A multiplicity of protein antigens in subcellular fractions of rat insulinoma tissue are able to stimulate T cells obtained from non-obese diabetic mice

Abstract

Type 1 (insulin-dependent) diabetes mellitus is a T-cell mediated autoimmune disease with a number of different proteins being implicated as target autoantigens. A 38 kDa protein residing in the insulin secretory granule of insulinoma tissue is recognized by T-cell clones from a newly-diagnosed Type 1 diabetic patient. We have investigated the capacity of normal rat pancreatic beta-cell extracts and various subcellular fractions of transplantable RIN tissue to induce proliferation of T cells from non-obese diabetic (NOD) mice and H-2 identical NON.NOD-H-2g7 control mice. Normal rat islet beta-cell protein fractions induced intense, dose-dependent proliferation of NOD splenic T cells, but only marginal proliferative responses of NON.NOD-H-2g7 splenic T cells. To further localize the target antigens, four different subcellular fractions from RIN tissue were used as a source of antigen; here in particular the cytosolic proteins showed dose-dependent activation capacity with splenic T cells in NOD animals. These activities were absent in control mice. There was no proliferation after incubation with microsome preparations from other rat endocrine tissues. Purified carboxypeptidase H did not have any stimulatory activity on NOD T cells. Fractionation of the RIN cytosolic proteins showed a large number of different fractions eliciting proliferative activity. These results demonstrate that NOD T cells respond to a large number of potential islet beta-cell target antigens and it will be necessary to utilize NOD T-cell clones to identify the number and nature of these antigens.

Imogen 38: a novel 38-kD islet mitochondrial autoantigen recognized by T cells from a newly diagnosed type 1 diabetic patient.

“…The majority of T cell responses in spontaneously diabetic animals or humans, however, appears to be to unidentified proteins of a broad range of molecular sizes (13)(14)(15)(16). Subcellular fractionation studies have suggested that the insulin-secretory granule may be a major source of such antigens (17)(18)(19)(20).…”

HLA-DR-restricted T cell lines from newly diagnosed type 1 diabetic patients specific for insulinoma and normal islet beta cell proteins: lack of reactivity to glutamic acid decarboxylase

“…The reason for selecting microsomal membranes secretory granule components from the transplantable rat RIN insulinoma as a source of antigens to study T cells in hutnan type 1 diabetic patients was based upon our previous observations in NOD mice that this preparation contains disease-related antigens important in the T cell mediated destruction of islet beta cells [10]. We used rat insulinoma tissue to study T cell reactivity in humans since large amounts of rat insulinoma tissue were available, in which secretory granules and different membrane fractions have been well characterized [18].…”

“…These antigens remain uncharacterized. Our earlier studies showed the presence of disease-related antigens residing in the transplantable RIN insulinoma membranes that stimulated T cctls from the unprimed NOD animals [10]. There have also been other detailed studies showing ihe presence of novel, early disease-related autoantigens in insulinoma tissues recognized by T cells from young NOD animals [11], Indeed, the antigens recognized by disease-inducing, diabetogenic T cell clones have also been localized to insulinoma secretory granules [12].…”

“…lOO^g/ml aprotinin. lO/ig/ml leupeptin and lO/fg/ml pepstatin) and differential centrifugation [10]. The washed pelleted microsomes were stored aliquoted at -70 C. Protein concentration was determined by Pierce assay.…”

“…Furlhermorc. considerable evidence has accumulated on the muttiplicly of islet cell antigens recognized by T cells in the NOD animals [10,11]. These antigens remain uncharacterized.…”