Aims: We discovered >40 K^b-peptide epitopes directly recognised by alloreactive CD8 T cells from B10.BR mice (H-2^k). Here, we integrated two approaches to profile the alloreactive T cell repertoire at an early stage in tolerance induction (Fig.1a).

Methods: B10.BR mice were primed with a K^b-expressing skin graft followed by inoculation with AAV-K^b. Nested PCR and Sanger sequencing of paired αβ TCR from single dextramer-positive cells was performed in parallel with BD Rhapsody library preparation/Illumina sequencing for paired TCR and targeted transcriptome analysis.

Results: Repertoires for 3 dominant epitopes (K^b-SNY, K^b-RTY and K^b-VGP) were determined (Fig.1b). TCR αβ diversity was significantly reduced among epitope-specific T cells (scores K^b-SNY 47.3, K^b-RTY 69.5, K^b-VGP 279.5) compared with PD-1^neg bystander cells (score 78730). Alloreactive repertoires were strongly skewed towards usage of particular V-J gene segments and comprised families of related TCRs including both private TCR clones and public meta-clonotypes (Fig.1b-d). One cross-reactive clone recognised both K^b-SNY and K^b-RTY (Fig.1c-d). A number of clones shared the same b chain paired with different alpha chains (Fig. 1d) while clones with dual alpha chains were also detected. The major meta-clonotypes were represented in multiple mice.

Rhapsody analysis of K^b-reactive (PD-1^hi), bystander (PD-1^neg) or K^b self-tolerant T cells (from the K^b-transgenic H-2^k strain 178.3) permitted profiling of a broader range of donor-reactive T cells. Comparison of V gene segment usage across the range of receptors expressed by PD-1^neg cells from Sanger-sequenced or Rhapsody samples (502 or 1151 cells respectively) did not reveal any biases in the repertoire obtained based on the amplification method (Fig. 2a). TRAV14 and TRAV16 were over-represented among the PD-1^hi cells compared to PD-1^neg or Kb-tolerant cells while cells expressing TRAV9 and TRAV12 were less frequent (Fig 2b). The top 10 clones from each mouse accounted for 32% of all K^b-reactive (PD-1^hi) cells. No clonal expansions were observed in the PD-1^neg or K^b self-tolerant populations, with negligible overlap between TCR sequences from PD-1^hi cells and these populations. K^b-reactive cells segregated into clusters corresponding to liver-resident or peripheral memory, precursor exhausted and proliferating cells, while PD-1^neg cells displayed a central memory phenotype (Fig. 2c-d). Individual clonotypes showed distinctive gene expression patterns following activation.

Conclusions: Directly-alloreactive CD8 T cell repertoires comprise families of closely-related clonotypes. Gene expression early during tolerance induction may determine the subsequent fate (deletion/exhaustion) of individual clones.