Novel insights into NK cell/leukemia molecular interactions and possible tools to potentiate the anti-leukemia NK cell activity

Natural killer (NK) cells are extremely promising for cellular antitumor therapy in terms of efficacy and safety, particularly in the control of hematological malignancies. The aim of this thesis was to provide deeper insights on receptor/ligand leukemia interactions and investigate possible tools to enhance the NK cell anti-leukemia activity. Two clinical contexts of haploidentical hematopoietic stem cell transplantation (haplo-HSCT) were under evaluation 1) \u3b1\u3b2T-cell and B-cell depleted haplo-HSCT in pediatric leukemia patients, and 2) haplo-HSCT with post-transplant cyclophosphamide (PT-Cy) in adult AML patients. Regarding the first platform, we tested the in vitro effect of different NK cell engagers (NKCEs), which can trigger either NKp46 or NKp30 together with CD16A, and target either CD19 or CD20 to induce killing of pediatric B cell precursor acute lymphoblastic leukemia (BCP-ALL). We used as target cells different BCP-ALL cell lines and primary leukemias, and as effector cells resting NK cells derived from healthy donors and pediatric leukemia patients after \u3b1\u3b2T/B-depleted haplo-HSCT. The NKCEs potentiated the killing of NK-cell resistant BCP-ALL cells and enhanced the degranulation and cytokine production of NK cells. These data strongly support the therapeutic use of NKp46/CD16A/CD19-NKCE to fight relapsed/refractory leukemia in pre- or post-transplantation setting. Regarding the other platform, we investigated the phenotype of NK cells after transplantation, and we evaluated the effect of immune checkpoint inhibitor (ICI), targeting NKG2A, to unleash NK-cell activity against leukemia. The data pave the way for the use of an anti-NKG2A antibody (as Monalizumab) at early-time points after transplantation to enhance the anti-leukemia activity of NK cells

Generation and characterization of KO lymphocyte cell line for Zn transporters

Curs 2015-2016La deficiencia en zinc puede ser debida a una malnutrición (afecta a >17% de la población mundial) o como consecuencia a enfermedades y al envejecimiento. Ésta deficiencia provoca retraso del crecimiento, problemas neurológicos y un mayor riesgo de infecciones, causado por la baja eficiencia del sistema inmunitario. Existe la suplementación de zinc, pero las bases biológicas de este efecto preventivo no son conocidas. A pesar del importante papel que juega el zinc en el sistema inmunitario, no se sabe en profundidad su forma de entrada, sobre todo a lo que se refiere a los linfocitos T, donde se ha visto que éstos son vulnerables a la deficiencia del zinc, afectando a la maduración, la activación, la diferenciación y envejecimiento. Este TFG es parte de un gran proyecto. En la primera fase, se caracterizaron los transportadores de zinc durante la maduración, activación, diferenciación y envejecimiento de los linfocitos T en diferentes condiciones fisiológicas. De los 14 candidatos, se seleccionaron los 4 transportadores más representativos: ZIP6, ZIP8, ZIP10 i ZIP14.Zinc has been the focus of considerable interest during the last two decades. This interest has been awakened by the impact of zinc deficiency in human health. Individuals at high risk of zinc deficiency include: infants, adolescents, women at reproductive age and the aged. A deficiency of this metal causes impairment in body growth, neurological disorder and immunosuppression, among others 1,2. Zinc functions have been organized into 3 categories: catalytic, structural and regulatory 3. There are several transporters and zinc binding proteins that operate coordinately in order to maintain zinc homeostasis. Although it is known the impact of zinc in the immune system, it is not clear how this element enters the immune system cells. This lack of knowledge is even greater in T lymphocytes, where it is described that zinc deficiency alters T cell maturation, influences Th subsets polarization and impairs cytokine production 4. The aim of the project is to identify the gate of zinc entry in T cells in order to have a better understanding of zinc transporters in T cells. The main objective of this final project and the second phase of the zinc project leaded by Dr. Rubén Vicente Garcia was to develop a tool by depleting Zip6, Zip8, Zip10 and Zip14 of the acute T cell leukemia Jurkat cell line using the revolutionary tool CRISPR-CAS9 technique. This tool would help us to study how the absence of these zinc transporters affects zinc kinetics and T cell function as activation, proliferation and differentiation

