Molecular analysis of the T-cell receptor V beta 5 and V beta 8 repertoire in pancreatic lesions of autoimmune diabetic NOD mice.

Abstract

The repertoire of V beta 5 and V beta 8 T-cell receptors in pancreatic lesions of autoimmune diabetic NOD mice was analysed by sequencing the CDR3 and adjacent regions. T-cell receptor mRNA isolated from four different cell populations (i.e. spleen, lymph node, infiltrated islets from male and female NOD mice) was amplified by PCR and cloned; out of these, 339 clones were sequenced. Of 170 beta chains sequenced from intra-islet T cells, nearly 90% were unique and six other sequences were found 2 to 4 times. These data argue against any oligoclonality of the islet infiltrate. Despite the lack of clonal restriction, we observed a bias in TcR usage which indicates the existence of some selective pressure with regard to TcR structure. Of the V beta 5 positive cells, 30% to 40% showed a rearrangement of V beta 5 to J beta 2.6 and a complete lack of V beta 5-J beta 1.6 combination. The selective J beta usage was not restricted to islets but was found in all tissues analysed. V beta 8 positive cells did not show such an overrepresentation of V beta-J beta combinations with the exception of clones of infiltrated islets of partially diabetes-resistant male NOD mice. There the rearrangement of V beta 8-J beta 1.1 was markedly over-expressed. Analysis of the CDR3 region did not show selection of specific TcR with regard to region length. However, we found a restricted use of amino acids in the second position of the CDR3 region. V beta 8 chains had conserved an aspartic acid from the germline configuration in about half of the cases in all tissues analysed. V beta 5 chains also showed diversity of position 2 but not islet specificity of rearrangements. Mutated chains had a clear bias towards proline indicating selective pressure in favour of this amino acid. In conclusion, sequence analysis of V beta 5 and V beta 8 TcRs excludes oligoclonality of T-cell receptors in pancreatic lesions. The bias found for J beta usage and CDR3 structure was seen also in extra-pancreatic tissues and thus probably is due to selective pressure during T-cell maturation in thymus or periphery.