*Purpose: Memory T cells primed with microbial antigen can cross-react with alloantigen and mediate graft rejection, a process termed allogeneic heterologous immunity. Human and murine studies have shown that a significant portion of pathogen-specific memory T cells can be alloreactive, and it is assumed that these cells drive heterologous graft rejection. CD45 is a transmembrane phosphatase that plays an important role in tuning T cell receptor (TCR) signaling and is regarded as a T cell differentiation marker. Naive peripheral T cells have high CD45RB isoform expression (denoted CD45RB^hi) while effector and memory CD8⁺ T cells are described as having low expression of the CD45RB isoform (CD45RB^lo). In this study, we sought to interrogate the role of CD45RB on memory CD8⁺ T cells during heterologous immunity.

*Methods: We assessed the phenotype and function of CD44^hi and tetramer⁺ CD8⁺ T cells following acute LCMV Armstrong infection at memory timepoints and following H-2^d Balb/c skin graft rechallenge of LCMV infected mice.

*Results: We found that following acutely cleared viral infection with LCMV, the viral-specific memory CD8⁺T cell population surprisingly included approximately equal populations of CD45RB^hi and CD45RB^lo cells. A 2D micropipette affinity assay revealed that CD45RB^hi memory cells had a 29-fold lower affinity for antigen relative to CD45RB^lo cells. CD45RB^lo and CD45RB^hi memory cells expressed similar levels of granzyme B and IFN-γ, but RNASeq analysis revealed that these populations differentially expressed over 150 genes. Phenotypically, the CD45RB^hi population contained a higher frequency of the CD62L^hiCD27^hiCD127^hiKLRG1^lo persistent memory phenotype compared to CD45RB^lo cells. We found that from early to late memory time-points, portions of each of the CD45RB^hi and CD45RB^lo populations underwent conversion to CD62L^hi, demonstrating that CD45RB status is distinct from the T_CM phenotype. Adoptive transfer of purified CD45RB^hi and CD45RB^lo populations into naïve mice revealed that CD45RB^lo memory cells underwent significant cell loss relative to CD45RB^hi memory cells. Over time, the majority of CD45RB^lo cells homeostatically converted to the CD45RB^hi persistent memory phenotype. Finally, following fully mismatched allogeneic skin graft in LCMV immune mice, approximately 80% of donor-specific memory CD8⁺T cells remained CD45RB^hi.

*Conclusions: Together, these data demonstrate a that CD45RB status correlates with memory persistence, which has implications for the therapeutic strategies aimed at eliminating heterologous immune responses.

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