Barbarella Lucarelli

HLA-haploidentical stem cell transplantation after removal of αβ+ T and B cells in children with nonmalignant disorders

Twenty-three children with nonmalignant disorders received HLA-haploidentical hematopoietic stem cell transplantation (haplo-HSCT) after ex vivo elimination of αβ(+) T cells and CD19(+) B cells. The median number of CD34(+), αβ(+)CD3(+), and B cells infused was 16.8 × 10(6), 40 × 10(3), and 40 × 10(3) cells/kg, respectively. No patient received any posttransplantation pharmacologic prophylaxis for graft-versus-host disease (GVHD). All but 4 patients engrafted, these latter being rescued by a second allograft. Three patients experienced skin-only grade 1 to 2 acute GVHD. No patient developed visceral acute or chronic GVHD. Cumulative incidence of transplantation-related mortality was 9.3%. With a median follow-up of 18 months, 21 of 23 children are alive and disease-free, the 2-year probability of disease-free survival being 91.1%. Recovery of γδ(+) T cells was prompt, but αβ(+) T cells progressively ensued over time. Our data suggest that this novel graft manipulation strategy is safe and effective for haplo-HSCT. This trial was registered at www.clinicaltrials.gov as #NCT01810120.

γδ T-cell reconstitution after HLA-haploidentical hematopoietic transplantation depleted of TCR-αβ+/CD19+ lymphocytes

We prospectively assessed functional and phenotypic characteristics of γδ T lymphocytes up to 7 months after HLA-haploidentical hematopoietic stem cell transplantation (haplo-HSCT) depleted of αβ(+) T cells and CD19(+) B cells in 27 children with either malignant or nonmalignant disorders. We demonstrate that (1) γδ T cells are the predominant T-cell population in patients during the first weeks after transplantation, being mainly, albeit not only, derived from cells infused with the graft and expanding in vivo; (2) central-memory cells predominated very early posttransplantation for both Vδ1 and Vδ2 subsets; (3) Vδ1 cells are specifically expanded in patients experiencing cytomegalovirus reactivation and are more cytotoxic compared with those of children who did not experience reactivation; (4) these subsets display a cytotoxic phenotype and degranulate when challenged with primary acute myeloid and lymphoid leukemia blasts; and (5) Vδ2 cells are expanded in vitro after exposure to zoledronic acid (ZOL) and efficiently lyse primary lymphoid and myeloid blasts. This is the first detailed characterization of γδ T cells emerging in peripheral blood of children after CD19(+) B-cell and αβ(+) T-cell-depleted haplo-HSCT. Our results can be instrumental to the development of clinical trials using ZOL for improving γδ T-cell killing capacity against leukemia cells. This trial was registered at www.clinicaltrials.gov as #NCT01810120.

Immunological Outcome in Haploidentical-HSC Transplanted Patients Treated with IL-10-Anergized Donor T Cells.

T-cell therapy after hematopoietic stem cell transplantation (HSCT) has been used alone or in combination with immunosuppression to cure hematologic malignancies and to prevent disease recurrence. Here, we describe the outcome of patients with high-risk/advanced stage hematologic malignancies, who received T-cell depleted (TCD) haploidentical-HSCT (haplo-HSCT) combined with donor T lymphocytes pretreated with IL-10 (ALT-TEN trial). IL-10-anergized donor T cells (IL-10-DLI) contained T regulatory type 1 (Tr1) cells specific for the host alloantigens, limiting donor-vs.-host-reactivity, and memory T cells able to respond to pathogens. IL-10-DLI were infused in 12 patients with the goal of improving immune reconstitution after haplo-HSCT without increasing the risk of graft-versus-host-disease (GvHD). IL-10-DLI led to fast immune reconstitution in five patients. In four out of the five patients, total T-cell counts, TCR-Vβ repertoire and T-cell functions progressively normalized after IL-10-DLI. These four patients are alive, in complete disease remission and immunosuppression-free at 7.2 years (median follow-up) after haplo-HSCT. Transient GvHD was observed in the immune reconstituted (IR) patients, despite persistent host-specific hypo-responsiveness of donor T cells in vitro and enrichment of cells with Tr1-specific biomarkers in vivo. Gene-expression profiles of IR patients showed a common signature of tolerance. This study provides the first indication of the feasibility of Tr1 cell-based therapy and paves way for the use of these Tr1 cells as adjuvant treatment for malignancies and immune-mediated disorders.

