C010 - Tracking Viral-Specific CD4⁺ T Cells That Cross-React to Alloantigen

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Author Block: A. Burg¹, A. Ferguson¹, E. Elfers¹, R. Caine¹, T. Shi¹, E. Khorki¹, J. Maltzman², B. Baker³, D. Hildeman¹, ¹Immunobiology, Cincinnati Children's Hospital Medical Center, Cincinnati, OH, ²Stanford University, Palo Alto, CA, ³University of Notre Dame, Notre Dame, IN
*Purpose: Heterologous immunity, exemplified by cross-reactive lymphocytes, can pose a threat to transplanted organs. We previously developed a mouse model to identify and characterize CD8⁺ T cells with inherent cross-reactivity to allogeneic, but not syngeneic, heart transplants. Here, we used this model to assess allo-specific potential of viral-specific CD4⁺ T cells and explored a potential mechanism underlying such inherent cross-reactivity.
*Methods: Nur77-GFP reporter mice, which transiently express GFP exclusively upon TCR engagement, were infected with lymphocytic choriomeningitis virus (LCMV) to generate a pool of memory CD4⁺ T cells and ~2 months later, were transplanted with hearts from allogeneic Balb/c (H2^d) or syngeneic BL/6 (H2^b) mice. Transplanted hearts were analyzed by flow cytometry using MHC tetramers specific for IA^b-gp(66-77) and using GFP expression as an indicator of alloreactivity. To determine the role of Bim-mediated negative selection, Bim^-/- mice were crossed to Nur77-GFP reporter mice and were subjected to LCMV infection followed by allogeneic or syngeneic heart transplant and responses measured as above.
*Results: As early as 2 days post-transplant, heightened CD4⁺ T cell infiltration is seen in allogeneic, relative to syngeneic allografts. While examining their level of cross-reactivity using the Nur77-GFP reporter we found that, unique to CD4⁺ T cell tetramer staining, binding of the I-A^b-gp(66-77) tetramer itself drove TCR signaling and induced substantial GFP expression. However, culturing with ponatinib, a tyrosine kinase inhibitor, during the staining procedure mitigated this artefact and allowed true quantitation of in vivo cross-reactivity. We found ~ 20% of LCMV-specific CD4⁺ T cells cross-reacted to alloantigens. Strikingly, in the absence of Bim, a significantly greater proportion of viral-specific CD4+ T cells cross-reacted to allogeneic heart transplants.
*Conclusions: We have established a model to probe the quality and quantity of heterologous immunity in both CD4⁺ and CD8⁺ T cells in real time during an alloresponse. Further, our data suggest that one purpose of negative selection is to limit the production of T cells whose TCRs possess substantial cross-reactivity. Defining the depth and breadth of heterologous immunity, could help stratify patient risk based on potential allograft mismatches that might favor such cross-reactivity.