TCRαß/CD19 cell-depleted HLA-haploidentical transplantation to treat pediatric acute leukemia: updated final analysis.

ABSTRACT: TCRαß/CD19 cell depletion is a promising graft manipulation technique frequently used in the context of human leukocyte antigen (HLA)-haploidentical hematopoietic stem cell transplantation (HSCT). We previously reported the results of a phase I-II clinical trial (NCT01810120) to assess the safety and the efficacy of this type of exvivo T-cell depletion in 80 children with acute leukemia, showing promising survival outcomes. We now report an updated analysis on a cohort of 213 children with a longer follow-up (median, 47.6 months for surviving patients). With a 5-year cumulative incidence of nonrelapse mortality of 5.2% (95% confidence interval [CI], 2.8%-8.8%) and a cumulative incidence of relapse of 22.7% (95% CI, 16.9%-29.2%), projected 10-year overall and disease-free survival (DFS) were 75.4% (95% CI, 68.6%-80.9%) and 71.6% (95% CI, 64.4%-77.6%), respectively. Cumulative incidence of both grade II-IV acute and chronic graft-versus-host disease were low (14.7% and 8.1%, respectively). In a multivariable analysis for DFS including type of disease, use of total body irradiation in the conditioning regimen (hazard ratio [HR], 0.5; 95% CI, 0.26-0.98; P = .04), disease status at HSCT (complete remission [CR] ≥3 vs CR 1/2; HR, 2.23; 95% CI, 1.20-4.16; P = .01), and high levels of pre-HSCT minimal residual disease (HR, 2.09; 95% CI, 1.01-4.33; P = .04) were independently associated with outcome. In summary, besides confirming the good outcome results already reported (which are almost superimposable on those of transplant from HLA-matched donors), this clinical update allows the identification of patients at higher risk of treatment failure for whom personalized approaches, aimed at reducing the risk of relapse, are warranted.

Mucosal-associated invariant T cells are functionally impaired in pediatric and young adult patients following allogeneic hematopoietic stem cell transplantation and their recovery correlates with clinical outcomes.

Mucosal-associated invariant T (MAIT) cells are innate-like T-cells implicated in the response to fungal and bacterial infections. Their contribution to restoring T-cell immunity and influencing hematopoietic stem cell transplant (HSCT) outcomes remains poorly understood. We retrospectively studied MAIT-cell recovery in 145 consecutive children and young adults with hematological malignancies undergoing allo-HSCT, between April/2019 and May/2022, from unrelated matched donor (MUD, n=52), with standard graft-versus-host-disease (GvHD) prophylaxis, or HLA-haploidentical (Haplo, n=93) donor after in vitro αßT/CD19-cell depletion, without post-HSCT pharmacological prophylaxis. With a median follow-up of 33 months (12-49), overall survival (OS), disease-free survival (DFS) and non-relapse mortality (NRM) were 79.5%, 72% and 7%, respectively; GvHD-free, Relapse-free Survival (GRFS) was 63%, while cumulative incidence of relapse was 23%. While WWT-cells reconstituted 1-2 years post-HSCT, MAIT-cells showed delayed recovery and prolonged functional impairment, characterized by expression of activation (CD25, CD38), exhaustion (PD1, TIM3) and senescence (CD57) markers, and suboptimal ex vivo response. OS, DFS and NRM were not affected by MAIT-cells. Interestingly, higher MAIT-cells at day+30 correlated with higher incidence of grade II-IV acute GvHD (19% vs 7%, p=0.06). Furthermore, a greater MAIT-cell count tended to be associated with a higher incidence of chronic GvHD (17% vs 6%, p=0.07) resulting in lower GRFS (55% vs 73%, p=0.05). Higher MAIT-cells also correlated with greater cytomegalovirus (CMV) reactivation and lower late blood stream infections (BSI) (44% vs 24%, p=0.02 and 9% vs 18%, p=0.08, respectively). Future studies are needed to confirm the impact of early MAIT-cell recovery on cGvHD, CMV reactivation and late BSI.

Outcome of children with acute leukemia given HLA-haploidentical HSCT after αß 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 αß 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 αß 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.

Long-term follow-up of IPEX syndrome patients after different therapeutic strategies: An international multicenter retrospective study.

