Recognition of human insulin in vitro by T cells from subjects treated with animal insulins.

Abstract

Structurally defined proteins and peptides have provided considerable information about the specificity and regulation of immune responses in inbred animals. Many diabetics require therapy with insulin; therefore, we used this defined protein as a model antigen to investigate immune responses in the outbred human population. In this report, we examine human T cell recognition of antigenic determinants on various insulins. A group of 25 subjects was selected from over 200 diabetics because of the magnitude of their in vitro responses. 13 of the 25 had significant T cell responses to human insulin despite treatment with only beef/pork insulin mixtures. This autoimmunity may be attributed to crossreactivity of lymphocytes highly reactive to "foreign" epitopes on therapeutic insulins. Alternatively, identical determinants shared by human and animal insulins may be recognized. By employing additional insulins not used therapeutically and isolated A and B chains, several potential mechanisms for lymphocyte autoreactivity to human insulin were demonstrated. Some epitopes are conformational and require recognition of an intact molecule, whereas other epitopes may arise from antigen processing at the cellular level. Studies using zinc-free insulins suggest that zinc-induced alterations of the molecular surface may result in some shared reactivities between animal and human insulin. Furthermore, T cell reactivity against "foreign" epitopes is more complex than anticipated from differences in amino acid sequence. The response patterns of many subjects indicate that the A-chain loop associates with the N-terminal B chain to form a complex determinant. This determinant is recognized more often than individual amino acids. We conclude that insulin therapy generates polyclonal T cell responses directed at multiple epitopes on the molecule. Many of these epitopes are not identified by amino acid exchanges and their presence on human insulin leads to apparent autoimmunity.

Spontaneous Hypoglycemia Associated With Autoimmunity Specific to Human Insulin

“…Together with the PBL proliferation data this shows a cross-reactivity of most of S.M. 's T-lymphocytes with common antigenic determinants that are expressed on all three insulin species (23).…”

“…An analogous situation is shown for S.M., whose B-lymphocytes produced only human insulin-specific antibody, but whose T-lympho' cytes were sensitized to epitopes shared by human, pork, and beef insulins. Similarly, in diabetics who were treated only with beef and pork insulins, lymphocyte proliferative re' sponses were induced with beef, pork, and human insulin species (23).…”

Helper T-cell clones that recognize autologous insulin are stimulated in nonresponder mice by pork insulin.

“…Approximately 20% of diabetics treated with heterologous insulin develop strong T-lymphocyte proliferative responses when stimulated with beef and pork insulin in vitro. A significant percentage of these patients' lymphocytes also responds to human insulin, thus demonstrating the presence of T cells that recognize autologous insulin (21). Moreover, T cells that react with autoantigens, such as the acetylcholine receptor (22) and autologous thymocytes (23), have been cloned from patients with spontaneous autoimmune diseases; T cells specific for autologous collagen (24), myelin basic protein (25), and thyroglobulin (26) have been cloned from animals in which autoimmunity was experimentally induced.…”

Immunological Responses to Exogenous Insulin

“…T cell responses are greater with the B chain than the A chain of insulin (363,364). Theoretically, insulin-specific T cells measured in these studies may be autoreactive T cells generated by endogenous insulin, autoreactive T cells expanded by exogenous insulin, and/or T cells initially generated by exogenous insulin; however, based on the assays used, it is not possible to differentiate among these possibilities.…”