IDENTIFICATION OF IMMUNODOMINANT PEPTIDES FOLLOWING DST AND REJECTION IN THE LEW.1W TO LEW.1A MODEL.

Abstract

255 Cardiac allograft tolerance can be induced in the Lew.1W (RT1AuBuDu) to Lew.1A (RT1AaBaDa) combination by pretreatment with two donor blood transfusions. Recently, we examined the TCR Vβ chain diversity at the CDR3 level of graft infiltrating cells (GIC) in both tolerant and rejecting animals. Tolerant animals displayed a predominant expansion of GIC with a conserved Vβ18-Dβ1-Jβ2.7 TCR gene rearrangement, whilst GIC from rejecting animals displayed preferential usage of Vβ2 and Vβ13 (J. Immunol 1996, 157:1250) This current study was designed to determine which regions of the donor MHC antigens are immunodominant in both rejection and tolerance, and to characterise which epitope(s) are recognised by recipient T cells bearing these Vβ rearrangements. To assess these questions, a series of overlapping 16-mer peptides corresponding to the polymorphic regions of the donor class I and class II molecules were synthesised. These included the entire coding region for class I (RT1.Au- 20 peptides), and the β1 domain of the β chain for both class II molecules (RT1.Bβu/RT1.Dβu- 20 and 21 peptides respectively). Splenocytes from two groups of animals; (a) Lew.1A rats rejecting a Lew.1W allograft (Rejecting group, n=5), and (b) Lew.1A rats treated at day −14 and −7 with 1ml of donor blood (DST group, n=6), were isolated and cultured in-vitro with the peptides in a standard 5-day proliferation assay. Splenocytes from rejecting animals proliferated in response to the majority of peptides. The greatest response was observed for peptides corresponding to residues 1-44 of the β1 domain of the Bβ chain. Likewise, peptides of the β1 domain of Dβ were in general stimulatory, however several peptides despite being clearly polymorphic were observed to inhibit proliferation. Several class I-derived peptides, corresponding to the α-helical and β-pleated sheet region of both α1 and α2 domains also induced proliferation. In general splenocytes from DST-treated animals displayed a similar response pattern with respect to stimulatory and non-stimulatory peptides, although the proliferative responses were lower than in rejecting animals. We are currently testing these immunodominant and inhibitory peptides for their ability to be specifically recognised by transfected T cell lines expressing the Vβ13, Vβ2 and Vβ18 TCR receptor when presented by purified recipient strain dendritic cells. This first series of results show that DST and allorejection result in a similar 'indirect' priming. Analysis of the peptide-mediated proliferation of splenocytes from DST-treated and grafted (tolerant) animals may allow us to determine whether the secondary 'direct' signal, provided by graft dendritic cells (Blood 1998, 92: 4539), is instrumental in the inhibition of this priming, and establishment of tolerance.