Poster Presentation Australasian Cytometry Society 43rd Annual Conference and Workshop

Single Cell Alloreactive TCR Repertoire Discovery (#113)

Moumita Paul 1 , Eric Son 1 , Martina Denkova 1 , Claerwen Jones 2 , Mario Leong 1 , Anthony W Purcell 3 , Nicole L La Gruta 3 , Nicole Mifsud 3 , Alexandra Sharland 1
  1. Transplantation Immunobiology Group, The University of Sydney, Sydney, New South Wales, Australia
  2. Monash Biomedicine Discovery Institute, Monash University, Melbourne, Victoria, Australia
  3. Infection and Immunity Program, Department of Biochemistry and Molecular Biology, Biomedicine Discovery Institute, Monash University, Melbourne, Victoria, Australia

In prior work, pMHC tetramers were used to screen self-peptides for recognition by alloreactive CD8+ T cells. We identified over 40 H-2Kb-peptide epitopes that are directly recognised by CD8+ T cells from allogeneic B10.BR (H-2k) or BALB/c (H-2d) mice. Some “super-epitopes” are strongly recognised by T cells from both strains. We aimed to characterise the TCR repertoire responding to these epitopes. B10.BR or BALB/c mice were primed with a Kb-bearing skin graft, then boosted by inoculation with AAV-Kb. Single-cell index sorting was followed by multiplex nested PCR and sequencing of PCR products. The TCR sequences from dextramer-positive and PD-1- bystander populations were compared. No clonal expansion was detected in any bystander population. For T cells recognising three “super-epitopes” (SNY, VGP, RTY), the repertoire of individual mice contained a small number of dominant clonotypes and multiple less frequent TCR species. Within each pMHC specificity, TCR Valpha segment usage was more variable that Vbeta usage. Strong segment pairing preferences marked the different specificities. The majority of TCRs recognising SNY utilised TRBV13-2, frequently paired with TRAV16D/DV11, 14-1, 14-2 or 12D-2. VGP-specific-T cells predominantly utilised TRBV3 with TRAV14-2, 14-3, 14D-1, 6-6 or 10, and RTY-specific TCRs favoured TRBV13-2 with TRAV16D/DV11 or TRBV 19 with TRAV6-7/DV9. A number of CDR3a and CDR3b clonotype consensus motifs were shared between individual mice of the same or different strains.

Access to the BD Rhapsody has opened the door to analysing the TCR repertoire of multiple pMHC specificities in parallel, simultaneous with the expression of hundreds of relevant genes. In our pilot study, we sorted activated (PD-1hi) and bystander (PD-1-) cells from liver leukocytes as outlined above, without using dextramers. We are moving towards incorporating a 48-dextramer panel following pilot experiments using 5 core pMHC tetramers to track changes in alloreactive T cell populations under different transplant conditions.