Processing without proteolytic cleavage is required for recognition of insulin by T cells

Abstract

Beef insulin as well as a chymotryptic A-chain fragment [BI-A1-14(SSO3-)3] need uptake by antigen-presenting cells (APC) for efficient presentation in combination with major histocompatibility complex class II molecules to insulin-specific T cells. This could be shown by the inability of aldehyde-fixed APC to present these antigens to T cells. Furthermore, presentation of the insulin fragment as well as presentation of ovalbumin (OVA) was inhibited by treatment of APC with chloroquine, cerulenin or tunicamycin. This was not the case for a processing-independent OVA peptide. Treatment of APC during antigen pulsing with various protease inhibitors, active on all classes of proteases, did not block presentation of insulin although some of these reagents did interfere with the presentation of OVA. Several inhibitors especially of serine or thiol proteases rather enhanced the presentation of insulin. This indicates that intracellular proteolytic cleavage of insulin does not seem to be required for generation of the antigenic determinant but, if it occurs, rather destroys the antigenic peptide. Insulin and its A-chain fragment may, therefore, represent a model for a processing-dependent antigen not requiring proteolytic cleavage but other modifications.

Reduction of disulfide bonds during antigen processing: evidence from a thiol-dependent insulin determinant.

“…We emphasize that the data with fixed APC reported by Naquet et al (8) suggest that even the minimal A(1-14)/B (7)(8)(9)(10)(11)(12)(13)(14)(15) peptide requires further processing. Evidence for further processing requirements of sulfonated and disulfide forms of A chain peptides were also provided by Miller et al (12) in studies with human loop-specific T cell clones and those of Gradehandt et al (11,32) using A~bAok-restricted clones. The first demonstration of efficient presentation of insulin peptides by fixed APC is provided by the current study.…”

“…This conclusion is consistent with the results of Falcioni et al (13), who demonstrated that >90% of a large panel of I-Ab-restricted beef insulin-reactive T cell clones responded to sulfonated A chain but not performic acid-oxidized insulin. Gradehandt et al (11,32) have demonstrated that A~bA~-restricted clones recognize an A (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14) determinant that is sensitive to performic acid oxidation. It was proposed that processing of insulin is not dependent on proteolysis, but rather on modification or conversion of disulfide bridges.…”

Freshly isolated mouse 4F7+ splenic dendritic cells process and present exogenous antigens to T cells

“…the presence or absence of Chq for additional 4 h. Following antigen pulsing they were fixed with 0.05 YO glutaraldehyde (EM grade) for 40 s. The reaction was stopped with L-lysine (0.1 M) [38] …”

Destructive proteolysis by cysteine proteases in antigen presentation of ovalbumin

“…A second approach to identifying enzymes involved in Ag processing is to digest Ag with purified proteases in vitro and test for recognition by Th in the presence of fixed APC. Such studies have implicated three different proteases in Ag processing: the aspartyl protease cathepsin D and the cysteine proteases cathepsins B and L [14,15,[17][18][19][20].…”