Conserved T-cell receptor CDR3 sequences involved in allorecognition of mutant H-2 antigens

GY Zhang, H Wu, JF Knight

Centre for Kidney Research, Royal Alexandra Hospital for Children, Westmead NSW, Australia

Mixed lymphocyte cultures (MLC) of spleen cells from mice with a well-characterised MHC gene mutation and otherwise genetically identical controls lacking the mutation were used to determine whether recognition of alloantigen by T-cell receptor (TCR) is restricted to a limited number of receptor types. C57BL/6(B6) mice were primed by in vivo stimulation with irradiated spleen cells obtained from B6 H-2 mutant strain, B6.C-H-2bm1 (bm1). This mutant and its parent strain B6 differ by only 3 amino acids in class I MHC. Spleen cells from the parent strains were cultured and after six rounds of weekly in vitro stimulation with irradiated spleen cells from the mutant strain bm1, the responder cells were analysed in two ways. Firstly, it was demonstrated by flow cytometry that more than 97% of the cells were CD8+. Secondly, rtPCR of responder cells demonstrated a restricted TCR V repertoire (only 3/18 V families - V 5, V 6 and V 8). In control stimulation with a full H-2 mismatch, 13 out of 18 V families were expressed. To further confirm the clonal expansion of the responder cells, the V 5, V 6 and V 8 PCR products were subcloned into pGEM vectors and DNA sequences were determined. For V 5, 10/10 clones had identical VDJ regions (CDR3). In the case of V 6, from the 9 clones studied, 7/9 shared one identical VDJ sequence and the remaining 2/9 shared another. In the case of V 8 three different VDJ combinations were identified from the 8 clones that were examined (5/8, 2/8, 1/8). Sequencing of 22 clones from V 5, 6 and 8 responding to a full H-2 mismatched control showed totally different CDR3 compared with the Bm1 responding cells. These data demonstrate that alloreactive CD8+ T-cells use a restricted set of TCR V genes with conserved VDJ combination in response to mutants of H-2 class I antigens.

Presented at the Transplantation Society of Australia and New Zealand Canberra May 1998
Presented at the American Society of Nephrology Philadelphia October 1998

Correspondence
Dr Geoffrey Zhang
GeoffZ@chw.edu.au