Exhaustive TCR Deep Sequencing Reveals That CMV Reactivation Fundamentally Resets Immune Reconstitution after Transplant and Results in Significant Deficits in the Effector Memory TCR Repertoire

Although CMV reactivation has long been implicated in post-transplant immune dysfunction, the molecular mechanisms by which even a single reactivation event can increase the risk of transplant-related mortality remains a mystery. To address this, we combined multiparameter flow cytometric analysis and T cell subpopulation sorting with high-throughput sequencing of the T cell repertoire, to produce an exhaustive evaluation of the impact of CMV reactivation on T cell reconstitution after BMT. These studies were performed in an initial cohort of 17 transplant patients, with an ongoing validation cohort consisting of an additional 19 patients (n = 36 total).  We discovered that a single CMV reactivation event was sufficient to drive a >50-fold specific expansion of Granzyme B^high/CD28^low/CD57^high/CD8+ effector-memory T cells (Figure 1A) and, surprisingly, to result in a linked contraction of all naive T cells, including CD31+/CD4+ putative thymic emigrants.  TCRb deep sequencing revealed that CMV reactivation drove a striking contraction of CD8+ Tem diversity compared to the diversity observed in patients that did not reactivate CMV (Figure 1B). Deep sequence analysis of clonally-expanded CD8+ Tem and of purified CMV-specific tetramer+ CD8+ T cells provided molecular confirmation that the CD8 Tem clonal expansion was driven by expansion of CMV-specific clones.

In addition to querying the topography of the expanding CMV-specific T cell clones, deep sequencing allowed us, for the first time, to exhaustively evaluate the rest of the TCR repertoire (in non-CMV-specific T cells). Our results reveal new evidence for significant holes in the underlying TCR repertoire in patients who reactivate CMV, suggesting that CMV reactivation is associated with a linked expansion of CMV-specific T cells and a contraction of other clones in the context of post-transplant immune reconstitution.

These data provide compelling evidence that key aspects of immunologic skewing during hematopoietic reconstitution are driven by CMV reactivation, and provide the first molecular evidence that in addition to driving expansion of virus-specific cells, CMV reactivation has a detrimental impact on the integrity and heterogeneity of the rest of the T cell repertoire.