Tracking T Cell Dynamics In The First Month After Haplo-HSCT With Post-Transplant Cyclophosphamide Reveals a Predominant Contribution Of Memory Stem T Cells To The Early Phase Of Immune Reconstitution

Haploidentical hematopoietic stem cell transplantation (HSCT) with T–replete grafts and post-transplant cyclophosphamide (PT-Cy) has gained much interest in the transplantation community for the low rates of GvHD, non-relapse mortality and opportunistic infections. This platform, devoid of anti-thymocyte globulins, allows a thorough analysis of circulating cells in the early phase post-HSCT. Indeed, several biological events that play a critical role for transplant outcome occur within the first month after HSCT; while engraftment and hematological reconstitution are carefully monitored, the shape of T cell dynamics within this timeframe remains largely unknown.

We characterized immune reconstitution (IR) during the first month post HSCT in 18 high-risk leukemia patients receiving myeloablative conditioning, T-replete haploidentical peripheral blood stem cell graft (PBSCs), and GvHD prophylaxis consisting of PT-Cy (day 3, 4), followed by mycophenolate mofetil and sirolimus from day 5. Infused PBSCs and blood samples harvested at day 1, 3, 5, 8, 15 and 30 post HSCT were analyzed by multiparametric flow cytometry.

T cells were infused in the absence of immunosuppressive agents, and by day 3, prior to PT-Cy, a large fraction of memory lymphocytes, possibly enriched for allo-specificities, proliferated (assessed by Ki-67 staining). Conversely, naïve T cells (T_N) were scantly Ki-67+ (P< 0.001). A high CD4:CD8 ratio was observed at this time-point (10). PT-Cy efficiently abated T cell proliferation and appeared to affect CD4 more than CD8 T cells. Nevertheless, T cell numbers progressively increased (mean CD3 counts, day 5: 19 cells/µL; day 8: 27 cells/µL; day 15: 97 cells/µL), suggesting that residual proliferation in extravascular sites was likely to fuel the surge in circulating T cells. Consistently, we observed an expansion of antigen-experienced T cells including central memory (T_CM), effector memory (T_EM), effectors (T_EFF) and the recently described stem memory T cells (T_SCM). T_SCM are a subset of memory cells hierarchically superior to T_CM and T_EM, for self-renewal, long-term persistence and functional capacity. Similarly to T_N, T_SCM coexpress CD45RA and CD62L but differently from T_N, T_SCM express CD95, a marker of memory cells. As early as day 8 post HSCT, the T cell compartment was predominantly composed by T_SCM cells (P < 0.01 compared to all other subsets). Such enrichment in T_SCM was not due to a selective resistance to PT-Cy, as suggested by the lack of activity of the ALDH enzyme, which converts Cy to a non-toxic metabolite, in T_SCM infused with the graft. Rather, we hypothesized that T_SCM expansion came directly from the differentiation of T_N infused within the graft, which escaped the purging effect of PT-Cy thanks to a delayed activation kinetics compared to alloreactive memory T cells. We demonstrated the in vivo differentiation of WT1 and PRAME specific TN cells, present in the graft, into memory lymphocytes, comprising T_SCM cells, in 4/7 patients suitable for dextramer tracking. Such tumor specific T cells were detected in the peripheral blood and bone marrow of treated patients, suggesting that PT-Cy did not hamper GvL players. Of note in the remaining patients for whom tumor-specific T_N were not detectable in the graft, no tumor response could be documented in vivo. From day 15 post HSCT, T_SCM were outnumbered by other memory subsets, suggesting their differentiation into more committed T_CM T_EM and T_EFF.

The quality of IR correlated with clinical events. The percentage of circulating Ki-67+ CD8 T_EMcells at day 8 post HSCT accurately predicted the occurrence of periengraftment syndrome, observed in 4 patients with a median time to onset of 15 days. Acute GvHD (Grade I/II in 6 patients, grade III/IV in 4) was accompanied by a rise in circulating Ki-67+ CD8 cells, and response to therapy resulted in a drop in Ki-67 expression. No immunological parameter correlated with chronic GvHD, observed in 2 patients. In all patients with a CMV-seropositive donor, CMV-specific T cells were tracked in the graft, at early time-points and up to 180 days post HSCT, indicating that virus-specific T cells escaped PT-Cy.

These results suggest that PT-Cy acts mainly on alloreactive memory T cells infused within the graft, while sparing infused virus-specific, non cross-reactive, memory cells and T_N, which can differentiate predominantly in T_SCM, but also in T_CM T_EM and T_EFF, thus promoting a rapid and broad IR.