Vkappa 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 TCRalpha 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 Vkappa 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 Igkappa locus, as these polymorphic sequences represent 43 distinct Vkappa genes which belong to 14 Vkappa families. Database searches reveal that a majority of polymorphic Vkappa genes identified in NOD are identical to Vkappa genes isolated from SLE-prone NZBxNZW F1 or MRL strains of mice, suggesting that a shared Igkappa 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.

Fig. 1

Insulin-binding Vκ genes are polymorphic to C57BL/6 germline sequences. Hybridomas generated from VH125Tg NOD splenic B cells were cloned at limiting dilution. a Hybridoma clone supernatants were tested by ELISA to identify insulin-binding antibody production. Tenfold excess insulin competitor was incubated in solution with the supernatants to detect an antigen-specific interaction. The percent inhibitable binding is shown. b–d Sequence alignment of insulin-binding hybridoma sequences with the closest C57BL/6 germline sequence matches. Kabat-defined CDR are boxed, putative polymorphisms are indicated by asterisks

Fig. 2

Polymorphisms are present in the majority of identified NOD Vκ genes and are spread throughout the Igκ locus. NOD Vκ genes for which three or more sequences that differed from each other by ≤2 nt changes are included in the analysis, the total number of sequences analyzed are indicated in Table 1. The closest C57BL/6 germline match was identified by IgBLAST and Mus musculus build 37.1. Chromosomal locations, and thus Vκ gene identity of NOD Vκ sequences are unconfirmed. Polymorphisms are defined as those nt which are conserved among NOD sequences and differ from the sequence of the closest C57BL/6 germline match. The total number of nt polymorphisms for each NOD Vκ gene is shown, aa replacement (black) and silent nt changes (white) are shown as portions of the total bar

Fig. 3

The frequency of NOD Vκ nucleotide changes is increased in CDR regions. The per site rate of nt changes in each FWR and CDR was calculated, e.g., the total number of nt changes in FWR1 was divided by the total number of nt in FWR1 for each of the NOD Vκ genes, and the average value observed for all 43 Vκ genes was calculated to produce the average frequency of nt changes in FWR1 (shown in parentheses). This was repeated for FWR2, FWR3, CDR1, CDR2, and CDR3. The average frequency of changes in FWR1 was divided by the sum of the average frequency of changes in all of the FWR and CDR to calculate the relative proportion of nt changes that were occurring in FWR1, which was repeated for the other regions, shown as percentage of the total

Fig. 4

Nucleotide polymorphisms result in amino acid changes in NOD Vκ genes. NOD Vκ aa sequences are based on predicted translations from NOD nt sequences. The number of aa changes in each NOD Vκ gene is shown, along with the putative IMGT Vκ gene identity along the top of the graph. These data are compiled from results presented in Table 3

Fig. 5

NOD Vκ sequence matches with NZB x NZW F1 Vκ cluster to the 5′ region, whereas matches with MRL Vκ cluster to the 3′ region of the Igκ locus. The closest C57BL/6 germline match was identified by IgBLAST, and NOD Vκ genes are positioned from the Jκ distal to Jκ proximal region of the locus as indicated, based on the positions of the C57BL/6 sequence matches in NCBI Map Viewer, M. musculus build 37.1. Chromosomal locations of NOD Vκ sequences are unconfirmed