T-cell tolerance induction is normal in the (NZB x NZW)F1 murine model of systemic lupus erythematosus.

The (New Zealand black (NZB) x New Zealand white (NZW))F1 (NZB/W) mouse strain spontaneously develops an autoimmune disease characterized by anti-dsDNA antibody production and glomerulonephritis. Although evidence suggests that production of pathogenic autoantibodies is T-cell dependent, the immunological defects that lead to activation of these autoreactive T cells are unknown. In particular, it has not been resolved whether autoreactive T cells become activated in these mice because of a generalized defect in T-cell tolerance induction. Previous work has demonstrated that thymic and peripheral tolerance to strongly deleting antigens are intact in NZB/W mice. In this study we investigate whether these mice possess a more subtle T-cell tolerance defect. To this end, we have produced NZB/W mice carrying a transgene encoding beef insulin (BI) which is expressed at levels close to the threshold for T-cell tolerance induction. In BALB/c mice this transgene produces a profound but incomplete state of BI-specific T-cell tolerance, mediated predominantly by clonal anergy. Comparison of BI-specific tolerance in NZB/W, major histocompatibility complex (MHC)-matched (BALB/c x NZW)F1, and BALB/c BI-transgenic mice clearly demonstrates that T-cell tolerance induction is normal in NZB/W mice. The data suggest that the loss of T-cell tolerance that ultimately supports nephritogenic autoantibody production in NZB/W mice does not result from a generalized defect in T-cell tolerance, and by extension likely results from aberrant activation of specific autoreactive T cells.

Genetic dissection of B cell traits in New Zealand black mice. The expanded population of B cells expressing up-regulated costimulatory molecules shows linkage to Nba2.

B cell abnormalities are a prominent feature of the immunologic derangement in NZB and NZB / W mice. We recently demonstrated that these mice have an increased proportion of splenic B cells expressing B7.1 and elevated levels of B7.2 and ICAM-1 that possess the characteristics of marginal zone B cells (CD23(low / -) CD5(-) CD44(hi) CD24(hi) IgD(- / low) IgM(hi)) and are found as early as 4 - 6 weeks of age. These findings suggest that activated B cells in NZB and NZB / W mice could serve a costimulatory function leading to activation of autoreactive T cells. However, it remains unclear whether there is any association between B abnormalities and nephritis in these mice. Here we have used genetic mapping techniques to address this issue. We show that increases in the proportion of B cells expressing costimulatory molecules, serum IgM levels, the number of IgM ELISpots, and IgG anti-single-stranded (ss) DNA antibody production, are significantly associated with a chromosomal region that overlaps with Nba2, a genetic locus previously linked to nephritis. Based on these findings we propose that immune mechanisms leading to polyclonal B cell activation and up-regulation of costimulatory molecules in these mice play a central role in the loss of tolerance that leads to production of pathogenic autoantibodies.

The orientation and nature of the interaction between beef insulin-specific TCRs and the insulin/class II MHC complex.

Recent crystallographic studies suggest that TCR interact with peptide/class I MHC complexes in a single preferred orientation. Although similar studies have not been performed for class II-restricted TCR, it has been proposed that T cell recognition of peptide/class II complexes has similar orientational restrictions. This study represents a functional approach to systematic analysis of this question. Twenty-one mutant A beta(d) molecules were produced by alanine scanning mutagenesis and assessed for their ability to present species variants of insulin to a panel of beef insulin-specific T cell hybridomas with limited TCR alpha- and/or beta-chain sequence differences. We demonstrate that all beef insulin-specific TCR have the same orientation on the insulin/Ad complex, such that the alpha-chain interacts with the carboxyl-terminal region of the A beta(d) alpha-helix, and the beta-chain complementarity-determining region 3 interacts with the carboxyl-terminal portion of the peptide, consistent with that observed for crystallized TCR-peptide/class I complexes. Despite this structural constraint, even TCR that share structural similarity show remarkable heterogeneity in their responses to the panel of MHC mutants. This variability appears to result from conformational changes induced by binding of the TCR to the complex and the exquisite sensitivity of the threshold for T cell activation.

Autoimmunity develops in lupus-prone NZB mice despite normal T cell tolerance.

NZB mice spontaneously develop an autoimmune disease characterized by production of anti-RBC, -lymphocyte, and -ssDNA Abs. Evidence suggests that the NZB mouse strain has all of the immunologic defects required to produce lupus nephritis but lacks an MHC locus that allows pathogenic anti-dsDNA Ab production. The capacity to produce diverse autoantibodies in these mice raises the possibility that they possess a generalized defect in self-tolerance. To determine whether this defect is found within the T cell subset, we backcrossed a transgene encoding bovine insulin (BI) onto the NZB background. In nonautoimmune BALB/c mice, the BI transgene induces a profound but incomplete state of T cell tolerance mediated predominantly by clonal anergy. Comparison of tolerance in NZB and BALB/c BI-transgenic mice clearly demonstrated that NZB T cells were at least as tolerant to BI as BALB/c T cells. NZB BI-transgenic mice did not spontaneously produce anti-BI Abs, and following antigenic challenge, BI-specific Ab production was comparably reduced in both BI-transgenic NZB and BALB/c mice. Further, in vitro BI-specific T cell proliferation and cytokine secretion were appropriately decreased for primed lymph node and splenic T cells derived from NZB BI-transgenic relative to their nontransgenic counterparts. These data indicate that a generalized T cell tolerance defect does not underlie the autoimmune disease in NZB mice. Instead, we propose that the T cell-dependent production of pathogenic IgG autoantibodies in these mice arises from abnormal activation of T cells in the setting of normal but incomplete tolerance.

