Murine islet allograft tolerance upon blockade of the B-lymphocyte stimulator, BLyS/BAFF.

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.

Acquisition of humoral transplantation tolerance upon de novo emergence of B lymphocytes.

A major obstacle to transplantation tolerance is humoral immunity. In this paper, we demonstrate that the intrinsic developmental propensity of the B lymphocyte compartment for acquisition of self-tolerance can be harnessed to induce humoral unresponsiveness to transplanted alloantigens. In the current study, when transitional B cells developed in the presence of donor lymphoid cells, the mature B lymphocyte compartment failed to mount a donor-specific alloantibody response to an organ transplant--despite unrestrained acute T cell-mediated allograft rejection. Specifically, we generated an experimental system wherein a B6 strain B cell compartment developed de novo in the presence of F1 (B6xBALB/c) lymphoid cells and in a T cell-deficient setting. Following establishment of a steady-state B cell compartment, these B6 mice were transplanted with heterotopic cardiac allografts from allogeneic BALB/c donors. The mice were then inoculated with purified syngeneic B6 T cells. As expected, all cardiac allografts were acutely rejected. However, the B lymphocyte compartment of these mice was completely inert in its capacity to form a BALB/c-specific alloantibody response. Using an alloantigen-specific Ig transgenic system, we demonstrated that this profound degree of humoral tolerance was caused by clonal deletion of alloreactive specificities from the primary B cell repertoire. Thus, de novo B cell compartment development at the time of transplantation is of critical importance in recipient repertoire "remodeling" to a humoral tolerant state.

A novel CD93 polymorphism in non-obese diabetic (NOD) and NZB/W F1 mice is linked to a CD4+ iNKT cell deficient state.

In the present study, we characterize a polymorphism in the CD93 molecule, originally identified as the receptor for the C1q complement component (i.e., C1qRp, or AA4.1) in non-obese diabetic (NOD) mice. This allele carries a coding polymorphism in the first epidermal growth factor-like domain of CD93, which results in an amino acid substitution from Asn-->His at position 264. This polymorphism does not appear to influence protein translation or ecto-domain cleavage, as CD93 is detectable in bone-marrow-derived macrophage and B-cell precursor lysates and in soluble form in the serum. The NOD CD93 isoform causes a phenotypic aberrancy in the early B-cell developmental stages (i.e., pro-, pre-, immature, and transitional), likely related to a conformational variation. Interestingly, the NZB/W F1 strain, which serves as a murine model of Lupus, also expresses an identical CD93 sequence polymorphism. Cd93 is located within the NOD Idd13 locus and is also tightly linked to the NZB/W F1 Wbw1 and Nkt2 disease susceptibility loci, which are thought to regulate natural killer T (NKT) cell homeostasis. Consistent with this genetic linkage, we found B6 CD93(-/-) and B6.NOD(Idd13) mice to be susceptible to a profound CD4(+) NKT cell deficient state. These data suggest that Cd93 may be an autoimmune susceptibility gene residing within the Idd13 locus, which plays a role in regulating absolute numbers of CD4(+) NKT cells.

In vivo BLyS/BAFF neutralization ameliorates islet-directed autoimmunity in nonobese diabetic mice.

B lymphocytes are required for the pathogenesis of autoimmune diabetes in NOD mice. Previous studies established that a lymphopenic transitional (TR) B cell compartment reduces the competitive constraint on the entry of newly emerging TR B cells into the splenic follicle (FO), thereby disrupting a peripheral negative selection checkpoint in NOD mice. Thus, development of clinically feasible immunotherapeutic approaches for restoration of appropriate negative selection is essential for the prevention of anti-islet autoimmunity. In this study we hypothesized that in vivo neutralization of the B lymphocyte stimulator (BLyS/BAFF) may enhance the stringency of TR-->FO selection by increasing TR B cell competition for follicular entry in NOD mice. This study demonstrated that in vivo BLyS neutralization therapy leads to the depletion of follicular and marginal zone B lymphocytes. Long-term in vivo BLyS neutralization caused an increased TR:FO B cell ratio in the periphery indicating a relative resistance to follicular entry. Moreover, in vivo BLyS neutralization: 1) restored negative selection at the TR-->FO checkpoint, 2) abrogated serum insulin autoantibodies, 3) reduced the severity of islet inflammation, 4) significantly reduced the incidence of spontaneous diabetes, 5) arrested the terminal stages of islet cell destruction, and 6) disrupted CD4 T cell activation in NOD mice. Overall, this study demonstrates the efficacy of B lymphocyte-directed therapy via in vivo BLyS neutralization for the prevention of autoimmune diabetes.

B lymphocyte-directed immunotherapy promotes long-term islet allograft survival in nonhuman primates.

We found that an induction immunotherapy regimen consisting of rabbit anti-thymocyte globulin (Thymoglobulin) and the monoclonal antibody to CD20 rituximab (Rituxan) promoted long-term islet allograft survival in cynomolgus macaques maintained on rapamycin monotherapy. B lymphocyte reconstitution after rituximab-mediated depletion was characterized by a preponderance of immature and transitional cells, whose persistence was associated with long-term islet allograft survival. Development of donor-specific alloantibodies was abrogated only in the setting of continued rapamycin monotherapy.

B cell-mediated antigen presentation is required for the pathogenesis of acute cardiac allograft rejection.

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.

Alloreactive CD4 T cell activation in vivo: an autonomous function of the indirect pathway of alloantigen presentation.

Activation of alloreactive CD4 T cells occurs via the direct and indirect pathways of alloantigen presentation. A novel TCR/alloantigen transgenic system was designed that permitted in vivo visualization of CD4 T cell priming through these pathways. When both pathways of alloantigen presentation were intact, CD4 T cell activation in response to cardiac allografts was rapid and systemic by day 4 after transplantation, in contrast to that seen in response to skin allografts, which was delayed until 10-12 days after transplantation. Despite this systemic CD4 T cell activation in response to cardiac allografts, there was a paucity of activated graft-infiltrating CD4 T cells at 4 days posttransplantation. This finding suggests that the initial priming of alloimmune CD4 T cell responses occurs within draining lymphoid organs. Furthermore, alloantigens derived from cardiac allografts failed to promote thymic negative selection of developing thymocytes expressing the alloreactive TCR clonotype. In the absence of a functional direct pathway, the kinetics of activation, anatomic localization, and effector function of alloreactive CD4 T cells remained unchanged. Overall, the present study defines the anatomic and temporal characteristics of CD4 T cell alloimmune responses and demonstrates that CD4 T cell priming via the indirect pathway proceeds optimally in the absence of the direct pathway of alloantigen presentation.