Exploiting Natural Killer Cell Engagers to Control Pediatric B-cell Precursor Acute Lymphoblastic Leukemia

International audience◥ Natural killer (NK) cells represent a promising cell type in antitumor immunotherapy for efficacy and safety, particularly in the treatment of hematologic malignancies. NK cells have been shown to exert antileukemia activity in the context of haploidentical hematopoietic stem cell transplantation (haplo-HSCT). Products have been developed to boost the activation of NK cells only when cross-linked by tumor cells, avoiding any off-target effect. Here, we tested the in vitro effect of different NK-cell engagers (NKCE), which trigger either NKp46 or NKp30 together with CD16A, and target either CD19 or CD20 to induce killing of pediatric B-cell precursor acute lymphoblastic leukemia (BCP-ALL). Target cells were NALM-16 and MHH-CALL-4 cell lines and four primary leukemias, while effector cells were resting NK cells derived from healthy donors and pediatric patients with leukemia after abT/B-depleted haplo-HSCT. The NK cell-resistant MHH-CALL-4 was efficiently killed using all NKCEs. Boosting of NK activity against MHH-CALL-4 was also evident by degranulation and IFNg production. Because of the lack of CD20 and high expression of CD19 on primary BCP-ALL, we focused on NKCEs targeting CD19. NKp46-and NKp30-based NKCEs displayed similar potency at inducing NK-cell activity, even when challenged with primary BCP-ALL blasts. Their efficacy was shown also using NK cells derived from transplanted patients. NKCE-induced activation against BCP-ALL can override HLA-specific inhibitory interactions, although the strongest response was observed by the alloreactive NK-cell subset. These data support the therapeutic use of NKp46/CD16A/ CD19-NKCE to fight refractory/relapsed leukemia in pretransplantation or posttransplantation settings

Characterization of KIR + NK cell subsets with a monoclonal antibody selectively recognizing KIR2DL1 and blocking the specific interaction with HLA-C

The phenotypic identification of different NK cell subsets allows more in-depth characterization of KIR repertoire and function, which are of potential interest in KIR and disease association studies. KIR genes are highly polymorphic, but a great homology exists among the various sequences and few monoclonal antibodies (mAbs) specifically recognize a single KIR. This is the case of HP-DM1 which was demonstrated by analysis of cell transfectants and epitope mapping to be exclusively KIR2DL1-specific, covering all allotypes identified to date, except for KIR2DL1*022 and *020, and also to react with KIR2DS1*013. Here, we compared in immunofluorescence analyses the staining of HP-DM1 with other available mAbs to precisely identify KIR2DL1+ NK cells in potential donors for αβT/B-depleted haplo-HSCT, with known KIR genotype. HP-DM1 mAb was used in combination with EB6 or 11PB6 (anti-KIR2DL1/S1 and anti-KIR2DL3*005), 143211 (anti-KIR2DL1/S5), and HP-MA4 (anti-KIR2DL1/S1/S3/S5) mAbs, allowing the accurate identification of different KIR+ NK cell subsets. These phenotypic evaluations appeared useful to dissect the expression pattern of various KIR2D in NK cells from KIR2DL3*005+ individuals, particularly if KIR2DS1 is present. HP-DM1 mAb remarkably refined NK cell phenotyping of donors carrying KIR2DS5, either in the centromeric or telomeric region. Functional assays with KIR2DL1+ /S1+ /S5+ NK cells confirmed that only HP-DM1 exclusively reacts with KIR2DL1. Finally, we demonstrated that HP-DM1 mAb blocked KIR2DL1 recognition of C2+ HLA-C. Altogether, the data support that HP-DM1 is a unique reagent valuable for characterizing KIR+ NK cell subsets.This work was supported by a network grant of the European Commission (H2020-MSCA-ITN-765104-MATURE-NK) to Miguel Lopez-Botet and Daniela Pende; Natalia Colomar-Carando and Anna Rea are fellows in the project. This work was also supported by Italian Ministry of Health (Ricerca Corrente G. Gaslini, Ricerca Corrente and 5xmille-2016 Ospedale Policlinico San Martino), Fondazione AIRC 5 per mille 2018 id 21147 to Franco Locatelli and Lorenzo Moretta

