag7-sufficient B cells developed diabetes spontaneously, NOD BCIID mice were resistant to the development of autoimmune diabetes. Despite their diabetes resistance, histologic examination of pancreata from NOD BCIID mice revealed foci of noninvasive peri-insulitis that could be intentionally converted into a destructive process upon treatment with cyclophosphamide. We conclude that I-Ag7-mediated Ag presentation by B cells serves to overcome a checkpoint in T cell tolerance to islet beta cells after their initial targeting has occurred. Overall, this work indicates that the full expression of the autoimmune potential of anti-islet T cells in NOD mice is intimately regulated by B cell-mediated MHC class II Ag presentation.

Anti-single stranded DNA (ssDNA) and anti-double stranded DNA (dsDNA) B cells are regulated in non-autoimmune mice. In this report we show that while both anti-ssDNA and anti-dsDNA B cells are blocked in their ability to differentiate into antibody-secreting cells, other phenotypic and functional characteristics distinguish them from one another. Splenic anti-ssDNA B cells are found distributed throughout the B cell follicle, and are phenotypically mature and long-lived. On the other hand, splenic anti-dsDNA B cells are short-lived, exhibit an immature and antigen-experienced phenotype, and localize to the T-B interface of the splenic follicle. Functionally, anti-ssDNA B cells proliferate, albeit suboptimally, in response to anti-IgM, lipopolysaccharide (LPS) and CD40L/IL-4 + anti-IgM stimulation, and tyrosine phosphorylate intracellular proteins upon mIgM cross-linking. Anti-dsDNA B cells, on the other hand, are functionally unresponsive to anti-IgM and LPS stimulation, and do not phosphorylate intracellular proteins, including Syk, upon mIg stimulation. Importantly, anti-DNA B cell anergy is maintained in the absence of T cells since both anti-ssDNA and anti-dsDNA B cells are as efficiently regulated in RAG2(-/-) mice as in their RAG2(+/+) counterparts. Interestingly, the severely anergic state of anti-dsDNA B cells is partially reversible upon stimulation with CD40 ligand and IL-4. In response to these signals, anti-dsDNA B cells remain viable, up-regulate cell surface expression of B7-2 and IgM, and restore their ability to proliferate and phosphorylate Syk upon mIg cross-linking. Collectively, these data suggest that anti-DNA B cell anergy encompasses distinct phenotypes which, even in its most severe form, may be reversible upon stimulation with T cell-derived factors.

The defining feature of autoimmune disease is the presence of specific autoreactive lymphocytes. Systemic lupus erythematosus (SLE), for example, is characterized by a discrete set of antibodies directed to nuclear antigens; these include autoantibodies to DNA and snRNPs that are diagnostic for SLE. The murine model of SLE, the MRL-lpr/lpr mouse, likewise, has a similar autoantibody profile. To understand how SLE-associated autoantibodies are regulated in healthy individuals and to identify mechanisms underlying their expression in autoimmunity, we have developed a transgenic (tg) model system using multiple sets of tgs. The development of B cells bearing these tgs has been studied in BALB/c and MRL-lpr/lpr autoimmune backgrounds, and the relative fates of anti-ssDNA and anti-dsDNA tg B cells when they are a part of a diverse as well as monoclonal B cell repertoire have been evaluated.

Nonobese diabetic (NOD) mice spontaneously develop an acute onset of hyperglycemia reminiscent of human type I diabetes. The disease is the end result of a mononuclear cell infiltration of pancreatic islets (insulitis), culminating in the selective destruction of islet beta-cells by autoreactive T-cells. NOD mice also exhibit defects in B-cell tolerance as manifested by the presence of autoantibodies against islet cell autoantigens. Based on the potential ability of B-cells to act as antigen presenting cells, we hypothesized that autoreactive B-cells of NOD mice may be necessary for the activation of islet reactive CD4+ T-cells. In the present study, we utilized an anti-mu antibody to induce in vivo depletion of B-cells and found that B-cell depletion completely abrogates the development of insulitis and sialitis in NOD mice. In contrast, control IgG-treated NOD mice developed insulitis and sialitis by 5 weeks of age. Additionally, the discontinuation of anti-mu chain antibody treatment led to the full restoration of the B-cell pool and the reappearance of insulitis and sialitis. Thus, we conclude that B-cells are a requisite cell population for the genesis of the inflammatory lesions of the islets of Langerhans. This finding suggests that autoreactive B-cells may act as the antigen presenting cells necessary for the initial activation of beta-cell-reactive CD4+ T-cells implicated in the pathogenesis of autoimmune diabetes.