BACKGROUND: Immunodysregulation polyendocrinopathy enteropathy x-linked (IPEX) syndrome is a monogenic autoimmune disease caused by FOXP3 mutations. Because it is a rare disease, the natural history and response to treatments, including allogeneic hematopoietic stem cell transplantation (HSCT) and immunosuppression (IS), have not been thoroughly examined. OBJECTIVE: This analysis sought to evaluate disease onset, progression, and long-term outcome of the 2 main treatments in long-term IPEX survivors. METHODS: Clinical histories of 96 patients with a genetically proven IPEX syndrome were collected from 38 institutions worldwide and retrospectively analyzed. To investigate possible factors suitable to predict the outcome, an organ involvement (OI) scoring system was developed. RESULTS: We confirm neonatal onset with enteropathy, type 1 diabetes, and eczema. In addition, we found less common manifestations in delayed onset patients or during disease evolution. There is no correlation between the site of mutation and the disease course or outcome, and the same genotype can present with variable phenotypes. HSCT patients (n = 58) had a median follow-up of 2.7 years (range, 1 week-15 years). Patients receiving chronic IS (n = 34) had a median follow-up of 4 years (range, 2 months-25 years). The overall survival after HSCT was 73.2% (95% CI, 59.4-83.0) and after IS was 65.1% (95% CI, 62.8-95.8). The pretreatment OI score was the only significant predictor of overall survival after transplant (P = .035) but not under IS. CONCLUSIONS: Patients receiving chronic IS were hampered by disease recurrence or complications, impacting long-term disease-free survival. When performed in patients with a low OI score, HSCT resulted in disease resolution with better quality of life, independent of age, donor source, or conditioning regimen.

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.

γδ T-cell reconstitution after HLA-haploidentical hematopoietic transplantation depleted of TCR-αß+/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 αß(+) 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 αß(+) 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.

HLA-haploidentical stem cell transplantation after removal of αß+ 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 αß(+) T cells and CD19(+) B cells. The median number of CD34(+), αß(+)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 αß(+) 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.

Longitudinal Evaluation of Immune Reconstitution and B-cell Function After Hematopoietic Cell Transplantation for Primary Immunodeficiency.

PURPOSE: Hematopoietic cell transplantation (HCT) provides a curative therapy for severe forms of primary immunodeficiencies (PID). While the timing and extent of T-cell reconstitution following transplant for PID has been studied in depth, less is known about the kinetics of B-cell development and long-term restoration of humoral functions, which been often reported to be suboptimal after HCT. METHODS: We studied longitudinally B-cell development and function in a cohort of 13 PID patients transplanted between 1997 and 2010, with a follow-up ranging from 0.7 to 15 years. Flow cytometric analysis of naïve and antigen-experienced B-cell subsets and in vitro functional responses to CpG were compared with data from healthy children and correlated with the degree of B-cell chimerism and in vivo antibody production. RESULTS: We found that total memory B-cells count remained below normal levels for the first 2 years of follow up and progressively normalized. Switched memory B-cells (CD19+CD27+IgD-IgM-) were restored early and better than IgM memory B-cells (CD19+CD27+IgD+IgM+), which remained significantly reduced long-term. The recovery of memory B-cells correlated with good in vivo humoral function and normalization of CpG-response. A complete B-cell reconstitution was usually associated with donor B-cells chimerism and pre-transplant conditioning. Donor source and the underlying genetic defect represented also important variables. CONCLUSION: Monitoring of phenotypic and functional changes on B-cells following HCT may prove clinically relevant to tailor patients' care. In particular the analysis of IgM memory and switched memory B-cells in addition to in vitro B-cells stimulation are recommended before Ig replacement therapy (IgRT) discontinuation.

Clinical tolerance in allogeneic hematopoietic stem cell transplantation.

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.

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

BACKGROUND: HLA-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-αß/CD19-depleted haploidentical HSCT, and how this agent affects the cellular graft composition. METHODS: Ninety 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. RESULTS: MZ 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. CONCLUSIONS: MZ facilitates the collection of mega-doses of CD34+ HSCs for haploidentical HSCT, while affecting graft composition.

Functional analysis and gene expression profile of umbilical cord blood regulatory T cells.

The aim of the study was to analyze and compare the functional properties and the gene expression profile of regulatory T cells (Tregs) isolated from cord blood (CB) units (n = 23) and from the peripheral blood (PB) of adult normal donors (n = 13). Tregs were purified from mononuclear cells and expanded for 6 days with anti-CD3, anti-CD28, and IL-2. CB and PB Tregs presented similar immunophenotypic features. However, Tregs isolated from CB presented a much higher expansion capacity; this was confirmed by the genomic characterization that showed in CB-derived Tregs significant enrichments of genes involved in cell proliferation, chromatin modification, and regulation of gene expression. All samples were positive for the FoxP3 gene and protein after expansion. CB and PB expanded Tregs exerted a comparable and potent suppressive function on the proliferative reaction of autologous T cells stimulated by allogeneic dendritic cells and presented a high in vitro IL-10 production capacity. Gene profile analysis also revealed for PB Tregs significant enrichments of genes involved in the adaptive immune response. These data offer further insights into the understanding of the biology of CB transplantation indicating a possible role played by CB Tregs in the suppression of the allogeneic T cell response.