Resting B cells from autoimmune lupus-prone New Zealand Black and (New Zealand Black x New Zealand White)F1 mice are hyper-responsive to T cell-derived stimuli.

To determine whether B cells from New Zealand Black (NZB) and (New Zealand Black x New Zealand White)F1 (NZB/W) mice possess intrinsic defects that lead to altered immune responsiveness, we purified resting B cells from these mice and compared their surface phenotype and function with those of resting B cells isolated from BALB/c and DBA/2 nonautoimmune mouse strains. Flow cytometric analysis of freshly isolated resting B cells revealed that NZB and NZB/W resting B cells are conventional B2-type cells similar to their nonautoimmune counterparts. Despite this, resting B cells from young NZB and NZB/W mice express lower levels of CD23 on their surface and aberrant levels of intracellular IgM. Upon stimulation, resting B cells from young NZB and NZB/W mice demonstrate increased proliferation, IgM secretion, or enhanced expression of costimulatory molecules in response to a variety of different T cell-derived stimuli, including cytokines and signals generated through CD40. Therefore, B cell hyper-responsiveness to T cell stimuli is immunodominant or codominant in NZB/W mice. Taken together, our results suggest that intrinsic B cell hyper-responsiveness may play a role in the pathogenesis of autoimmune disease in NZB and NZB/W mice. The increased clonal expansion of these B cells together with increased Ig production and enhanced costimulatory capacity serve to amplify the immune response. In the context of normal but incomplete T cell tolerance, B cell hyperresponsiveness to the limited signals provided by partially tolerant T cells may be sufficient to yield an autoantibody response.

Tolerance is overcome in beef insulin-transgenic mice by activation of low-affinity autoreactive T cells.

To gain insight into the factors controlling the maintenance or loss of T cell self tolerance we produced beef insulin (BI)-transgenic BALB/c mice. Transgenic mice express BI under control of the human insulin promoter and secrete physiological amounts of beef insulin. Although these mice are tolerant to BI, as evidenced by the lack of insulin-specific IgG antibody production following intraperitoneal immunization, tolerance is not complete. Footpad immunization results in a weak antigen-specific T cell proliferative response, indicating the presence of self-reactive BI-specific T cell in the periphery. These T cells are functional in vivo, providing support for IgG1, IgG2a, and IgG2b BI-specific antibody production, but require higher higher concentrations of antigen than nontransgenic T cells (both in vivo and following recall responses in vitro) to become activated. In vitro, BI-specific T cell proliferation in BI-transgenic mice can be largely restored by addition of interleukin-2, indicating that a significant component of T cell tolerance is mediated by anergy. To characterize the autoreactive T cells that become activated when tolerance is broken, BI-specific T cell hybridomas were generated from transgenic mice and compared to a panel of hybridomas previously derived from nontransgenic BALB/c mice. The majority of BI-transgenic hybridomas recognized the immunodominant A1-14 beef insulin peptide but with lower avidity than BALB/c hybridomas. Consistent with this, none of the dominant T cell receptor rearrangements found in the BALB/c BI-specific T cell receptor repertoire were found in the transgenic hybridomas. These results indicate that, despite evidence for clonal inactivation of many BI-specific T cells in BI-transgenic mice, loss of tolerance results from activation of low-affinity antigen-specific T cells that appear to have escaped this process.

Both MHC and background gene heterozygosity alter T cell receptor repertoire selection in an antigen-specific response.

Many autoimmune diseases are associated with specific class II MHC alleles; however, this association is not complete. One explanation for the variable expression of disease in susceptible individuals is that variability in the TCR repertoire may alter the potential to generate pathogenic autoreactive T cells. The current study was undertaken to examine the possibility that MHC and background heterozygosity, which is the norm in the outbred human population, alters the expressed TCR repertoire and, if so, whether this has an impact on peptide recognition and antigenic specificity. We, therefore, systematically analysed the beef insulin-specific TCR repertoire in inbred BALB/c mice before and after introduction of MHC heterozygosity (BALB/c x BALB.K)F1 mice, or MHC and background gene heterozygosity (BALB/c x A/J)F1 mice. We show that T cells from all three repertoires are predominantly Ad-restricted and recognize the same immunodominant peptide. Despite this, the beef insulin-specific TCR repertoires in F1 mice differ from those seen in BALB/c mice with the most dramatic changes seen in (BALB/c x A/J)F1 mice. These changes are accompanied by subtle differences in the antigenic specificity of the T cells. The results demonstrate that both MHC and background gene heterozygosity affect TCR repertoire selection, suggesting that the variable expression of autoimmune disease in individuals with a susceptible MHC allele may result, in part, from variability in the TCR repertoire introduced by this heterozygosity.