Nicoletta Cieri

IL-7 and IL-15 instruct the generation of human memory stem T cells from naive precursors

Long-living memory stem T cells (T(SCM)) with the ability to self-renew and the plasticity to differentiate into potent effectors could be valuable weapons in adoptive T-cell therapy against cancer. Nonetheless, procedures to specifically target this T-cell population remain elusive. Here, we show that it is possible to differentiate in vitro, expand, and gene modify in clinically compliant conditions CD8(+) T(SCM) lymphocytes starting from naive precursors. Requirements for the generation of this T-cell subset, described as CD62L(+)CCR7(+)CD45RA(+)CD45R0(+)IL-7Rα(+)CD95(+), are CD3/CD28 engagement and culture with IL-7 and IL-15. Accordingly, T(SCM) accumulates early after hematopoietic stem cell transplantation. The gene expression signature and functional phenotype define this population as a distinct memory T-lymphocyte subset, intermediate between naive and central memory cells. When transplanted in immunodeficient mice, gene-modified naive-derived T(SCM) prove superior to other memory lymphocytes for the ability to expand and differentiate into effectors able to mediate a potent xenogeneic GVHD. Furthermore, gene-modified T(SCM) are the only T-cell subset able to expand and mediate GVHD on serial transplantation, suggesting self-renewal capacity in a clinically relevant setting. These findings provide novel insights into the origin and requirements for T(SCM) generation and pave the way for their clinical rapid exploitation in adoptive cell therapy.

In vivo tracking of T cells in humans unveils decade-long survival and activity of genetically modified T memory stem cells

Genetically engineered T memory stem cells preserve differentiation activity for decades after patient infusion. Sealing T cell fate Clinical trials are a relatively untapped source of experimental data that can be leveraged to explore both basic and pathological biology in humans. Now, Biasco et al. take advantage of two different gene therapy trials for inherited immunodeficiency to track in the long term T cell fate in humans. They find that the recently described T memory stem cells (TSCM) are able to persist and preserve their precursor potential in human recipients for up to 12 years after genetic correction and infusion into patients. The safety and long-term survival of these cells not only strengthen our knowledge of human immunology but also support the use of TSCM cells for adoptive immunotherapy. 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.

Post-transplantation Cyclophosphamide and Sirolimus after Haploidentical Hematopoietic Stem Cell Transplantation Using a Treosulfan-based Myeloablative Conditioning and Peripheral Blood Stem Cells

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.

Generation of human memory stem T cells after haploidentical T-replete hematopoietic stem cell transplantation.

Memory stem T cells (TSCM) 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 TSCM 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 TSCM are highly enriched early after HSCT. We showed at the antigen-specific and clonal level that TSCM lymphocytes can differentiate directly from naive precursors infused within the graft and that the extent of TSCM 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 TSCM as relevant novel players in the diversification of immunological memory after allogeneic HSCT.

Improving the safety of cell therapy with the TK-suicide gene.

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.

Adoptive immunotherapy with genetically modified lymphocytes in allogeneic stem cell transplantation

Hematopoietic stem cell transplantation from a healthy donor (allo‐HSCT) represents the most potent form of cellular adoptive immunotherapy to treat malignancies. In allo‐HSCT, donor T cells are double edge‐swords: highly potent against residual tumor cells, but potentially highly toxic, and responsible for graft versus host disease (GVHD), a major clinical complication of transplantation. Gene transfer technologies coupled with current knowledge on cancer immunology have generated a wide range of approaches aimed at fostering the immunological response to cancer cells, while avoiding or controlling GVHD. In this review, we discuss cell and gene therapy approaches currently tested in preclinical models and in clinical trials.

Tracking genetically engineered lymphocytes long-term reveals the dynamics of T cell immunological memory

