Single-Cell Analysis of Adaptive NK and CMV-Specific T Cell Dynamics after Allogeneic HSCT

Cytomegalovirus (CMV) reactivation reshapes immune reconstitution and remains a significant cause of morbidity and mortality following allogeneic hematopoietic stem cell transplantation (HSCT). To understand the impact of asymptomatic CMV viremia on immune reconstitution, we profiled longitudinal blood samples (n=32) from donor-recipient pairs pre- and post-HSCT with single-cell RNA and TCR sequencing for three months after transplantation. To analyze the development of antigen-specific T-cell responses in detail, we sorted and TCR-sequenced T cells specific to the immunodominant CMV epitope pp65. After HSCT, an early increase in CD14+ monocytes was observed, followed by expansion of T and NK cells. Persisting patient cell-chimerism was observed in the CD4+ T cell fraction. CMV-specific T cells and CMV-associated adaptive NK cells matured slower in grafts from seronegative donors compared to seropositive donors. The early maturation of adaptive NK cells was markedly more rapid in patients with CMV seropositive donors with enhanced recall-function; however, adaptive NK cell cytotoxicity was higher in patients with CMV seronegative donors. Large oligoclonal CD8+ T cell expansions against the immunodominant CMV pp65NLV epitope comprised up to 63.6% of the CD8+ T cell repertoire, showing terminal maturation with upregulation of canonical NK cell receptors. Our study presents the first comprehensive single-cell transcriptomic characterization of adaptive immune recovery following allogeneic HSCT, with a particular focus on CMV reactivation. Our findings highlight the impact of donor CMV serostatus on immune maturation in recipients with CMV reactivation, which may help predict the timing of immune reconstitution and inform strategies for management of post-transplant complications.