B cell activation potential of insulin‐reactive T cells in H‐2^b mice

Abstract

The murine T cell response to heterologous insulins provides a good model system for studying the mechanism of immune response (Ir)-gene function, since insulin is a small, chemically well-defined molecule. H-2b mice respond predominantly to A chain loop determinants of beef insulin, presented by the I-A epitope Ia. W39. However, using a library of insulin-specific T cell hybridomas (THy), we previously found that immunization of H-2b mice with beef insulin activates a much wider population of T cells than are detected in T cell proliferation assays. Using such cloned THy we were able to study Ir-gene control at the level of antigen presentation. We compared the ability of the various THy to induce differentiation in I-A-matched B cells in response to antigen. Although both A and B chain-reactive clones respond with interleukin 2 production, they differ markedly in their potential to activate B cells in that only the former are able to induce B cell differentiation in the presence of the intact beef insulin molecule. The latter, however, can serve as helper cells in the presence of isolated B chain, and can synergize with a suboptimal concentration of A chain-reactive THy to induce an optimal B cell response. These results suggest that the insulin molecule is presented by I-Ab antigen-presenting cells in a very specific configuration that allows more effective T cell recognition of the A chain loop than the B chain determinants. To explain the discrepancy between the interleukin 2 assay and the induction of polyclonal activation, it can be assumed that in the former assay antigen is presented by macrophages, while presentation by B cells is necessary for induction of polyclonal activation. Macrophages are able to process the intact beef insulin molecule and, therefore, present B chain determinants, while nonimmune B cells may be unable to process antigen and could present B chain determinants only when the isolated B chain is given as antigen.

Identification of an autologous insulin B chain peptide as a target antigen for H-2Kb-restricted cytotoxic T lymphocytes.

“…A number of studies have demonstrated that maintenance of an intact interchain disulfide linkage between the A and B chains is necessary for the effective Th response to several dominant insulin epitopes (1)(2)(3)(4)(5). However, other investigators have shown that a class II H-2-restricted Th response can be induced against both insulin A chain-and insulin B chain-derived peptides in the absence of the disulfidelinked B and A chain fragments, respectively (6)(7)(8)(9). It is tempting to speculate that the Th response to one or more of these insulin-derived peptides may be relevant to the pathogenesis of insulin-dependent (type I) diabetes mellitus (IDDM).…”

Molecular analysis of antigen recognition by insulin-specific T-cell hybridomas from B6 wild-type and bm12 mutant mice.

“…Insulin is a chemically well-defined peptide hormone (molecular weight, -5,000), which consists of an A and a B chain that are linked by two disulfide bridges; an intrachain disulfide bond creates a loop in the A chain which contains the amino acids at positions A7, A8, A9, and A10. Using the various species of insulin which differ at single amino acids, we were able to determine the fine specificity of our T cells (30,31,34).…”

“…Insulin is a chemically well-defined peptide hormone (molecular weight, -5,000), which consists of an A and a B chain that are linked by two disulfide bridges; an intrachain disulfide bond creates a loop in the A chain which contains the amino acids at positions A7, A8, A9, and A10. Using the various species of insulin which differ at single amino acids, we were able to determine the fine specificity of our T cells (30,31,34).To define the MHC restriction of our insulin-reactive T helper hybridoma (THy) lines precisely, we made use of the class II mutant bml2 mouse and the B6 wild type. The I-Ap gene of the bml2 mouse differs from that of the wild type at three nucleotide positions, which give rise to three amino acid changes clustered within the first extracellular domain of the Ap molecule (43).…”

Insulin‐specific T cell help to TNP‐specific B cells requires activation via the antigen T cell receptor

“…The following cell lines, transformed cells and hybrids were cultured in 10% fetal calf serum (FCS)/RPMI 1640 medium supplemented as described below: CTL-L (IL 2-dependent C57BL/6 anti-DBN2 CTL line [19]); EL4-16 (H-2b) thymoma [18]; ELWPG (H-2b) thymoma cells are EL4 cell variant that is sensitive to HAT-containing medium (P. Golstein, unpublished); BW5147 (H-2k) thymoma cells; L12R4 (H-2b) T lymphoma cells [20]; and two C57BLi6 anti-Ins T cell X BW5147 hybrid cell lines: 52H10Fll with specificity against the bovine Ins A chain and 51A4C10 with specificity against ungulate Ins B chains [21].…”

“…Thus, the Ins-specific Th cell clones needed about lo4-fold higher TNP-Ins concentration for induction of IL 2 release compared to the TNP-Ins concentration needed for maximal helper activity. Analysis of helper activity and IL 2 release by two Ins-specific Th cell hybridoma clones (Dr. B. Huber) showed that these cells needed the same high concentration of TNP-Ins for both induction of help and IL2 release (not shown, see [21]). On the other hand, chicken gamma-globulin (CGG)-specific T cell lines [12] and clones (unpublished) showed maximal helper activity also in the range of 1-10 ng TNP-CGG/ml for TNP-LPS primed B/ MQ, cells in the same culture system.…”