Molecular analysis of the T-cell receptor V beta 5 and V beta 8 repertoire in pancreatic lesions of autoimmune diabetic NOD mice.
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.
Berschick
P
P
Diabetes-Forschungsinstitut, Universität Düsseldorf, FRG.
Fehsel
K
K
Weltzien
H U
HU
Kolb
H
H
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D003922:Diabetes Mellitus, Type 1 / Q000235:genetics* / Q000473:pathology*; D005260:Female; D015333:Gene Rearrangement, beta-Chain T-Cell Antigen Receptor / Q000235:genetics; D007515:Islets of Langerhans / Q000276:immunology / Q000473:pathology*; D008297:Male; D051379:Mice; D016688:Mice, Inbred NOD; D008969:Molecular Sequence Data; D009928:Organ Specificity; D016693:Receptors, Antigen, T-Cell, alpha-beta / Q000737:chemistry / Q000145:classification / Q000235:genetics*; D013601:T-Lymphocytes / Q000276:immunology / Q000502:physiology* / Q000648:ultrastructure
D016693:Receptors, Antigen, T-Cell, alpha-beta