Processing Requirements for the Recognition of Insulin Fragments by Murine T cells

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Abstract
In this study we investigated aspects of antigen processing using insulin and insulin A chain-derived fragments as model antigens in Ab alpha Ak beta-restricted T-cell stimulation. Similarly to other proteins, the immunodominant region of insulin recognized by these T cells is limited in size. It is located on the insulin A chain and encompasses a portion of the molecule that is represented faithfully by peptide A1-14(SSO3-)3. Efficient presentation of intact insulin and its entire A chain is dependent on uptake and processing by APC. Whereas peptides stemming from various globular proteins are known to be presented to T cells by APC without requiring processing, this is not the case with A-chain fragment A1-14 (SSO3-)3. This observation suggested that, in addition to proteolytic degradation, other mechanisms might play a role in the processing of these antigens. Three cys-residues are located in close proximity to those amino acid residues of the insulin A chain that are inferred to participate in the specific interaction with MHC class II molecules and the TcR. In A-chain derivatives that are stimulatory for the T cells or in intact insulin these cys residues are engaged in disulfide bonds or are S-sulfonated. Both linkages can be reversibly modified by reaction with thiols. Functional data indicate that from intact insulin and from structurally distinct A-chain derivatives a closely similar or identical peptide is formed and bound to class II molecules for recognition by the T cells. The question arises as to whether, in this processed peptide, the cys residues are present in reduced form, engaged in disulfide bonds, or are modified in some other way. Taken together, these findings suggest that modification of cys residues or isomerization of disulfide bonds may play a role in insulin processing. It can be expected that other proteins carrying cys residues in their immunodominant peptides may show similar processing requirements. The inhibition of N-glycosylation of proteins by tunicamycin in APC blocked the processing and presentation of insulin and OvA whereas, under the same conditions, the presentation of a processing-independent peptide was not affected. Furthermore, an autoreactive T-cell clone was capable of recognizing tunicamycin-treated APC. Since the expression of class II molecules was found to be unaltered as demonstrated by cytofluorometric analysis the deficient N-glycosylation appears to have little influence on class II antigen-mediated T-cell recognition but interferes with uptake of antigen and/or its processing by APC.