Development of T cell clones reactive to two defined restriction elements in conjunction with two defined epitopes of antigen

Abstract

A previously described pig insulin (PI)-specific T cell line of (B10 X B10.BR)F1 origin was assayed for its reactivity with species variants of insulin in the presence of antigen-presenting cells (APC) of various H-2 haplotypes. In addition to its reactivity with PI and bovine insulin (BI) in the context of syngeneic F1 (H-2b X k)-APC, a weak cross-reactivity was observed with parental B10 (H-2b)-APC and BI but not PI. The cross-reactive cells could be selected out by several restimulations with the combination of BI and B10-APC. From the resulting, strongly cross-reactive T cell line several interleukin 2-dependent sublines were developed which did not require antigen-specific restimulations for further propagation. All such sublines had retained the original cross-reactivity with BI and B10-APC but showed significant differences in their fine specificity patterns, which indicates that each subline represents a clonal population. One of the sublines was cloned by limiting dilution at one cell/culture with a cloning efficiency of 76%. Five of the clones that were tested for reactivity had the same cross-reactivity as the original subline and upon recloning at 0.1 cells/culture the pattern again remained unchanged. From an analysis of the two antigen combinations (PI/F1 and BI/B10) it can be concluded that single cells can react with different restriction elements in the context of distinct epitopes of insulin. The implications of this finding for the mechanism of T cell recognition are discussed and a model for the function of major histocompatibility complex molecules in T cell recognition is proposed.

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

“…The authors concluded that the T cells recognized a confor-mational determinant containing essential residues from both the A and B chains of insulin. Others have described experiments in which mouse and human A chain loop-reactive T cells were stimulated by A chain peptides with no associated B chain residues (9)(10)(11)(12)(13). These results indicate that at least some loop-reactive T cells recognize determinants within the A chain.…”

“…3). Fixed TH2.2 B cells were pulsed with various concentrations of A (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14) in the absence of serum, washed, and cultured with T cell hybridomas. The presence of DTT during the peptide pulse did not affect the capacity of the treated APC to stimulate T cells, in contrast to our results with insulin.…”

“…However, it seems ~2~ a unlikely that a disulfide bond between the loop cysteines, A6 and All, would form in reduced A (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14) in the presence of DTT. Results similar to those obtained with air-oxidized A (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14) were observed in experiments using sulfonated A chain. Fixed APC pulsed with sulfonated A chain in the presence, but not the absence, of DTT were capable of stimulating Bd-l.4 and Bb-6 ( Fig.…”

“…Disulfide bond-containing peptides of insulin of minimal length to maintain antigenicity still require processing by the operational definition. Naquet et al (8) have described a minimal determinant comprised of residues A(1-14) disulfide linked to B (7)(8)(9)(10)(11)(12)(13)(14)(15) that was recognized by I-A d-and I-Ab-restricted, loop-reactive T cell hybridomas. Further truncation or cleavage of disulfide bonds in this peptide destroyed its antigenicity.…”

Tcgfiii/p40 is produced by naive murine cd4+ t cells but is not a general t cell growth factor*

“…ST2/K9.4a2 is a subclone of the CD4+ Tcell clone ST2/K9 specific for insulin and I-A: Af; [9] which can be propagated in TCGF 111-containing medium. CSP2.1 is an IL 2-dependent [lo], DA-1 an IL 3-dependent [11], and 7TD1 an IL 6-dependent cell line [ 121.…”