Arun Fotedar

Unusually diverse T cell response to a repeating tripeptide epitope

The immune system utilizes a diverse T cell repertoire for the recognition of foreign antigens in the context of self MHC gene products. We have examined the potential diversity of the T cell response directed to a immunodominant repeating tripeptide epitope (EYA)5. This peptide represents one of the two T cell epitopes on the synthetic alpha-helical polypeptide antigen Poly 18, Poly EYK(EYA)5 in H-2d mice and does not require antigen processing prior to presentation to Poly 18-specific T cell hybridomas. The T cell response directed to the repeating tripeptide epitope (EYA)5 is extremely heterogenous even though the epitope has a relatively simple amino acid sequence. We have analyzed the fine specificity of 21 randomly chosen Poly 18-reactive, (EYA)5-specific and H-2d-restricted T cell hybridomas derived from H-2d, H-2bxd, and H-2b----H-2bxd Poly 18-responding mice to determine the number of unique antigen reactivity patterns represented by this T cell population. We used alanine- and/or lysine-substituted (EYA)5 peptides and a panel of haplotype-varied splenocytes and observed a great deal of microheterogeneity in response. We find that 13 of the 21 hybridomas have a distinct fine antigen specificity and T cell receptors. The binding of (EYA)5 to the antigen-binding groove of I-Ad appears to generate a highly diversified T cell response. Therefore, (EYA)5-I-Ad complex allows the activation of unrelated T cell clonotypes with the same overall antigen specificity and MHC restriction, but with distinct microheterogeneity in response and receptor usage.

Gene conversion may be responsible for the generation of the alloreactive repertoire

Meiotic gene conversion in the major histocompatibility complex (MHC) appears to be involved in the generation of MHC polymorphism. Here the authors suggest that mitotic gene conversion of MHC may generate somatic variants which sensitize the immune system. This could lead to an MHC-skewed repertoire with a high background alloreactive potential crossreacting with foreign antigens in the context of self MHC. Allodeterminant-bearing autologous cells may be deleted by the immune cells, thereby maintaining the observed genotype/phenotype correlation.

Characterization of agretopes and epitopes involved in the presentation of beef insulin to T cells

Beef insulin-specific I-Ad-restricted T cell hybridomas were derived from the fusion of antigen-primed (BALB/c X B6)F1 T cells with BW5147 thymoma. Specificity analysis revealed that the A-chain loop region is involved in antigen recognition. Hybridoma A20.2.15 is specific for beef insulin and cross-reacted with sheep insulin, but not with pork insulin. Using synthetic peptides we showed that the A-chain loop containing peptide A1-A14 jointed to the B7-B15 peptide by a disulfide bond can activate this hybridoma. Fragments generated by enzyme digest further suggest that the peptide recognized on beef insulin appears to involve A-chain loop residues A5-A12 and B-chain residues B7-B13 that are linked by the A7-B7 disulfide bridge. We found that beef insulin needs to be processed prior to T cell activation. Glutaraldehyde fixation and chloroquine treatment of presenting cells abolished their capacity to present insulin. Beef insulin denatured by pH changes cannot activate, thus suggesting that simple denaturation is not sufficient for presentation by antigen presenting cells. Finally, the agretope on beef insulin is comprised of two functional regions B7-B13 on the B chain and the A-chain loop in the A-chain, while residues A8 and A10 are probably involved in interaction with the T cell receptor.

Immunological competence and host-specific tolerance of antibody-facilitated bone marrow chimeras.

We have generated murine antibody-facilitated (AF) bone marrow chimeras in the genetic combination P1----(P1 X P2)F1 by the simultaneous injection of P1 bone marrow cells and anti-P2 MHC monoclonal antibody into normal (unirradiated) adult (P1 X P2)F1 recipients. These mice have normal life spans and appear to be healthy, with no overt signs of graft-versus-host disease. We have undertaken an extensive survey of the ability of stable, long-term AF chimeras to generate immune responses in vitro and in vivo. Both T and B lymphocyte functions have been analyzed in proliferative and effector cell assays. The AF chimeras respond normally to mitogenic as well as antigenic stimuli, and exhibit normal capacities for cellular collaboration in the generation of immune responses. However, splenic lymphocytes from AF chimeras are substantially and specifically hyporesponsive or nonresponsive to host, P2-encoded, alloantigens in in vitro assays of cell-mediated immunity. This host-specific tolerance is exhibited by the cytotoxic T lymphocyte lineage; T helper cells necessary for the generation of a cytotoxic response may also have decreased reactivity to host determinants. We conclude that our protocol for the production of AF chimeras does not compromise the immune system of chimeric animals but does allow the maintenance of host-specific tolerance, after stable equilibrium has been attained.

Antigen-Specific T-Cell Activation

Extensive investigations into the phenomenon of activation of antigen-specific ‘IA’-restricted T cells have led to the emergence of two main research areas. One is antigen processing by antigen-presenting cells (APC), prior to actual presentation to T cells in the context of IA on the APC surface. The second involves the study of the ternary interaction between antigen, IA and the T cell receptor. This Note concerns some of our results which impinge on these two overlapping areas of interest. We have generated T cell hybridomas by fusing antigen-primed T-cell blasts with BW5147, using procedures essentially described earlier [1]. T-cell hybridomas specific for beef insulin [2] selected for their ability to release IL2 in the presence of appropriate APC and antigen [3] were used in these studies.