Donor selection for adoptive immunotherapy with NK cells in AML patients: Comparison between analysis of lytic NK cell clones and phenotypical identification of alloreactive NK cell repertoire

Natural killer (NK) cell-based adoptive immunotherapy in leukemia patients is an emerging field of interest based on clinical evidence of efficacy and safety. Elderly acute myeloid leukemia (AML) patients have been successfully treated with NK cells from HLA-haploidentical donors, especially when high amounts of alloreactive NK cells were infused. The aim of this study was comparing two approaches to define the size of alloreactive NK cells in haploidentical donors for AML patients recruited in two clinical trials with the acronym "NK-AML" (NCT03955848), and "MRD-NK". The standard methodology was based on the frequency of NK cell clones capable of lysing the related patient-derived cells. The alternative approach consisted of the phenotypic identification of freshly derived NK cells expressing, as inhibitory receptors, only the inhibitory KIR(s) specific for the mismatched KIR-Ligand(s) (HLA-C1, HLA-C2, HLA-Bw4). However, in KIR2DS2(+) donors and HLA-C1(+) patients, the unavailability of reagents staining only the inhibitory counterpart (KIR2DL2/L3) may lead to an underestimated identification of the alloreactive NK cell subset. Conversely, in the case of HLA-C1 mismatch, the alloreactive NK cell subset could be overestimated due to the ability of KIR2DL2/L3 to recognize with low-affinity also HLA-C2. Especially in this context, the additional exclusion of LIR1-expressing cells might be relevant to refine the size of the alloreactive NK cell subset. We could also associate degranulation assays, using as effector cells IL-2 activated donor peripheral blood mononuclear cells (PBMC) or NK cells upon co-culture with the related patient target cells. The donor alloreactive NK cell subset always displayed the highest functional activity, confirming its identification accuracy by flow cytometry. Despite the phenotypic limitations and considering the proposed corrective actions, a good correlation was shown by the comparison of the two investigated approaches. In addition, the characterization of receptor expression on a fraction of NK cell clones revealed expected but also few unexpected patterns. Thus, in most instances, the quantification of phenotypically defined alloreactive NK cells from PBMC can provide data similar to the analysis of lytic clones, with several advantages, such as a shorter time to achieve the results and, perhaps, higher reproducibility/feasibility in many laboratories

Phenotypic and Functional Characterization of NK Cells in αβT-Cell and B-Cell Depleted Haplo-HSCT to Cure Pediatric Patients with Acute Leukemia

NK cells can exert remarkable graft-versus-leukemia (GvL) effect in HLA-haploidentical hematopoietic stem cell transplantation (haplo-HSCT). Here, we dissected the NK-cell repertoire of 80 pediatric acute leukemia patients previously reported to have an excellent clinical outcome after αβT/B-depleted haplo-HSCT. This graft manipulation strategy allows the co-infusion of mature immune cells, mainly NK and γδT cells, and hematopoietic stem cells (HSCs). To promote NK-cell based antileukemia activity, 36/80 patients were transplanted with an NK alloreactive donor, defined according to the KIR/KIR-Ligand mismatch in the graft-versus-host direction. The analysis of the reconstituted NK-cell repertoire in these patients showed relatively high proportions of mature and functional KIR+NKG2A−CD57+ NK cells, including the alloreactive NK cell subset, one month after HSCT. Thus, the NK cells adoptively transfused with the graft persist as a mature source of effector cells while new NK cells differentiate from the donor HSCs. Notably, the alloreactive NK cell subset was endowed with the highest anti-leukemia activity and its size in the reconstituted repertoire could be influenced by human cytomegalovirus (HCMV) reactivation. While the phenotypic pattern of donor NK cells did not impact on post-transplant HCMV reactivation, in the recipients, HCMV infection/reactivation fostered a more differentiated NK-cell phenotype. In this cohort, no significant correlation between differentiated NK cells and relapse-free survival was observed