Francesca Moretta

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.

Human Natural Killer Cells: Origin, Receptors, Function, and Clinical Applications

Natural killer (NK) cells are important effectors playing a relevant role in innate immunity, primarily in tumor surveillance and in defenses against viruses. Human NK cells recognize HLA class I molecules through surface receptors (KIR and NKG2A) that inhibit NK cell function and kill target cells that have lost (or underexpress) HLA class I molecules as it occurs in tumors or virus-infected cells. NK cell activation is mediated by an array of activating receptors and co-receptors that recognize ligands expressed primarily on tumors or virus-infected cells. In vivo anti-tumor NK cell activity may be suppressed by tumor or tumor-associated cells. Alloreactive NK cells (i.e. those that are not inhibited by the HLA class I alleles of the patient) derived from HSC of haploidentical donors play a major role in the cure of high-risk leukemia, by killing leukemia blasts and patients DC, thus preventing tumor relapses and graft-versus-host disease. The expression of the HLA-C2-specific activating KIR2DS1 may also contribute to NK alloreactivity in patients expressing C2 alleles. A clear correlation has been proven between the size of the alloreactive NK cell population and the clinical outcome. Recently, haplo-HSCT has been further improved with the direct infusion, together with HSC, of donor-derived, mature alloreactive NK cells and TCRγδ+ T cells - both contributing to a prompt anti-leukemia effect together with an efficient defense against pathogens during the 6- to 8-week interval required for the generation of alloreactive NK cells from HSC.

Negative depletion of α/β+ T cells and of CD19+ B lymphocytes: A novel frontier to optimize the effect of innate immunity in HLA-mismatched hematopoietic stem cell transplantation

In recent years, infusion of T-cell depleted hematopoietic stem cells from an HLA-haploidentical relative has been shown to represent a suitable and effective, alternative option in patients in need of an allograft who lack an HLA-identical relative. In particular, this type of allograft is associated with the enormous advantage of offering an immediate transplant treatment to virtually all pediatric patients without an HLA-matched donor, whether related or unrelated, or a suitable umbilical cord blood unit. Several studies have shown that in patients given a T-cell depleted transplant relevant part of the anti-leukemia effect is mediated by alloreactive (i.e. KIR/HLA mismatched) Natural Killer cells originated from donor hematopoietic stem cells. After infusion of positively selected hematopoietic stem cell, fully functioning Natural Killer cells emerge in the recipient peripheral blood, persisting over time, only several weeks after the allograft. We have developed a new method of T-cell depletion (based on the physical elimination of mature T cells carrying α and β chains of the T-cell receptor), which permits to maintain mature donor-derived alloreactive Natural Killer cells and γδ(+) T cells in the graft. We, thus, started a formal study in children with hematological disorders aimed at evaluating the safety and efficacy of this approach. Preliminary results on 60 children transplanted so far after this type of graft manipulation are particularly promising.

Natural killer cells in the treatment of high-risk acute leukaemia

Several studies have shown that in patients with acute leukaemia given allogeneic haematopoietic stem cell transplantation (allo-HSCT) large part of the therapeutic effect lies on the anti-tumour effect displayed by cells of both adaptive and innate immunity. This evidence has also opened new scenarios for the treatment of patients with other haematological malignancies/solid tumours. In particular, donor-derived natural killer (NK) cells play a crucial role in the eradication of cancer cells in patients given an allograft from an HLA-haploidentical relative, especially when there is a killer inhibitory-receptor (KIR)-KIR ligand mismatched in the donor-recipient direction. Alloreactive donor-derived NK cells have been also demonstrated to kill recipient antigen-presenting cells and cytotoxic T lymphocytes, thus preventing graft-versus-host disease (GvHD) and graft rejection and to largely contribute to the defence against cytomegalovirus infection in the early post-transplant period. Several clinical studies have recently focused also on the influence of NK-cell activating receptors on the outcome of allo-HSCT recipients; in particular, B/x haplotype donors offer clinical advantages compared with A/A donors, even when the donor is an HLA-identical volunteer. Altogether, these data have provided the rationale for implementing phase I/II clinical trials based on adoptive infusion of either selected or ex vivo activated NK cells from an HLA-mismatched donor. This review summarizes the biological and clinical data on the role played by NK cells in patients with high-risk acute leukaemia, focusing also on the still unsolved issues and the future perspectives related to the approaches of adoptive NK cell therapy.

