Raghavanpillai Raju

T cell recognition of human pre-proinsulin peptides depends on the polymorphism at HLA DQ locus: A study using HLA DQ8 and DQ6 transgenic mice

HLA DQ8 (DQ A1*0301/DQB1*0302) molecule is implicated in the susceptibility to insulin dependent diabetes mellitus whereas, HLA DQ6 (DQ A1*0103/DQB1*0601) molecule may have a protective effect. In this study we used mice transgenic to HLA DQ8 and HLA-DQ6 to elucidate the T cell determinants on a putative islet cell target antigen, insulin. These mice do not express endogenous mouse class II heterodimers on cell surface. Using overlapping synthetic peptides spanning the complete sequence of huma pre-proinsulin, we identified the sequences recognized by T cells in DQ8 transgenic mice and compared these to those in DQ6 transgenic mice. We observed a differential pattern of recognition of epitopes on human pre-proinsulin (HPI) polypeptide presented by the HLA DQ8 allele as compared to HLA DQ6. The sequences 1-24 and 44-63 were immunodominant in DQ8 transgenic mice while DQ6 transgenic mice primarily recognized sequences 14-33 and 74-93 of HPI. We found that the immune response generated in HLA DQ8 transgenic mice against HPI 1-24 cross-reacted to the mouse pre-proinsulin sequence 1-24. The T cell response were specifically inhibited using anti-CD4 and anti-DQ8 monoclonal antibodies. This cross-recognition of self sequences raises the possibility of modulation of experimental diabetes using this peptide.

Acetylcholine Receptor Peptide Recognition in HLA DR3-Transgenic Mice: In Vivo Responses Correlate with MHC-Peptide Binding

HLA DR3 is an MHC molecule that reportedly predisposes humans to myasthenia gravis (MG). Though MG is an Ab-mediated autoimmune disease, CD4+ T cells are essential for the generation of high-affinity Abs; hence the specificities of autoreactive CD4+ T cells are important. In this study we report the HLA DR3-restricted T cell determinants on the extracellular region sequence of human acetylcholine receptor α subunit. We find two promiscuous determinants on this region 141–160 and 171–190 as defined by their immunogenicity in HLA DR3-, HLA DQ8-, and HLA DQ6-transgenic mice in the absence of endogenous mouse class II molecules. We also studied the minimal determinants of these two regions by truncation analysis, and the MHC binding affinity of a set of overlapping peptides spanning the complete sequence region of human acetylcholine receptor α subunit. One of the peptide sequences strongly immunogenic in HLA DR3-transgenic mice also had the highest binding affinity to HLA DR3. Identification of T cell determinants restricted to an MHC molecule known to predispose to MG may be an important step toward the development of peptide-based immunomodulation strategies for this autoimmune disease.

Evidence of a diverse T cell receptor repertoire for acetylcholine receptor, the autoantigen of myasthenia gravis.

We utilized two methods to look for T cell clonal expansions in myasthenia gravis (MG). We analyzed TCRBV CDR3 length polymorphism (spectratyping) to look for evidence of clonal expansion of CD4 or CD8 T cells directly from peripheral blood of MG patients. No statistically significant differences were found between the diversity of TCR repertoires in MG patients compared to normal control individuals when analyzed as groups. Rare oligoclonal expansions were detected in some individual MG patients but the significance of these findings is unclear. Next, we analyzed a panel of T cell hybridomas from acetylcholine receptor (AChR) immunized, MG-susceptible HLA-DR3 transgenic mice. The epitope specificity, TCRBV gene usage and CDR3 sequences of these hybridomas were highly diverse. We conclude there is only limited evidence for restricted TCR repertoire usage in human MG and suggest this may be due to the inability of HLA-DR molecules to select for restricted TCR recognition of AChR epitopes.

Cryptic determinants and promiscuous sequences on human acetylcholine receptor: HLA-dependent dichotomy in T-cell function.

Experimental autoimmune myasthenia gravis can be induced in some strains of mice and rats by immunizing with acetylcholine receptor. Also, epidemiologic studies demonstrate an MHC linkage of myasthenia gravis in the man. In order to obtain direct experimental evidence for the influence of the genes of the MHC complex in the development of myasthenia gravis, we used mice transgenic to individual HLA molecules. We observed an increased susceptibility to the disease in HLA DQ8 transgenic mice compared to HLA DQ6 transgenic mice ( J. Immunol. 160:4169; 1998). These mice lacked endogenous mouse class II molecules. In the present study we mapped the cryptic and dominant sequences on the extra cellular region of human acetylcholine receptor. Although some epitopes (e.g., alpha11-30, alpha141-160, alpha171-190) were common between DQ8 and DQ6 transgenic mice, several others were disparately recognized. We also found a functional dichotomy in T cells from mice differing by one MHC molecule (HLA DQ8 or DQ6) when primed by sequences immunodominant in DQ8 and DQ6 tg mice. Differential disease manifestation in the two different HLA transgenic mice could be explained not only by differential recognition of peptides by these antigen presenting molecules, but also by the difference in the functional profile of T cells generated when primed by promiscuous sequence regions.

Anti-Acetylcholine Receptor CD4+ T Cells in Myasthenia Gravis: Epitope repertoire and T cell receptor gene usage

Although the symptoms of myasthenia gravis (MG) are due to the binding of auto-antibodies to the nicotinic acetylcholine receptor (AChR) at the neuromuscular junction (reviewed in [1]), the anti-AChR CD4+T cells have been intensely investigated, because they may be the prime movers in the pathogenesis of MG. Furthermore, specific immunosuppressive treatments targeted on the autoimmune CD4+ cells have proven successful in experimental autoimmune diseases, including the experimental model of MG, mouse experimental autoimmune MG (EAMG) ([2–4] and references therein).