Anti-insulin B cell receptors display skewed immunoglobulin gene usage and limited mutation in pre-symptomatic type 1 diabetes donors

Abstract

Abstract Antibodies targeting insulin and other islet antigens predict type 1 diabetes (T1D) onset. Insulin autoantibody (IAA)-positive individuals progress to diabetes more rapidly, yet little is known about how IAA-precursor anti-insulin B cells (AIBCs) recognize insulin. Complementarity-determining regions (CDRs) of the B cell receptor (BCR) are structurally diverse loops that constitute the antigen-binding site, and amino acid variability in BCRs increases the breadth of repertoire antigen-specificities. To investigate anti-insulin BCRs during the earliest detectable stages of T1D, we enrolled eight insulin therapy-naïve Type 1 Diabetes TrialNet Pathway to Prevention participants who were positive for ≥2 islet autoantibodies and thus at high risk for diabetes. We stimulated donor peripheral blood mononuclear cells to drive BCR secretion as antibody, screened for AIBC-containing wells by ELISA, and immortalized these cells as hybridomas. We validated 25 monoclonal anti-insulin hybridoma lines and sequenced 16 heavy chain (IgH) and 14 light chain (IgL) immunoglobulin genes. 25% of VH and 50% of VL sequences contained at least one mutation within a CDR compared to germline, with 13% of VH and 29% of VL sequences having ≥6 mutations within the V-region. We surveyed previously and currently reported AIBC VH (n=25 BCRs), which revealed skewed VH and JH gene use compared to the total polyclonal IgH repertoire (n= 8790 BCRs, p < 0.001 and p < 0.05, respectively, chi-squared test). These data expose unique features of insulin recognition among AIBCs isolated from pre-symptomatic T1D donors, including V and J gene usage bias and potential for germline IgH insulin recognition, providing genetic and molecular insight into AIBC origins.