Biological, functional and genetic characterization of bone marrow-derived mesenchymal stromal cells from pediatric patients affected by acute lymphoblastic leukemia

Alterations in hematopoietic microenvironment of acute lymphoblastic leukemia patients have been claimed to occur, but little is known about the components of marrow stroma in these patients. In this study, we characterized mesenchymal stromal cells (MSCs) isolated from bone marrow (BM) of 45 pediatric patients with acute lymphoblastic leukemia (ALL-MSCs) at diagnosis (day+0) and during chemotherapy treatment (days: +15; +33; +78), the time points being chosen according to the schedule of BM aspirates required by the AIEOP-BFM ALL 2009 treatment protocol. Morphology, proliferative capacity, immunophenotype, differentiation potential, immunomodulatory properties and ability to support long-term hematopoiesis of ALL-MSCs were analysed and compared with those from 41 healthy donors (HD-MSCs). ALL-MSCs were also genetically characterized through array-CGH, conventional karyotyping and FISH analysis. Moreover, we compared ALL-MSCs generated at day+0 with those isolated during chemotherapy. Morphology, immunophenotype, differentiation potential and in vitro life-span did not differ between ALL-MSCs and HD-MSCs. ALL-MSCs showed significantly lower proliferative capacity (p<0.001) and ability to support in vitro hematopoiesis (p = 0.04) as compared with HD-MSCs, while they had similar capacity to inhibit in vitro mitogen-induced T-cell proliferation (p = N.S.). ALL-MSCs showed neither the typical translocations carried by the leukemic clone (when present), nor other genetic abnormalities acquired during ex vivo culture. Our findings indicate that ALL-MSCs display reduced ability to proliferate and to support long-term hematopoiesis in vitro. ALL-MSCs isolated at diagnosis do not differ from those obtained during treatment.

Markers and function of human NK cells in normal and pathological conditions

Natural killer (NK) cells, the most important effectors of the innate lymphoid cells (ILCs), play a fundamental role in tumor immune‐surveillance, defense against viruses and, in general, in innate immune responses. NK cell activation is mediated by several activating receptors and co‐receptors able to recognize ligands on virus‐infected or tumor cells. To prevent healthy cells from auto‐aggression, NK cells are provided with strong inhibitory receptors (KIRs and NKG2A) which recognize HLA class I molecules on target cells and, sensing their level of expression, allow killing of targets underexpressing HLA‐class I. In vivo, NK cell‐mediated anti‐tumor function may be suppressed by tumor or tumor‐associated cells via inhibitory soluble factors/cytokines or the engagement of the so called immune‐check point molecules (e.g., PD1‐PDL1). The study of these immune check‐points is now offering new important opportunities for the therapy of cancer. In haemopoietic stem cell transplantation, alloreactive NK cells (i.e., those that express KIRs, which do not recognize HLA class I molecules on patient cells), derived from HSC of haploidentical donors, are able to kill leukemia blasts and patients DC, thus preventing both tumor relapses and graft‐versus‐host disease. A clear correlation exists between size of the alloreactive NK cell population and clinical outcome. Thus, in view of the recent major advances in cancer therapy based on immuno‐mediated mechanisms, the phenotypic analysis of cells and molecules involved in these mechanisms plays an increasingly major role.

Human innate lymphoid cells

The interest in innate lymphoid cells (ILC) has rapidly grown during the last decade. ILC include distinct cell types that are collectively involved in host protection against pathogens and tumor cells and in the regulation of tissue homeostasis. Studies in mice enabled a broad characterization of ILC function and of their developmental requirements. In humans all mature ILC subsets have been characterized and their role in the pathogenesis of certain disease is emerging. Nonetheless, still limited information is available on human ILC development. Indeed, only the cell precursors committed toward NK cells or ILC3 have been described. Here, we review the most recent finding on human mature ILC, discussing their tissue localization and function. Moreover, we summarize the available data regarding human ILC development.