Concurrent search for unrelated cord and volunteer donor in high-risk acute lymphoblastic leukemia.

To assess the effectiveness of the search for an unrelated donor on the outcome of patients with high-risk acute lymphoblastic leukemia, we analyzed prospectively 136 patients who underwent a search for cord blood (CB) and an unrelated volunteer donor (UD) at the same time. The probability of finding a donor was 58.2%, 70.3%, and 75.7% at 3, 6, and 12 months, respectively. The median time to find a donor was 1.8 months for CB and 3.5 months for UD. Of the 99 patients with a donor, 38.4% failed to undergo the transplant because of a relapse observed at a median of 4 months from the start of the search. In univariate analysis, absence of relapse during the search (p < 0.0001) and transplant (p = 0.004) showed a positive impact on long-term survival. In multivariate analysis, relapse during the search remained the key factor affecting survival (p < 0.0001). Since an extension of the search beyond 3 months enables only a slight increase in the probability of finding a donor compared to the increased risk of relapse, the time of the search should not exceed the 3-month time point. The simultaneous search for CB and UD increases the likelihood of performing a timely transplant.

Prognostic impact of pre-transplantation transfusion history and secondary iron overload in patients with myelodysplastic syndrome undergoing allogeneic stem cell transplantation: a GITMO study.

BACKGROUND: Transfusion-dependency affects the natural history of myelodysplastic syndromes. Secondary iron overload may concur to this effect. The relative impact of these factors on the outcome of patients with myelodysplastic syndrome receiving allogeneic stem-cell transplantation remains to be clarified. DESIGN AND METHODS: We retrospectively evaluated the prognostic effect of transfusion history and iron overload on the post-transplantation outcome of 357 patients with myelodysplastic syndrome reported to the Gruppo Italiano Trapianto di Midollo Osseo (GITMO) registry between 1997 and 2007. RESULTS: Transfusion-dependency was independently associated with reduced overall survival (hazard ratio=1.48, P=0.017) and increased non-relapse mortality (hazard ratio=1.68, P=0.024). The impact of transfusion-dependency was noted only in patients receiving myeloablative conditioning (overall survival: hazard ratio=1.76, P=0.003; non-relapse mortality: hazard ratio=1.70, P=0.02). There was an inverse relationship between transfusion burden and overall survival after transplantation (P=0.022); the outcome was significantly worse in subjects receiving more than 20 red cell units. In multivariate analysis, transfusion-dependency was found to be a risk factor for acute graft-versus-host disease (P=0.04). Among transfusion-dependent patients undergoing myeloablative allogeneic stem cell transplantation, pre-transplantation serum ferritin level had a significant effect on overall survival (P=0.01) and non-relapse mortality (P=0.03). This effect was maintained after adjusting for transfusion burden and duration, suggesting that the negative effect of transfusion history on outcome might be determined at least in part by iron overload. CONCLUSIONS: Pre-transplantation transfusion history and serum ferritin have significant prognostic value in patients with myelodysplastic syndrome undergoing myeloablative allogeneic stem cell transplantation, inducing a significant increase of non-relapse mortality. These results indicate that transfusion history should be considered in transplantation decision-making in patients with myelodysplastic syndrome.

T-cell depleted HLA-haploidentical HSCT in a child with neuromyelitis optica.

Neuromyelitis optica is an immune-mediated disease characterized by a relapsing course, resulting in progressive disability. In children, given the long life expectancy, a disease-modifying treatment could be particularly desirable. Unfortunately, the currently available treatment strategies with this potential are scarce. Very limited data are available about the use of allogeneic hematopoietic stem cell transplantation (HSCT) for autoimmune neurological diseases. In this report, we present a pediatric case successfully treated with allogeneic HSCT from an HLA-haploidentical donor, after ex vivo TCR/CD19-depletion of the graft. To the best of our knowledge, this is the first case of a pediatric patient to benefit from such a treatment.

B-cell concentration in the apheretic product predicts acute graft-versus-host disease and treatment-related mortality of allogeneic peripheral blood stem cell transplantation.

