Key Points• Identification of the biologic requirements for memory stem T cell (T SCM ) generation and expansion from naive precursors ex vivo.• Differentiation, expansion, and genetic manipulation of human T SCM for cancer adoptive cellular therapy. Long-living memory stem T cells (T SCM) IntroductionAdaptive immunity is a potent and flexible system able to combat microbes and cancer cells. 1,2 In the presence of infections or cancer, antigen-specific lymphocytes expand and differentiate into effectors devoted to rapidly clearing the pathogen and memory cells able to persist long-term to patrol the entire organism for recurrence and minimal residual disease. 3,4 However, the mechanism and hierarchical differentiation path underlying the generation of memory precursors and terminal effector cells remain to be fully elucidated. 5 This process has been proposed to involve a self-renewing, stem cell-like memory T-cell subset capable of differentiating into effectors on antigen reencounter. 6,7 This T-cell subset, referred to as memory stem T cells (T SCM ), and initially described in mice, 8,9 begins to be unveiled in humans. 10 T SCM potential biodistribution and long-term persistence represent appealing features to overcome the current limitations of cancer adoptive immune-gene therapy. [11][12][13] At present, clinical-grade protocols able to obtain or preserve T SCM functional and phenotypic characteristics remain to be defined. We previously showed that costimulation of unselected T cells and culture with ␥-chain cytokines allow the preferential generation of gene-modified T cells with a functional central memory (T CM ) phenotype, superior to effector/effector memory (T EM ) counterparts for expansion potential and antitumor activity. 14,15 Compared with T CM and T EM lymphocytes, naive T cells (T N ) are endowed with the highest developmental plasticity and are unique in the ability to generate daughter cells with potential to enter the entire spectrum of immunologic memory, including T SCM . We thus hypothesized that, starting from naive precursors, we could differentiate and genetically engineer human T SCM . We report that IL-7 and IL-15 support the generation of postmitotic costimulated CD8 ϩ T cells with molecular and functional features of T SCM cells. These cells-defined by the expression of CD45RA, CD45R0, CD62L, CCR7, IL-7R␣, and CD95-can be identified among healthy subjects, are selectively enriched in hematopoietic stem cell transplant (HSCT) recipients, and reveal a phenotypic and functional profile distinct from that of T CM and T EM cells for extensive expansion capacity and ability Submitted May 22, 2012; accepted October 25, 2012. Prepublished online as Blood First Edition paper, November 15, 2012; DOI 10.1182 DOI 10. /blood-2012 There is an Inside Blood commentary on this article in this issue.The online version of this article contains a data supplement.The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this arti...

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The use of posttransplant cyclophosphamide (PT-Cy) as graft-versus-host disease (GVHD) prophylaxis has revolutionized haploidentical hematopoietic stem cell transplantation (HSCT), allowing safe infusion of unmanipulated T cell-replete grafts. PT-Cy selectively eliminates proliferating alloreactive T cells, but whether and how it affects natural killer (NK) cells and their alloreactivity is largely unknown. Here we characterized NK cell dynamics in 17 patients who received unmanipulated haploidentical grafts, containing high numbers of mature NK cells, according to PT-Cy-based protocols in 2 independent centers. In both series, we documented robust proliferation of donor-derived NK cells immediately after HSCT. After infusion of Cy, a marked reduction of proliferating NK cells was evident, suggesting selective purging of dividing cells. Supporting this hypothesis, proliferating NK cells did not express aldehyde dehydrogenase and were killed by Cy in vitro. After ablation of mature NK cells, starting from day 15 after HSCT and favored by the high levels of interleukin-15 present in patients' sera, immature NK cells (CD62LNKG2AKIR) became highly prevalent, possibly directly stemming from infused hematopoietic stem cells. Importantly, also putatively alloreactive single KIR NK cells were eliminated by PT-Cy and were thus decreased in numbers and antileukemic potential at day 30 after HSCT. As a consequence, in an extended series of 99 haplo-HSCT with PT-Cy, we found no significant difference in progression-free survival between patients with or without predicted NK alloreactivity (42% vs 52% at 1 year, = NS). Our data suggest that the majority of mature NK cells infused with unmanipulated grafts are lost upon PT-Cy administration, blunting NK cell alloreactivity in this transplantation setting.

Key Points• T SCM lymphocytes are preferentially generated from naive precursors in vivo early after haploidentical HSCT.• T SCM represent relevant novel players in the diversification of immunological memory after haploidentical HSCT.Memory stem T cells (T SCM ) have been proposed as key determinants of immunologic memory. However, their exact contribution to a mounting immune response, as well as the mechanisms and timing of their in vivo generation, are poorly understood. We longitudinally tracked T SCM dynamics in patients undergoing haploidentical hematopoietic stem cell transplantation (HSCT), thereby providing novel hints on the contribution of this subset to posttransplant immune reconstitution in humans. We found that donor-derived T SCM are highly enriched early after HSCT. We showed at the antigenspecific and clonal level that T SCM lymphocytes can differentiate directly from naive precursors infused within the graft and that the extent of T SCM generation might correlate with interleukin 7 serum levels. In vivo fate mapping through T-cell receptor sequencing allowed defining the in vivo differentiation landscapes of human naive T cells, supporting the notion that progenies of single naive cells embrace disparate fates in vivo and highlighting T SCM as relevant novel players in the diversification of immunological memory after allogeneic HSCT. (Blood. 2015;125(18):2865-2874

