Regulation of anti-double-stranded DNA B cells in nonautoimmune mice: localization to the T-B interface of the splenic follicle.

Systemic lupus erythematosus (SLE) and the MRL-lpr/lpr murine model for SLE are characterized by the presence of serum anti-double-stranded (ds)DNA antibodies (Abs), whereas nonautoimmune individuals have negligible levels of these Abs. To increase the frequency of anti-DNA B cells and identify the mechanisms involved in their regulation in nonautoimmune mice, we have used Ig transgenes (tgs). In the present study, we used the VH3H9 heavy (H) chain tg which expresses an H chain that was repeatedly isolated from anti-dsDNA Abs from MRL-lpr/lpr mice. Because the VH3H9 H chain can pair with endogenous L chains to generate anti-single-stranded DNA, anti-dsDNA, and non-DNA B cells, this allowed us to study the regulation of anti-dsDNA B cells in the context of a diverse B cell repertoire. We have identified anti-dsDNA B cells that are located at the T-B interface in the splenic follicle where they have an increased in vivo turnover rate. These anti-dsDNA B cells exhibit a unique surface phenotype suggesting developmental arrest due to antigen exposure.

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.

Self-reactive B cells in nonautoimmune and autoimmune mice.

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.

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.

Tracking alloreactive cell division in vivo.

BACKGROUND: The study of alloimmune responses has been limited by a lack of assays that can track the behavior of alloreactive lymphocytes in vivo. Here we utilize an experimental system that allows the identification and study of alloreactive CD4+ lymphocytes responding to major histocompatibility antigens in vivo. METHODS: Responder mouse lymphocytes were labeled with a fluorescein-based dye, adoptively transferred into irradiated allogeneic stimulator mice, and recovered at serial time points for analysis by flow cytometry. RESULTS: Discrete generations of CD4+ responder lymphocytes proliferating specifically in response to allogeneic MHC class II were distinguished by fluorescein intensity. Successive division of alloreactive CD4+ lymphocytes was traced up to six generations after 60 hr. CONCLUSIONS: This experimental system provides information on the division kinetics of alloreactive CD4+ cells. Other applications include immunophenotyping of alloreactive lymphocyte subsets. Further study of systems such as this will allow the detailed characterization of how alloimmune responses are initiated and proceed in vivo.

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.

Sustained reduction of alloantibody secreting plasma cells and donor specific antibody with proteasome inhibition in mice.

The long-lived plasma cells, which develop after alloantigen sensitization, produce donor specific alloantibodies (DSAs) that generate a positive serum cross-match and preclude transplantation. Bortezomib, a proteasome inhibitor, is being investigated in clinical desensitization protocols, however preclinical studies in a transplant model are nonexistent. We hypothesized that sustained treatment with only a proteasome inhibitor would eliminate plasma cells and reduce DSA over time. Cardiac allografts were transplanted into murine recipients. Eight weeks after allograft rejection the proteasome inhibitor, bortezomib, was injected intravenously twice weekly for 60 days. Serum alloantibody responses were assayed using flow cross-match. Total and alloreactive plasma cell numbers were enumerated using flow cytometry and ELISPOT. All recipients of cardiac allografts rejected their graft promptly within 16 days and demonstrated alloantibody by flow cross-match. DSA was sustained in the control mice while mice treated with bortezomib had sustained elimination of DSA and a marked reduction in plasma cell population. Also, bortezomib was associated with an increased level of BLyS. Within a murine model, proteasome inhibition can eliminate alloantibody secreting plasma cells, and reduce alloantibody. Cessation of bortezomib is not associated with return of DSA.

Cutting edge: alloimmune responses against major and minor histocompatibility antigens: distinct division kinetics and requirement for CD28 costimulation.

Comparative study of alloimmune responses against major and minor histocompatibility Ags has been limited by the lack of suitable assays. Here, we use a bioassay that permits tracking of alloreactive CD4+ T cell populations as they proliferate in response to major or minor histocompatibility Ags in vivo. Division of alloreactive CD4+ T cells proceeded more rapidly in response to major histocompatibility Ags than minor Ags, although CD4+ T cells alloreactive to minor Ags had a similar capacity to divide successively up to eight times after stimulation. Allorecognition of minor histocompatibility Ags was highly dependent on CD28 costimulation, with the frequency of CD4+ T cells proliferating in response to minor Ags in the absence of CD28 costimulation reduced up to 20-fold. These findings highlight differences in signaling processes that lead to allorecognition of major and minor histocompatibility Ags and have implications on the design of interventions aimed at abrogating these responses.

Contribution of the innate immune system to autoimmune diabetes: a role for the CR1/CR2 complement receptors.

B lymphocytes are required for diabetogenesis in nonobese diabetic (NOD) mice. The complement component of the innate immune system regulates B cell activation and tolerance through complement receptors CR1/CR2. Thus, it is important to assess the contribution of complement receptors to autoimmune diabetes in NOD mice. Examination of the lymphoid compartments of NOD mice revealed striking expansion of a splenic B cell subset with high cell surface expression of CR1/CR2. This subset of B cells exhibited an enhanced C3 binding ability. Importantly, long-term in vivo blockade of C3 binding to CR1/CR2 prevented the emergence of the CR1/CR2(hi) B cells and afforded resistance to autoimmune diabetes in NOD mice. These findings implicate complement as an important regulatory element in controlling the T cell-mediated attack on islet beta cells of NOD mice.

Characterization of anergic anti-DNA B cells: B cell anergy is a T cell-independent and potentially reversible process.

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.

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.

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.

Impaired CD4 T cell activation due to reliance upon B cell-mediated costimulation in nonobese diabetic (NOD) mice.

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.