BACKGROUND: The influence of the graft composition on the clinical outcome after allogeneic peripheral blood stem cell (PBSC) transplantation is not well established. METHODS: The cellular composition of the apheretic products obtained from 63 human leukocyte antigen-identical siblings was prospectively correlated with the outcome of patients with hematological malignancies undergoing an allogeneic PBSC transplant after myeloablative conditioning. The concentration of nuclear, mononuclear, CD34+, T-cell subsets, B cells, and natural killer cells in the graft has been analyzed. RESULTS: In univariate analysis, acute graft-versus-host disease (GVHD) correlated with the disease (P=0.002), with the phase of disease at transplant (P=0.01), and with the number of CD20+ cells infused (P=0.05). In multivariate analysis, a dose of CD20+ cells in the graft higher than the median dose remained the only factor negatively affecting the incidence of acute GVHD (P=0.01; 95% confidence interval [CI]: 0.12-0.78). In univariate analysis, treatment-related mortality (TRM) correlated with the disease (P=0.04) and was negatively affected by a dose of infused B cells greater than the median value (28% versus 50%; P=0.02). In multivariate analysis, TRM was close to statistical correlation with the dose of CD20+ cells (P=0.06; 95% CI: 0.02-1.05). No other clinical parameter was influenced by the composition of the graft. CONCLUSIONS: Our results suggest that the concentration of B cells in the apheretic product may predict the incidence of acute GVHD and TRM in patients undergoing an allogeneic PBSC transplantation and open the way to the new preventive and therapeutic strategies for the management of GVHD.

Unrelated cord blood transplant in children with high-risk acute lymphoblastic leukemia: a long-term follow-up.

BACKGROUND AND OBJECTIVES: The aim of this single center study was to assess the impact of pre-transplant factors on long-term follow-up in young patients affected by high-risk acute lymphoblastic leukemia (ALL) who underwent an unrelated cord blood transplant (CBT). The conditioning regimens, graft-versus-host disease (GVHD) prophylaxis and supportive policies were uniform for all patients. DESIGN AND METHODS: We analyzed the results of CBT performed in 30 patients, aged <18 years, affected by high risk ALL. As conditioning regimen, all patients received 12 Gy fractionated total body irradiation, etoposide, cyclophosphamide and horse anti-lymphocyte globulin. GVHD prophylaxis consisted of 6-methylprednisolone and cyclosporine A. RESULTS The cumulative incidence of engraftment was 93% (95% CI:0.85-0.93). The cumulative incidence of grade III-IV acute and chronic GVHD was 7% (95% CI:0.01-0.19) and 33% (95% CI: 0.17-0.64), respectively. The 9-year cumulative incidence of transplant-related mortality and relapse was 34% (95% CI:0.13-0.45) and 31% (95% CI:0.16-0.61), respectively. The 9-year overall survival, leukemia-free survival and event-free survival were 42% (95% CI:0.52-0.93), 47% (95% CI:0.25-0.61) and 46% (95% CI:0.33-0.61), respectively. A number of CFU-GM <1 x 10(4)/Kg of recipient body weight was the only factor that negatively affected all outcome parameters both in univariate and multivariate analyses. INTERPRETATION AND CONCLUSIONS: The infused cell dose expressed as in vitro progenitor cell growth represents the most important pre-transplant factor affecting the long-term outcome after an unrelated CBT in young patients with high risk ALL. The number of CFU-GM should thus be considered in the selection process of cord blood units for transplant.

Preservation of Antigen-Specific Functions of αß T Cells and B Cells Removed from Hematopoietic Stem Cell Transplants Suggests Their Use As an Alternative Cell Source for Advanced Manipulation and Adoptive Immunotherapy.

Hematopoietic stem cell transplantation is standard therapy for numerous hematological diseases. The use of haploidentical donors, sharing half of the HLA alleles with the recipient, has facilitated the use of this procedure as patients can rely on availability of a haploidentical donor within their family. Since HLA disparity increases the risk of graft-versus-host disease, T-cell depletion has been used to remove alloreactive lymphocytes from the graft. Selective removal of αß T cells, which encompass the alloreactive repertoire, combined with removal of B cells to prevent EBV-related lymphoproliferative disease, proved safe and effective in clinical studies. Depleted αß T cells and B cells are generally discarded as by-products. Considering the possible use of donor T cells for donor lymphocyte infusions or for generation of pathogen-specific T cells as mediators of graft-versus-infection effect, we tested whether cells in the discarded fractions were functionally intact. Response to alloantigens and to viral antigens comparable to that of unmanipulated cells indicated a functional integrity of αß T cells, in spite of the manipulation used for their depletion. Furthermore, B cells proved to be efficient antigen-presenting cells, indicating that antigen uptake, processing, and presentation were fully preserved. Therefore, we propose that separated αß T lymphocytes could be employed for obtaining pathogen-specific T cells, applying available methods for positive selection, which eventually leads to indirect allodepletion. In addition, these functional T cells could undergo additional manipulation, such as direct allodepletion or genetic modification.

Strategies to accelerate immune recovery after allogeneic hematopoietic stem cell transplantation.

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.

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.