*Purpose: Cytomegalovirus (CMV) remains an important threat to immunocompromised transplant recipients, leading to graft injury and mortality. CMV elicits sustained T cell responses with accumulation of memory T cells occupying up to 30% of total CD8 T cells. Despite this knowledge, the heterogeneity and mechanistic underpinnings of CMV-driven memory T cell inflation are incompletely understood. This study aims to comprehensively map CMV-induced CD8 T cells longitudinally at high resolution.

*Methods: We profiled T cells in 31 CMV PCR+ kidney transplant recipients (KTRs), with/without prior CMV exposure, and 31 matched, PCR- controls. Blood was sampled 3- (Baseline, BL) and 12-months (Long-term, LT) post-transplant, with additional samples 1-wk (1W) and 1-mo (1M) post-viremia for PCR+ patients. Patient PBMCs were profiled by 17-color immunophenotyping and single-cell RNA-Seq and single-cell CITE-Seq on sorted CD8 T cells from 4 PCR+ patients, 2 CMV seropositive (S/P) and 2 seronegative (S/N), and their controls.

*Results: Immunophenotyping revealed contraction of naïve and central memory T cells and expansion of terminal effector memory cells post-viremia. ScRNA-Seq of CD8 T cells further classified cells at early (CD28+, CCR7+), transitional (CD28+, GZMK+), or advanced (CD28-, KLRG1+) stages of differentiation (Fig 1A). Pseudotime analyses performed to model CD8 T cell differentiation revealed pseudotemporally distinct transcriptomic modules (Fig 1B). CD8 T cells from S/N KTRs were enriched for early and transitional states, whereas CD8 T cells from S/P KTRs failed to maintain early differentiated states and their terminal differentiation profiles were further amplified by CMV viremia (Fig 1C). Notably, the differentiated KLRG1⁺CD28^– CD8 T cell population comprised a rarer population of dysfunctional cells with exhausted-like markers and a prevalent effector memory-like subset lacking inhibitory receptors but with a remarkable presence of stemness and anti-apoptotic markers.

*Conclusions: In contrast to previous dogma of strictly segregated pathways of generating either memory or exhausted T cells in chronic viral infections, we identify a hybrid T cell memory subpopulation with underlying transcriptional bifurcation pertaining to exhaustion and stem-like progenitors.

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