ACTIVATION AND ROLE OF T REGULATORY CELLS

K. Saeb-parsy¹, T. Conlon¹, M. Negus¹, S. Sivaganesh¹, E.M. Bolton¹, J.A. Bradley², G.J. Pettigrew^3
1Department Of Surgery, University of Cambridge, Cambridge/UNITED KINGDOM, ²Transplant Surgery, Addenbrooke's Hospital NHS Trust, Cambridge/UNITED KINGDOM, ³Department Of Surgery, Addenbrooke's Hospital, University of Cambridge, Cambridge/UNITED KINGDOM

Body: Introduction: Indirect CD4 T cell allorecognition is fundamental to allograft rejection, but how this primes development of effector mechanisms for graft destruction is not clear. We investigate how indirect-pathway CD4 T cells that recognise processed alloantigen presented by recipient APCs provide 'unlinked' help for direct pathway cytotoxic CD8 T cells recognising intact allo-MHCI on donor cells. Methods: Female Mar (B6 RAG2^-/-) mice, whose monoclonal CD4 T cells recognise self-restricted male H-Y peptide, were reconstituted with effector female B6 CD8 T cells and received BALB/c or B6xBALB/c heart transplants. CD8 and CD4 T cell activation was analysed using IFN-γ ELISPOT and division of CFSE-labelled Mar cells. Results: Mar recipients rejected male BALB/c heart grafts acutely (Fig 1A), but only if reconstituted with WT CD8 T cells. Female BALB/c grafts survived >50d, confirming that CD8 T cell-mediated rejection was dependent on indirect help from Mar CD4 T cells. Surprisingly, although the epitopes for Mar CD4 and CD8 T cell recognition are on different APCs (recipient vs donor), CD8 T cell-reconstituted Mar recipients challenged with male B6 APCs (to activate Mar CD4 T cells) mounted minimal cytotoxic CD8 T cell responses and did not reject female BALB/c grafts. Effective help was thus only generated when H-Y and MHCI alloantigens were co-expressed on graft cells. We hypothesised that, analogous to the provision of cognate T cell help to B cells, this requirement for co-expression reflects acquisition of H-Y antigen from graft cells by allospecific CD8 T cells, with subsequent processing and presentation in the context of MHCII for Mar CD4 T cell recognition. In support, Mar recipients reconstituted with MHCII^-/- CD8 T cells rejected male BALB/c grafts more slowly and reconstitution with CD8 T cells from H-2DMa mice (which are unable to process antigen) resulted in indefinite graft survival. In contrast, male B6xBALB/c grafts (which enable 'linked' help via direct allorecognition of both Mar CD4 and CD8 T cell epitopes on donor APCs) were rejected rapidly when reconstituted with WT, MHCII^-/- or H-2DMa CD8 T cells (fig 1A), indicating that antigen processing and MHCII expression by CD8 T cells is necessary only for indirect-pathway CD4 T cell help. Finally, mouse CD8 T cells, which do not normally express MHCII, acquired MHCII upon culture with WT APCs (fig 1B) and also when adoptively transferred to MHCII^+/+ TCR^-/- mice which lack T cells (fig 1C). Conclusion: Indirect pathway CD4 T cells provide help to allospecific CD8 T cells through recognition of alloantigen that is internalised by CD8 T cells via the TCR and presented as processed allopeptide on acquired MHC II.

Disclosure: All authors have declared no conflicts of interest.