A definitive understanding of survival and differentiation potential in humans of T cell subpopulations is of paramount importance for the development of effective T cell therapies. In particular, uncovering the dynamics in vivo in humans of the recently described T memory stem cells (TSCM) would be crucial for therapeutic approaches that aim at taking advantage of a stable cellular vehicle with precursor potential. We exploited data derived from two gene therapy clinical trials for an inherited immunodeficiency, using either retrovirally engineered hematopoietic stem cells or mature lymphocytes to trace individual T cell clones directly in vivo in humans. We compared healthy donors and bone marrow-transplanted patients, studied long-term in vivo T cell composition under different clinical conditions, and specifically examined TSCM contribution according to age, conditioning regimen, disease background, cell source, long-term reconstitution, and ex vivo gene correction processing. High-throughput sequencing of retroviral vector integration sites (ISs) allowed tracing the fate of more than 1700 individual T cell clones in gene therapy patients after infusion of gene-corrected hematopoietic stem cells or mature lymphocytes. We shed light on long-term in vivo clonal relationships among different T cell subtypes, and we unveiled that TSCM are able to persist and to preserve their precursor potential in humans for up to 12 years after infusion of gene-corrected lymphocytes. Overall, this work provides high-resolution tracking of T cell fate and activity and validates, in humans, the safe and functional decade-long survival of engineered TSCM, paving the way for their future application in clinical settings.

The major cause of death after allogeneic Hematopoietic Stem Cell Transplantation (HSCT) for acute myeloid leukemia (AML) is disease relapse. We investigated the expression of Inhibitory Receptors (IR; PD-1/CTLA-4/TIM-3/LAG-3/2B4/KLRG1/GITR) on T cells infiltrating the bone marrow (BM) of 32 AML patients relapsing (median 251 days) or maintaining complete remission (CR; median 1 year) after HSCT. A higher proportion of early-differentiated Memory Stem (TSCM) and Central Memory BM-T cells express multiple IR in relapsing patients than in CR patients. Exhausted BM-T cells at relapse display a restricted TCR repertoire, impaired effector functions and leukemia-reactive specificities. In 57 patients, early detection of severely exhausted (PD-1+Eomes+T-bet−) BM-TSCM predicts relapse. Accordingly, leukemia-specific T cells in patients prone to relapse display exhaustion markers, absent in patients maintaining long-term CR. These results highlight a wide, though reversible, immunological dysfunction in the BM of AML patients relapsing after HSCT and suggest new therapeutic opportunities for the disease.

Haploidentical hematopoietic stem cell transplantation (HSCT) performed using bone marrow (BM) grafts and post-transplantation cyclophosphamide (PTCy) has gained much interest for the excellent toxicity profile after both reduced-intensity and myeloablative conditioning. We investigated, in a cohort of 40 high-risk hematological patients, the feasibility of peripheral blood stem cells grafts after a treosulfan-melphalan myeloablative conditioning, followed by a PTCy and sirolimus-based graft-versus-host disease (GVHD) prophylaxis (Sir-PTCy). Donor engraftment occurred in all patients, with full donor chimerism achieved by day 30. Post-HSCT recovery of lymphocyte subsets was broad and fast, with a median time to CD4 > 200/μL of 41 days. Cumulative incidences of grade II to IV and III-IV acute GVHD were 15% and 7.5%, respectively, and were associated with a significant early increase in circulating regulatory T cells at day 15 after HSCT, with values < 5% being predictive of subsequent GVHD occurrence. The 1-year cumulative incidence of chronic GVHD was 20%. Nonrelapse mortality (NRM) at 100 days and 1 year were 12% and 17%, respectively. With a median follow-up for living patients of 15 months, the estimated 1-year overall and disease-free survival (DFS) was 56% and 48%, respectively. Outcomes were more favorable in patients who underwent transplantation in complete remission (1-year DFS 71%) versus patients who underwent transplantation with active disease (DFS, 34%; P = .01). Overall, myeloablative haploidentical HSCT with peripheral blood stem cells (PBSC) and Sir-PTCy is a feasible treatment option: the low rates of GVHD and NRM as well as the favorable immune reconstitution profile pave the way for a prospective comparative trial comparing BM and PBSC in this specific transplantation setting.

While opening new frontiers for the cure of malignant and non-malignant diseases, the increasing use of cell therapy poses also several new challenges related to the safety of a living drug. The most effective and consolidated cell therapy approach is allogeneic hematopoietic stem cell transplantation (HSCT), the only cure for several patients with high-risk hematological malignancies. The potential of allogeneic HSCT is strictly dependent on the donor immune system, particularly on alloreactive T lymphocytes, that promote the beneficial graft-versus-tumor effect (GvT), but may also trigger the detrimental graft-versus-host-disease (GvHD). Gene transfer technologies allow to manipulate donor T-cells to enforce GvT and foster immune reconstitution, while avoiding or controlling GvHD. The suicide gene approach is based on the transfer of a suicide gene into donor lymphocytes, for a safe infusion of a wide T-cell repertoire, that might be selectively controlled in vivo in case of GvHD. The herpes simplex virus thymidine kinase (HSV-TK) is the suicide gene most extensively tested in humans. Expression of HSV-TK in donor lymphocytes confers lethal sensitivity to the anti-herpes drug, ganciclovir. Progressive improvements in suicide genes, vector technology and transduction protocols have allowed to overcome the toxicity of GvHD while preserving the antitumor efficacy of allogeneic HSCT. Several phase I-II clinical trials in the last 20 years document the safety and the efficacy of HSV-TK approach, able to maintain its clear value over the last decades, in the rapidly progressing horizon of cancer cellular therapy.