Clinical tolerance in allogeneic hematopoietic stem cell transplantation

Summary:  Allogeneic hematopoietic stem cell transplantation (HSCT) has been a curative therapeutic option for a wide range of immune hematologic malignant and non‐malignant disorders including genetic diseases and inborn errors. Once in the host, allogeneic transplanted cells have not only to ensure myeloid repopulation and immunological reconstitution but also to acquire tolerance to host human leukocyte antigens via central or peripheral mechanisms. Peripheral tolerance after allogeneic HSCT depends on several regulatory mechanisms aimed at blocking alloimmune reactivity while preserving immune responses to pathogens and tumor antigens. Patients transplanted with HSCT represent an ideal model system in humans to identify and characterize the key cellular and molecular players underlying these mechanisms. The knowledge gained from these studies has allowed the development of novel therapeutic strategies aimed at inducing long‐term peripheral tolerance, which can be applicable not only in allogeneic HSCT but also in autoimmune diseases and solid‐organ transplantation. In the present review, we describe Type 1 regulatory T cells, initially discovered and characterized in chimeric patients transplanted with human leukocyte antigen‐mismatched HSCT, and how their presence correlates to tolerance induction and maintenance. Furthermore, we summarize different cell therapy approaches with regulatory T cells, designed to facilitate tolerance induction, minimizing pharmaceutical interventions.

Outcome of children with acute leukemia given HLA-haploidentical HSCT after αβ T-cell and B-cell depletion

Allogeneic hematopoietic stem cell transplantation (HSCT) from an HLA-haploidentical relative (haplo-HSCT) is a suitable option for children with acute leukemia (AL) either relapsed or at high-risk of treatment failure. We developed a novel method of graft manipulation based on negative depletion of αβ T and B cells and conducted a prospective trial evaluating the outcome of children with AL transplanted with this approach. Eighty AL children, transplanted between September 2011 and September 2014, were enrolled in the trial. All children were given a fully myeloablative preparative regimen. Anti-T-lymphocyte globulin from day -5 to -3 was used for preventing graft rejection and graft-versus-host disease (GVHD); no patient received any posttransplantation GVHD prophylaxis. Two children experienced primary graft failure. The cumulative incidence of skin-only, grade 1-2 acute GVHD was 30%; no patient developed extensive chronic GVHD. Four patients died, the cumulative incidence of nonrelapse mortality being 5%, whereas 19 relapsed, resulting in a 24% cumulative incidence of relapse. With a median follow-up of 46 months for surviving patients, the 5-year probability of chronic GVHD-free, relapse-free survival (GRFS) is 71%. Total body irradiation-containing preparative regimen was the only variable favorably influencing relapse incidence and GRFS. The outcomes of these 80 patients are comparable to those of 41 and 51 children given transplantation from an HLA-identical sibling or a 10/10 allelic-matched unrelated donor in the same period. These data indicate that haplo-HSCT after αβ T- and B-cell depletion represents a competitive alternative for children with AL in need of urgent allograft. This trial was registered at www.clinicaltrials.gov as #NCT01810120.

Current and future approaches to treat graft failure after allogeneic hematopoietic stem cell transplantation

Introduction: One significant obstacle to the success of allogeneic hematopoietic stem cell transplantation (HSCT) is represented by graft failure, defined as either lack of initial engraftment of donor cells (primary graft failure) or loss of donor cells after initial engraftment (secondary graft failure). Graft failure mediated by host immune cells attacking donor stem cells is named graft rejection. Factors associated with graft failure include HLA disparity in the donor/recipient pair, underlying disease, viral infections, type of conditioning regimen and stem cell source employed. Areas covered: In this article, the experts summarize current approaches to treat graft failure/rejection after HSCT, and they discuss new strategies of graft manipulation and immune therapy of particular interest for preventing/treating this complication. Expert opinion: A limited array of options is available to treat graft failure. The experts believe that re-transplantation from another donor or the same donor (if there is no evidence of immunologically mediated graft failure) is the treatment of choice for patients with primary graft failure or acute graft rejection. The experts think that strategies based on innovative approaches of graft manipulation, new agents or cellular therapies could render in the future graft failure a much less relevant problem for HSCT recipients.