Antigen exposure and differentiation phenotype influence long-term persistence of memory T cells after hematopoietic stem cell transplant. Committing T cells to memory Adoptive cell transfer is an increasingly successful therapy for a variety of diseases; however, little is known about what regulates the survival of these cells in humans. Now, Oliveira et al. leverage patients who have received genetically modified hematopoietic stem cells to track T cells over time. They found labeled effector memory, central memory, and stem memory T cells 2 to 14 years after infusion in all patients. Antigen recognition was critical in driving persistence and expansion. The clones that survived long-term appeared to initiate preferentially from central and stem cell memory T cell populations. These data suggest that the original phenotype of infused cells may influence long-term persistence of adoptively transferred cells. Long-lasting immune protection from pathogens and cancer requires the generation of memory T cells able to survive long-term. To unravel the immunological requirements for long-term persistence of human memory T cells, we characterized and traced, over several years, T lymphocytes genetically modified to express the thymidine kinase (TK) suicide gene that were infused in 10 patients after haploidentical hematopoietic stem cell transplantation (HSCT). At 2 to 14 years after infusion and in the presence of a broad and resting immune system, we could still detect effectors/effector memory (TEM/EFF), central memory (TCM), and stem memory (TSCM) TK+ cells, circulating at low but stable levels in all patients. Longitudinal analysis of cytomegalovirus (CMV)– and Flu-specific TK+ cells indicated that antigen recognition was dominant in driving in vivo expansion and persistence at detectable levels. The amount of infused TSCM cells positively correlated with early expansion and with the absolute counts of long-term persisting gene-marked cells. By combining T cell sorting with sequencing of integration (IS), TCRα and TCRβ clonal markers, we showed that T cells retrieved long-term were enriched in clones originally shared in different memory T cell subsets, whereas dominant long-term clonotypes appeared to preferentially originate from infused TSCM and TCM clones. Together, these results indicate that long-term persistence of gene-modified memory T cells after haploidentical HSCT is influenced by antigen exposure and by the original phenotype of infused cells. Cancer adoptive immunotherapy might thus benefit from cellular products enriched in lymphocytes with an early-differentiated phenotype.

CD3+ graft cell count influence on chronic GVHD in haploidentical allogeneic transplantation using post-transplant cyclophosphamide

The effects of graft or donor characteristics in haploidentical hematopoietic cell transplantation (HCT) using post-transplant cyclophosphamide (PT-Cy) are largely unknown. In this multicenter retrospective study we analyzed the correlations between graft cell composition (CD34+, CD3+) and donor features on transplant outcomes in 234 patients who underwent HCT between 2010 and 2016. On multivariate analysis, the use of peripheral blood stem cells (PBSC) was associated with an increased incidence of grade 2–4 acute GVHD [HR 1.94, 95% confidence Interval (CI) = 1.01–3.98, p = 0.05]. An elevated CD3+ graft content was associated with an increased incidence of all-grade chronic GVHD [HR 1.36 (95% CI = 1.06–1.74), p = 0.01]. This effect was confirmed only for the PBSC graft group. A higher CD34+ graft content had a protective role on non-relapse mortality [HR 0.78 (95% CI = 0.62–0.96), p = 0.02] but this was confirmed only for the bone marrow (BM)-derived graft cohort. Donor characteristics did not influence any outcomes. GVHD prophylaxis should be modulated accordingly to CD3+ graft content, especially when a PBSC graft is used. These results need further validation in prospective trials.

Immune monitoring in allogeneic hematopoietic stem cell transplant recipients: a survey from the EBMT-CTIWP

Allogeneic hematopoietic stem cell transplantation (alloHSCT) is the most effective curative treatment for several high-risk hematological malignancies (high-risk leukemia, myelodysplastic syndromes, advanced myeloproliferative disorders, high-risk lymphomas, and multiple myeloma). Sixty years after the first clinical application, it is currently applied in more than 15,000 patients per year in Europe [1]. The post-transplant immune restoration is one of the main factors influencing the clinical outcome of allo-HSCT. Even though fast hematopoietic engraftment and chimerism can usually be documented, severe immune defects commonly occur early after transplant and might persist for several months. The kinetics and quality of immune reconstitution depend on several pre and post-transplant variables. Well established pre-transplant parameters include the age of the recipient, diagnosis, degree of HLA antigen mismatches and non-HLA antigens, origin and manipulation of the graft, conditioning regimen, infectious serostatus of the donor and recipient. Several clinical parameters, including infectious complications, disease relapse, occurrence of graft-versus-host disease (GvHD), impaired thymic function, contribute to post-transplant immuno-incompetence [2–7]. Considerable efforts have been made in recent years to boost immune reconstitution with innovative transplant procedures [8, 9] and experimental cellular therapies [10, 11]. In addition post-transplant immune interventions become standard of care. In parallel, a large number of clinical studies have been devoted to identify biomarkers predictive of a fast and robust immune reconstitution and of a favorable outcome [7, 12–15].

Second malignancies and cardiovascular diseases in long-term Hodgkin lymphoma survivors: The Istituto Nazionale Tumori of Milan (INT) experience

SECOND MALIGNANCIES AND CARDIOVASCULAR DISEASES IN LONG‐ TERM HODGKIN LYMPHOMA SURVIVORS: THE ISTITUTO NAZIONALE TUMORI OF MILAN (INT) EXPERIENCE S. Viviani* | M. Soldarini | N. Cieri | I. Arendar | C. Materazzo | A. Busia | P. Matteucci | A. Di Russo | P. Corradini Hematology and Pediatric Onco‐Hematology, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy; 2Division of Cardiology and Pneumology, Fondazione IRCCS Istituto Tumori dei Tumori, Milan, Italy; Radiotherapy, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy