Revealing the Generation of Human Memory Stem T Cells in Haploidentical T-Replete Hematopoietic Stem Cell Transplantation
Authors: Cieri Nicoletta , Oliveira Giacomo , Greco Raffaella , Forcato Mattia , Taccioli Cristian , Valtolina Veronica , Noviello Maddalena , Vago Luca , Bondanza Attilio , Lunghi Francesca , Marktel Sarah , Bellio Laura , Bordignon Claudio , Bicciato Silvio , Peccatori Jacopo , Ciceri Fabio , Bonini Chiara 
Published: October 2019

Abstract
Introduction: The T cell memory compartment is multi-faceted and encompasses multiple subsets with divergent properties. In addition to central memory (TCM) and effector memory (TEM) cells, the spectrum of immunological memory has been recently extended with the identification of memory stem T cells (TSCM). Gene expression profiling, corroborated by in vitro and in vivo experimental results, posits TSCM upstream TCM and TEM in T-cell ontogeny. However, TSCM role in immune reconstitution (IR) following allogeneic HSCT remains largely unknown. Here, we tracked TSCM dynamics in patients undergoing haploidentical HSCT. By this process, we unconventionally exploited HSCT as model system to provide novel insights into the contribution of TSCM to a mounting immune response.
Patients and Methods: We characterized T cell dynamics during the first month post HSCT in 20 patients receiving myeloablative conditioning, T-replete haploidentical peripheral blood stem cell graft, and GvHD prophylaxis consisting of cyclophosphamide (PT-Cy day 3,4), MMF and sirolimus from day 5.
Results: Upon infusion, naïve T cells (TN) cells gradually disappeared from circulation, and by day 8 post HSCT the peripheral compartment was composed only by memory lymphocytes. At day 8, TSCM cells were the most represented circulating subset, and their frequency was significantly higher than that of the infused graft (LK). To investigate whether such high TSCM frequency was due to expansion of TSCM cells infused or to their direct in vivo generation, T cell subset proliferation was analyzed. At day 3 upon infusion (in the absence of immunosuppression), all memory subsets robustly proliferated, with TSCM cells displaying significantly higher frequencies of Ki-67+ cells compared to all other subsets. In sharp contrast, TN cells were scantly Ki-67+, in line with the longer timeframe required for their priming. PT-Cy abrogated proliferation in all subsets. T cell counts however did not drop between day 5 and 8 post HSCT, and TSCM cells increased in percentages and absolute numbers. We thus explored whether TSCM cells were resistant to PT-Cy, but failed to record ALDH activity, the major mechanism of Cy inactivation, in CD3+ cells. neither within LK nor upon infusion. Accordingly, we detected high percentages of apoptotic cells within all memory subsets at day 5 post HSCT. Nonetheless, at day 8 significantly lower percentages of TSCM cells were Annexin V+ compared to TCM/TEM, while by day 15 post-HSCT all memory subsets displayed very low levels of apoptosis. Thus, we reasoned that direct conversion of TN into TSCM could be the preferential mechanism underlying TSCM expansion. To prove this, we exploited the TCR sequences harbored by each individual T cell as surrogate clonal markers. Purified T cell subsets from LK and harvested 30 days post HSCT in 3 consecutive patients were subjected to TCR sequencing. Sequences harbored by LK-TN were retrieved in all memory subsets at day 30 after HSCT, showing that TN were able to generate the complete spectrum of immunological memory, including TSCM. Quantitative analysis revealed that only clonotypes originally harbored by LK-TN were significantly increased in counts at day 30 post-HSCT. In contrast, sequences unique to LK-TSCM, LK-TCM or LK-TEM were similar or reduced in counts at day 30 post-HSCT compared to LK, indicating that either PT-Cy efficiently dampened their expansion and/or that such memory lymphocytes persisted unchallenged upon in vivo infusion. Overall, in vivo fate mapping through TCR sequencing allowed defining the in vivo differentiation landscapes of human naïve T cells upon HSCT, highlighting TSCM as privileged players in the diversification of immunological memory. We next validated these results at the antigen-specific level. In suitable 5 patients, we recorded the presence of WT1 or PRAME-specific TN cells within donor LK. Upon HSCT, tumor-specific T cells increased in frequency and displayed a memory phenotype, comprising TSCM. Finally, by quantifying patient serum cytokines, we found that the degree of IL-7 availability at day 1 post HSCT correlated with the extent of TSCM expansion at day 8.
Conclusions: These data provide novel insights into TSCM biology and ongoing analyses will define correlations with clinical events. Longer immunological follow-up will advance our understanding of the contribution of early TSCM generation to the overall success of post HSCT IR.

Disclosures
Bordignon: MolMed: Employment. Bonini:MolMed S.p.A.: Consultancy.