Repertoire of Rat MBP-Reactive T Cells: DNA Sequencing Analysis Further Demonstrates the Clonal Heterogeneity of Rat T Cells Reactive against Encephalitogenic Epitopes

Reports of the expression of very similar TCR structures by disparate rodent encephalitogenic T cells reactive with regions of MBP have aroused much interest for both theoretical and practical reasons. To ascertain the extent to which structural requirements of epitope recognition constrain TCR expression by MBP-reactive T cells, we set out to estimate the size of the overall repertoire of TCR β-chain Vβ-Dβ-Jβ (VDJ) assemblies in T cells of Lewis rats specific for MBP(68-88) as well as those specific for MBP(87-99). We previously reported that such T cells can express a diversity of Vβ genes as revealed by PCR analysis. In this study, we have used direct sequencing of PCR products amplified from encephalitogenic T-cell clones and pauciclonal T-cell lines to demonstrate that VDJ structures of the rat T cells specific for either residues 68 to 88 or 87 to 99 of MBP are highly heterogeneous. Our results showed that (1) no pattern is evident in the utilization of germline J gene segments by individual T-cell clones; from a total of over 100 successfully sequenced clones displaying in-frame rearrangements, all the Jβ segments have been demonstrated. (2) Even among the T-cell clones which share the Vβ expression, Jβ is variable. (3) Due to joining variations between the Vβ, Dβ, and Jβ gene segments, no two of the T-cell clones examined share entire VDJ structures. Our study is the first report of nucleotide and amino acid sequences of TCR β-chains from rat encephalitogenic T cells expressing Vβ genes other than Vβ8.2. It demonstrates that the TCR repertoire of the MBP-reactive as well as encephalitogenic T cells is heterogeneous, even though a certain T-cell subset frequently dominated by the mechanism needs to be clarified.