We studied a human leukocyte antigen—identical pancreas graft transplanted into an insulin-dependent (type l) diabetic patient shortly after onset of recurrent diabetes to characterize the putative autoreactive T lymphocytes mediating the lesion. The immunohistopathological analysis revealed the presence of isletitis and a selective loss of β-cells. The isletitis was mostly constituted by CD8+/T-lymphocyte receptor α,β (TCR α,β+) T lymphocytes surrounding and infiltrating the affected islets. CD4/CD8/TCRγ,δ+ T lymphocytes were observed within the islets. Incubation of the tissue in 15% interleukin 2 induced the migration and initial expansion of the infiltrating cells (66% CD3+lymphocytes) for up to 2 wk; most T lymphocytes in this initial isolate were CD4+ (92% CD4+ and 7% CD8+). Long-term anti-CD3 stimulation of this T-lymphocyte population induced the selectivegrowth of CD8+/TCRαβ+ (75%) and CD4/CD8/TCRγδ+ v(all V1δ+) (17%) T lymphocytes. Therefore, this strategy selectively expanded the T lymphocytes, found to be the predominantly islet-infiltrating cells, rather than the lymphocytes predominating in the initial isolate. Anti-CD3 did not stimulate growth of T lymphocytes in cultures of three isletitis-free pancreas graft biopsies. In a control experiment with a CD4+-rich T-lymphocyte population, long-term anti-CD3 stimulation and cloning of cytomegalovirus (CMV)-primed peripheral blood mononuclear cells from a CMV+ subject selectively induced the growth of CD4+ T-lymphocyte clones, all CMVspecific.Application of the same strategy to a CD8+ and CD3+/CD4/CD8/TCRγδ+ T-lymphocyterich population from a Graves' disease thyroid resulted in expansion of CD8+ but not CD4/CD8 lymphocytes. We conclude that 1) the selective T-lymphocyte expansionproperties of anti-CD3 monoclonal antibody in long-term cultures is not dependent on thephenotype of the T lymphocytes but most likely on their activation state and 2) application of this strategy to T lymphocytes isolated from pancreas grafts transplanted into diabetic patients undergoing recurrent diabetes may allow the characterization of the putative CD8+ cells mediating type l diabetes.

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