Vκ polymorphisms in NOD mice are spread throughout the entire immunoglobulin kappa locus and are shared by other autoimmune strains

Abstract

The diversity of immunoglobulin (Ig) and T cell receptor (TCR) genes available to form the lymphocyte repertoire has the capacity to produce a broad array of both protective and harmful specificities. In type 1 diabetes (T1D), the presence of antibodies to insulin and other islet antigens predicts disease development in both mice and humans, and demonstrate that immune tolerance is lost early in the disease process. Anti-insulin T cells isolated from T1D-prone non-obese diabetic (NOD) mice use polymorphic TCRα chains, suggesting that the available T cell repertoire is altered in these autoimmune mice. To probe whether insulin-binding B cells also possess polymorphic V genes, Ig light chains were isolated and sequenced from NOD mice that harbor an Ig heavy chain transgene. Three insulin-binding Vκ genes were identified, all of which were polymorphic to the closest germline sequence matches present in the GenBank database. Additional analysis of over 300 light chain sequences from multiple sources, including germline DNA, shows that polymorphisms are spread throughout the entire NOD Igκ locus, as these polymorphic sequences represent 43 distinct Vκ genes which belong to 14 Vκ families. Database searches reveal that a majority of polymorphic Vκ genes identified in NOD are identical to Vκ genes isolated from SLE-prone NZBxNZW F1 or MRL strains of mice, suggesting that a shared Igκ haplotype may be present. Predicted amino acid changes preferentially occur in CDR, and thus could alter antigen recognition by the germline B cell repertoire of autoimmune versus non-autoimmune mouse strains.

Reversing Tolerance in Isotype Switch–Competent Anti-Insulin B Lymphocytes

“…Resulting cDNA was used as PCR template to amplify Igκ genes using a murine Vκ primer, 5′−ATT GTK MTS ACM CAR TCT CCA-3′, and murine Cκ primer, 5′−GGA TAC AGT TGG TGC AGC ATC-3′, where K = G or T, M = A or C, S = C or G, and R = A or G with a 44°C annealing temperature. Appropriately sized PCR products were gel purified using the MinElute Gel Extraction Kit (Qiagen) and Igκ were cloned as described previously (37, 38). Vκ and Jκ gene segment identities were assigned using Ig BLAST (http://www.ncbi.nlm.nih.gov/igblast/) with CDR boundaries defined by the KABAT V domain delineation system.…”

Bruton’s Tyrosine Kinase Synergizes with Notch2 To Govern Marginal Zone B Cells in Nonobese Diabetic Mice

“…Expression of the HC transgene alone (V H 125) generates a broad repertoire in which endogenous light chains pair with the V H 125Tg (21, 31, 32). In NOD, but not C57BL/6 mice, two of these L chains generate insulin-binding BCRs that enter the mature repertoire (22).…”

“…Thus, fine-tuned differences in BCR specificity affect reliance on BTK for MZ selection, and further affects reliance on BTK for survival. B cells from autoimmune-prone mice have altered signaling thresholds, which reduce tolerance and generate increased numbers of BCRs with specificities that may be suitable for entry into the MZ (21, 22, 24). This may simply provide a numeric advantage that allows discernment of a partial effect of Notch2 haploinsufficiency that may be present, but less visible, in strains with smaller MZs.…”

“…7 Aminoactinomycin-D (BD Biosciences) was used for the exclusion of dead cells. Insulin-binding B cells were detected with biotinylated insulin, prepared as previously described(21), followed by streptavidin-flourochrome. Inhibition studies were completed using duplicate samples to which ten-fold quantities of unlabeled insulin were added.…”

“…Components of the extracellular matrix newly discovered to contribute to MZ development are also overexpressed in the MZ of NOD mice (20). Our group has shown that polymorphisms in Igκ genes contribute to increased propensity for autoreactivity in NOD B cells and that these polymorphisms are shared with SLE-prone mice (21, 22). B cell intrinsic tolerance defects in the NOD strain also allow increased numbers of autoreactive B cells to reach maturity (22-24).…”