Spectratyping analysis of the islet-reactive T cell repertoire in diabetic NOD Igμ^null mice after polyclonal B cell reconstitution

Non Obese Diabetic mice lacking B cells (NOD.Igμ^null mice) do not develop diabetes despite their susceptible background. Upon reconstitution of B cells using a chimera approach, animals start developing diabetes at 20 weeks of age.

We have used the spectratyping technique to follow the T cell receptor (TCR) V beta repertoire of NOD.Igμ^null mice following B cell reconstitution. This technique provides an unbiased approach to understand the kinetics of TCR expansion. We have also analyzed the TCR repertoire of reconstituted animals receiving cyclophosphamide treatment and following tissue transplants to identify common aggressive clonotypes.

We found that B cell reconstitution of NOD.Igμ^null mice induces a polyclonal TCR repertoire in the pancreas 10 weeks later, gradually diversifying to encompass most BV families. Interestingly, these clonotypic BV expansions are mainly confined to the pancreas and are absent from pancreatic lymph nodes or spleens. Cyclophosphamide-induced diabetes at 10 weeks post-B cell reconstitution reorganized the predominant TCR repertoires by removing potential regulatory clonotypes (BV1, BV8 and BV11) and increasing the frequency of others (BV4, BV5S2, BV9, BV16-20). These same clonotypes are more frequently present in neonatal pancreatic transplants under the kidney capsule of B-cell reconstituted diabetic NOD.Igμ^null mice, suggesting their higher invasiveness. Phenotypic analysis of the pancreas-infiltrating lymphocytes during diabetes onset in B cell reconstituted animals show a predominance of CD19⁺ B cells with a B:T lymphocyte ratio of 4:1. In contrast, in other lymphoid organs (pancreatic lymph nodes and spleens) analyzed by FACS, the B:T ratio was 1:1. Lymphocytes infiltrating the pancreas secrete large amounts of IL-6 and are of Th1 phenotype after CD3-CD28 stimulation in vitro.

Diabetes in NOD.Igμ^null mice appears to be caused by a polyclonal repertoire of T cell accumulation in pancreas without much lymphoid organ involvement and is dependent on the help by B cells.