Abstract
BackgroundNon Obese Diabetic mice lacking B cells (NOD.Igμnull 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.MethodsWe have used the spectratyping technique to follow the T cell receptor (TCR) V beta repertoire of NOD.Igμnull 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.ResultsWe found that B cell reconstitution of NOD.Igμnull 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.Igμnull 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.ConclusionsDiabetes in NOD.Igμnull 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.
Highlights
Type 1 diabetes (T1D) is a T cell mediated disease in which both CD4 and CD8 lymphocytes infiltrate the islets of Langerhans, causing destruction of insulin-producing beta cells and hyperglycemia
Profiles of T1D in Non ObeseDiabetic (NOD).Igμnull mice reconstituted with NOD splenic B cells We studied the progression of diabetes in > 100 NOD
10% of these mice kept for long-term observation did develop diabetes very late in life, beyond 12 months of age! onset of T1D following B cell reconstitution was roughly equivalent to that as seen for spontaneous disease in the NOD founder strain
Summary
Type 1 diabetes (T1D) is a T cell mediated disease in which both CD4 and CD8 lymphocytes infiltrate the islets of Langerhans, causing destruction of insulin-producing beta cells and hyperglycemia. In NOD mice, T cell islet infiltration starts within 3-4 weeks of life, producing overt diabetes in 80% of female mice beyond 30 weeks of age. NOD.Igμnull mice (which are B cell deficient) do not become diabetic [1], but develop disease if reconstituted with B cells [2]. It is known that to cause disease, the B cells are required to possess the I-Ag7 MHC class II molecule [5] and that the specificity of the B cells is important, as reconstitution of HEL-specific transgenic B cells in NOD.Igμnull mice did not cause diabetes [6]. Igμnull mice have been shown to contain a functional autoimmune T cell repertoire (in the absence of B cells) capable of causing diabetes if transferred into NOD.scid mice [8]. Upon reconstitution of B cells using a chimera approach, animals start developing diabetes at 20 weeks of age
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