Mobilization of healthy donors with plerixafor affects the cellular composition of T-cell receptor (TCR)-αβ/CD19-depleted haploidentical stem cell grafts

BackgroundHLA-haploidentical hematopoietic stem cell transplantation (HSCT) is suitable for patients lacking related or unrelated HLA-matched donors. Herein, we investigated whether plerixafor (MZ), as an adjunct to G-CSF, facilitated the collection of mega-doses of hematopoietic stem cells (HSC) for TCR-αβ/CD19-depleted haploidentical HSCT, and how this agent affects the cellular graft composition.MethodsNinety healthy donors were evaluated. Single-dose MZ was given to 30 ‘poor mobilizers’ (PM) failing to attain ≥40 CD34+ HSCs/μL after 4 daily G-CSF doses and/or with predicted apheresis yields ≤12.0x106 CD34+ cells/kg recipient’s body weight.ResultsMZ significantly increased CD34+ counts in PM. Naïve/memory T and B cells, as well as natural killer (NK) cells, myeloid/plasmacytoid dendritic cells (DCs), were unchanged compared with baseline. MZ did not further promote the G-CSF-induced mobilization of CD16+ monocytes and the down-regulation of IFN-γ production by T cells. HSC grafts harvested after G-CSF + MZ were enriched in myeloid and plasmacytoid DCs, but contained low numbers of pro-inflammatory 6-sulfo-LacNAc+ (Slan)-DCs. Finally, children transplanted with G-CSF + MZ-mobilized grafts received greater numbers of monocytes, myeloid and plasmacytoid DCs, but lower numbers of NK cells, NK-like T cells and Slan-DCs.ConclusionsMZ facilitates the collection of mega-doses of CD34+ HSCs for haploidentical HSCT, while affecting graft composition.

The generation of human innate lymphoid cells is influenced by the source of hematopoietic stem cells and by the use of G‐CSF

NK cells play a central role in the haploidentical HSC transplantation (HSCT) to cure high‐risk leukemias. Other innate lymphoid cells (ILCs) have been proposed to exert a protective role in graft‐versus‐host disease and could also contribute to anti‐microbial defence and to lymphoid tissue remodeling. Thus, we investigated the ILC differentiation potential of HSCs isolated from BM, mobilized peripheral blood (PB), and umbilical cord blood (UCB). BM CD34+ cells are enriched in lymphoid‐committed precursors, while PB CD34+ cells preferentially contain myeloid precursors. In vitro differentiation experiments revealed that the highest and the lowest CD56+CD161+ ILC recovery was detected in UCB and PB HSC cultures, respectively. Among CD56+CD161+ ILCs, the ratio between NK cells and ILC3s was similar for all HSC analyzed. ILC recovery in PB CD34+ cultures was lower for G‐CSF‐mobilized HSCs (good mobilizers) than for G‐CSF+plerixafor‐mobilized HSC (poor mobilizers). Moreover, G‐CSF inhibited in vitro ILC recovery and the degree of inhibition was proportional to the time of exposure to the cytokine. Thus, although all common sources of HSC for transplant differentiate towards ILCs, substantial differences exist among different sources and G‐CSF may influence ILC recovery. These data offer new clues for a better understanding of the immune reconstitution after HSCT.

Strategies to accelerate immune recovery after allogeneic hematopoietic stem cell transplantation

ABSTRACT The interplay existing between immune reconstitution and patient outcome has been extensively demonstrated in allogeneic hematopoietic stem cell transplantation. One of the leading causes of infection-related mortality is the slow recovery of T-cell immunity due to the conditioning regimen and/or age-related thymus damage, poor naïve T-cell output, and restricted T-cell receptor (TCR) repertoires. With the aim of improving posttransplantation immune reconstitution, several immunotherapy approaches have been explored. Donor leukocyte infusions are widely used to accelerate immune recovery, but they carry the risk of provoking graft-versus-host disease. This review will focus on sophisticated strategies of thymus function-recovery, adoptive infusion of donor-derived, allodepleted T cells, T-cell lines/clones specific for life-threatening pathogens, regulatory T cells, and of T cells transduced with suicide genes.

