Impaired activation of islet-reactive CD4 T cells in pancreatic lymph nodes of B cell-deficient nonobese diabetic mice.

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.

Origin, kinetics, and function of chimeric B lymphocytes in liver allografts.

BACKGROUND: Despite the well-recognized concordance of chimerism with spontaneous acceptance of rat liver allografts, the active role and the identity of chimeric cells mediating liver allograft tolerance are unknown. Because resting B cells are endowed with a tolerogenic antigen-presenting capacity, we assessed whether donor B cells propagated from the grafted liver may be responsible for liver allograft tolerance. METHODS: Dark Agouti or Lewis rats were grafted with Lewis or Dark Agouti livers as a tolerogenic or a rejection combination, respectively. We followed the kinetics of donor B cells in recipients by flow cytometry, and we examined the fate of liver allografts depleted of passenger B cells in either B cell-sufficient or -deficient recipients. B-cell depletion was achieved by treatment of animals with polyclonal goat anti-rat IgM antibody from birth. RESULTS: During the first 3 days after liver allografting, donor B cells rapidly migrated from graft-infiltrating cells and appeared in systemic circulation in both the tolerogenic and rejection combinations. However, systemic chimerism was detectable in the tolerogenic combination by day 14, whereas it was undetectable in the rejection combination by day 7. In graft-infiltrating cells, a significant expansion of chimeric IgM+ (newly formed) B cells was observed on day 5 in the tolerogenic, but not in the rejection, combination. However, depletion of B cells from liver grafts and the absence of antibodies failed to alter the outcome of liver allograft survival in the tolerogenic or immunogenic combination. CONCLUSION: Although intragraft chimeric B cells proliferated in tolerogenic liver allografts, their clonal expansion does not seem to be essential for the promotion of liver allograft tolerance.

I-Ag7-mediated antigen presentation by B lymphocytes is critical in overcoming a checkpoint in T cell tolerance to islet beta cells of nonobese diabetic 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.

A direct method for the calculation of alloreactive CD4+ T cell precursor frequency.

BACKGROUND: Direct measurement of the precursor frequency of alloreactive CD4+ T cells has been impossible due to the lack of a specific means of determining the absolute number of daughter cells generated with each division in a repertoire of stimulated T cells. METHODS: Responder lymphocytes were fluorescently labeled and adoptively transferred into irradiated allogeneic stimulator mice or incubated in vitro with irradiated stimulator splenocytes. After a 65- to 70-hr stimulation period, responder cells were analyzed by flow cytometry. RESULTS: The precursor frequency of dividing CD4+ T cells was determined both in vivo and in vitro. The observed number of alloreactive daughter cells generated with each round of division was used to calculate the frequency of alloantigen-specific CD4+ T cells. CONCLUSIONS: A novel method for the direct calculation of the frequency of alloreactive CD4+ T cells is described. This technique allows the determination of changes in the frequency of alloreactive T cells that might underlie tolerance to alloantigens.

B-cells are required for the initiation of insulitis and sialitis in nonobese diabetic mice.

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.