NK Cells and Other Innate Lymphoid Cells in Hematopoietic Stem Cell Transplantation

Natural killer (NK) cells play a major role in the T-cell depleted haploidentical hematopoietic stem cell transplantation (haplo-HSCT) to cure high-risk leukemias. NK cells belong to the expanding family of innate lymphoid cells (ILCs). At variance with NK cells, the other ILC populations (ILC1/2/3) are non-cytolytic, while they secrete different patterns of cytokines. ILCs provide host defenses against viruses, bacteria, and parasites, drive lymphoid organogenesis, and contribute to tissue remodeling. In haplo-HSCT patients, the extensive T-cell depletion is required to prevent graft-versus-host disease (GvHD) but increases risks of developing a wide range of life-threatening infections. However, these patients may rely on innate defenses that are reconstituted more rapidly than the adaptive ones. In this context, ILCs may represent important players in the early phases following transplantation. They may contribute to tissue homeostasis/remodeling and lymphoid tissue reconstitution. While the reconstitution of NK cell repertoire and its role in haplo-HSCT have been largely investigated, little information is available on ILCs. Of note, CD34+ cells isolated from different sources of HSC may differentiate in vitro toward various ILC subsets. Moreover, cytokines released from leukemia blasts (e.g., IL-1β) may alter the proportions of NK cells and ILC3, suggesting the possibility that leukemia may skew the ILC repertoire. Further studies are required to define the timing of ILC development and their potential protective role after HSCT.

Autoimmunity and infection in common variable immunodeficiency (CVID)

Common variable immunodeficiency (CVID) is a heterogeneous group of diseases, characterized by primary hypogammaglobulinemia. B and T cell abnormalities have been described in CVID. Typical clinical features of CVID are recurrent airway infections; lymphoproliferative, autoinflammatory, or neoplastic disorders; and autoimmune diseases among which autoimmune thrombocytopenia (ITP) is the most common. The coexistence of immunodeficiency and autoimmunity appears paradoxical, since one represents a hypoimmune state and the other a hyperimmune state. Considering both innate and adaptive immune response abnormalities in CVID, it is easier to understand the mechanisms that lead to a breakdown of self-tolerance. CD21(low) B cells derive from mature B cells that have undergone chronic immune stimulation; they are increased in CVID patients. The expansion of CD21(low) B cells is also observed in certain autoimmune diseases. We have studied CD21(low) B cells in patients with CVID, CVID, and ITP and with ITP only. We observed a statistically significant increase in the CD21(low) population in the three pathological groups. Moreover, we found statistical differences between the two groups of CVID patients: patients with ITP had a higher percentage of CD21(low) cells. Our data suggest that CD21(low) cells are related to autoimmunity and may represent a link between infection and autoimmunity.

Human natural killer cells: news in the therapy of solid tumors and high-risk leukemias

It is well established that natural killer (NK) cells play an important role in the immunity against cancer, while the involvement of other recently identified, NK-related innate lymphoid cells is still poorly defined. In the haploidentical hematopoietic stem cell transplantation for the therapy of high-risk leukemias, NK cells have been shown to exert a key role in killing leukemic blasts residual after conditioning. While the clinical results in the cure of leukemias are excellent, the exploitation of NK cells in the therapy of solid tumors is still limited and unsatisfactory. In solid tumors, NK cell function may be inhibited via different mechanisms, occurring primarily at the tumor site. The cellular interactions in the tumor microenvironment involve tumor cells, stromal cells and resident or recruited leukocytes and may favor tumor evasion from the host’s defenses. In this context, a number of cytokines, growth factors and enzymes synthesized by tumor cells, stromal cells, suppressive/regulatory myeloid and lymphoid cells may substantially impair the function of different tumor-reactive effector cells, including NK cells. The identification and characterization of such mechanisms may offer clues for the development of new immunotherapeutic strategies to restore effective anti-tumor responses. In order to harness NK cell-based immunotherapies, several approaches have been proposed, including reinforcement of NK cell cytotoxicity by means of specific cytokines, antibodies or drugs. These new tools may improve NK cell function and/or increase tumor susceptibility to NK-mediated killing. Hence, the integration of NK-based immunotherapies with conventional anti-tumor therapies may increase chances of successful cancer treatment.