Treatment of disease recurrence after allogeneic hematopoietic stem cell transplantation in children with juvenile myelomonocytic leukemia: A great challenge still to be won

T he last three decades have witnessed a dramatic improvement in the prognosis of many childhood cancers, some tumors, including retinoblastoma, Wilms tumor, Hodgkin disease, and acute lymphoblastic leukemia, having now an expected survival rate above 90% [1]. However, there are still some childhood cancers which have a dismal prognosis, especially when disease relapse occurs. Among them, there is no doubt that juvenile myelomonocytic leukemia (JMML) occupies a prominent position. JMML is a unique clonal hematopoietic disorder, typical of infancy and early childhood, that occurs with an estimated incidence of 1.2 cases per million; it is mainly characterized by hepato-splenomegaly and organ infiltration due to excessive proliferation of cells of the monocytic and granulocytic lineages [2]. Because of the rarity of the disease and the heterogeneous nature of the clinical manifestations, the diagnosis of JMML is frequently challenging for the average general practitioner/pediatrician and many children are diagnosed weeks or even months after the appearance of the first symptoms. Approximately, 90% of JMML patients harbor either somatic or germ-line mutations in genes coding for SHP-2, RAS, CBL, or NF1 proteins in their leukemic cells [3]. These genetic aberrations are largely mutually exclusive and all activate the Ras/mitogenactivated protein kinase (MAPK) signaling pathway, this resulting in hypersensitivity of JMML progenitors to granulocyte-macrophage colony-stimulating factor (GM-CSF). While some children with JMML (especially those with either Noonan syndrome or CBL mutations or specific RAS mutations) may experience spontaneous resolution or improvement of hematological abnormalities [4,5], the median survival time of patients who do not receive an allograft is only 10–12 months [2]. Thus, allogeneic hematopoietic stem cell transplantation (HSCT) remains the therapy of choice for the majority of patients. Indeed, allogeneic HSCT from either a histocompatible relative or from an HLA-matched/1-locus disparate unrelated donor has been demonstrated to be able to cure more than 50% of affected children [6–8]. Disease recurrence still remains the main cause of treatment failure, the relapse rate being as high as 40–50% [6–8]. Relapse occurs early, at a median of 2–4 months after transplantation and generally within the first year. The prognosis of children with JMML experiencing leukemia relapse after allogeneic HSCT remains unsatisfactory despite the fact that salvage therapy is employed. While donor lymphocyte infusion (DLI) was proved to be largely ineffective [9], a second allograft can rescue about one third of the patients [10]. In this issue of Pediatric Blood & Cancer, Inagaki et al. substantially confirm these data, since only one of the five patients given DLI obtained hematological remission, while 5 of the 11 children receiving a second allograft are alive and disease free. They also confirm other previously published data, namely that serial monitoring of quantitative chimerism identifies patients with increasing mixed chimerism as having the highest risk of relapse [11]. Immediate withdrawal of immunosuppressive therapy can lead to the eradication of the malignant cells re-growing/ persisting after the conditioning regimen used in preparation to the allograft, this finding highlighting that the graft-versusleukemia (GvL) plays an important role for a successful posttransplant outcome [11]. How can we further increase the proportion of children with JMML rescued after a relapse following HSCT? This crucial question involves more generally the issues of how to render both early detection of leukemia cell re-growth more effective and approaches of immunotherapy safer and more capable of eradicating the malignant clone. Concerning the first issue, allele-specific quantitative PCR approaches aimed at detecting mutation-specific clones have been reported to be a sensitive tool for identifying minimal residual disease, thus facilitating earlier withdrawal of immunosuppression [12]. While DLI is the treatment of choice for patients with Philadelphia-positive chronic myeloid leukemia relapsing after allogeneic HSCT, it resulted of limited efficacy in children with JMML. Moreover, DLI can be associated with the development of life-threatening acute or chronic graft-versus-host disease (GvHD). The biological/ immunological reasons why JMML recurrence has limited sensitivity to DLI remain obscure: the population doubling of the neoplastic cells could be too fast to allow donor immune cells to control leukemia re-growth, but we cannot exclude that mechanisms of immune escape peculiar of JMML cells contribute to disease progression. Further investigations in this area are more