Natural killer cells are the first lymphocyte population to reconstitute early after non-myeloablative and T cell-replete haploidentical hematopoietic stem cell transplantation with post-transplant infusion of cyclophosphamide. The study herein characterizes the transient and predominant expansion starting from the second week following haploidentical hematopoietic stem cell transplantation of a donor-derived unconventional subset of NKp46neg-low/CD56dim/CD16neg natural killer cells expressing remarkably high levels of CD94/NKG2A. Both transcription and phenotypic profiles indicated that unconventional NKp46neg-low/CD56dim/CD16neg cells are a distinct natural killer cell subpopulation with features of late stage differentiation, yet retaining proliferative capability and functional plasticity to generate conventional NKp46pos/CD56bright/CD16neg-low cells in response to interleukin-15 plus interleukin-18. While present at low frequency in healthy donors, unconventional NKp46neg-low/CD56dim/CD16neg cells are greatly expanded in the seven weeks following haploidentical hematopoietic stem cell transplantation, and express high levels of the activating receptors NKG2D and NKp30 as well as of the lytic granules Granzyme-B and Perforin. Nonetheless, NKp46neg-low/CD56dim/CD16neg cells displayed a markedly defective cytotoxicity that could be reversed by blocking the inhibitory receptor CD94/NKG2A. These data open new and important perspectives to better understand the ontogenesis/homeostasis of human natural killer cells and to develop a novel immune-therapeutic approach that targets the inhibitory NKG2A check-point, thus unleashing natural killer cell alloreactivity early after haploidentical hematopoietic stem cell transplantation.

Haploidentical hematopoietic stem cell transplantation (h-HSCT) represents an efficient curative approach for patients affected by hematologic malignancies in which the reduced intensity conditioning induces a state of immunologic tolerance between donor and recipient. However, opportunistic viral infections greatly affect h-HSCT clinical outcomes. Natural Killer (NK) cells are the first lymphocytes recovering after transplant and provide a prompt defense against human Cytomegalovirus (HCMV) infection/reactivation. By undertaking a longitudinal single cell computational profiling of multiparametric flow cytometry, we show that HCMV accelerates NK cell immune-reconstitution together with the expansion of CD158b1b2j pos /NKG2A neg /NKG2C pos /NKp30 low NK cells. The frequency of this subset correlates with HCMV viremia, further increases in recipients experiencing multiple episodes of viral reactivations and persists for months after the infection. The transcriptional profile of FACS-sorted CD158b1b2j pos NK cells confirmed the ability of HCMV to de-regulate NKG2C, NKG2A and NKp30 gene expression, thus inducing the expansion of NK cells with adaptive traits.These NK cells are characterized by the down-modulation of several gene pathways associated with cell migration, cell-cycle, effector-functions and by a state of metabolic/cellular exhaustion. This profile reflects the functional impairments of adaptive NK cells to produce IFN-, a phenomenon also due to the viral-induced expression of LAG-3 and PD-1 checkpoint-inhibitors.

Natural Killer (NK) cells are innate immune cells with a primary role in the immune surveillance against non‐self‐cells. NK cell recognition of “self” relies on the surface expression on autologous cells of MHC class I (MHC‐I) molecules. Either the absence or the down‐modulation of MHC‐I on target cells “license” NK cells to kill threatening tumor‐transformed or virally infected cells. This phenomenon is controlled by a limited repertoire of activating and inhibitory NK receptors (aNKRs and iNKRs) that tunes NK cell activation and effector functions. Hence, the calibration of NK cell alloreactivity depends on the ability of iNKRs to bind MHC‐I complex and these interactions are key in regulating both NK cell differentiation and effector functions. Indeed, the presence of iNKRs specific for self‐MHC haplotypes (i) plays a role in the “licensing/education” process that controls the responsiveness of mature NK cells and prevents their activation against the “self” and (ii) is exploited by tumor cells to escape from NK cell cytotoxicity. Herein, we review our current knowledge on function and clinical application of NKG2A, a C‐type lectin iNKR that binds specific haplotypes of human leukocyte antigens early during the NK cell maturation process, thus contributing to modulate the terminal maturation of NK cells as potent effectors against cancers cells. These NKG2A‐mediated mechanisms are currently being exploited for developing promising immune‐therapeutic strategies to improve the prognosis of solid and blood tumors and to ameliorate the clinical outcome of patients undergone allogeneic hematopoietic stem cell transplantation to treat high‐risk hematologic malignancies.

Immune checkpoints refer to a plethora of inhibitory pathways of the immune system that play a crucial role in maintaining self-tolerance and in tuning the duration and amplitude of physiological immune responses to minimize collateral tissue damages. The breakdown of this delicate balance leads to pathological conditions, including cancer. Indeed, tumor cells can develop multiple mechanisms to escape from immune system defense, including the activation of immune checkpoint pathways. The development of monoclonal antibodies, targeting inhibitory immune checkpoints, has provided an immense breakthrough in cancer therapy. Immune checkpoint inhibitors (ICI), initially developed to reverse functional exhaustion in T cells, recently emerged as important actors in natural killer (NK)-cell-based immunotherapy. Moreover, the discovery that also helper innate lymphoid cells (ILCs) express inhibitory immune checkpoints, suggests that these molecules might be targeted on ILCs, to modulate their functions in the tumor microenvironment. Recently, other strategies to achieve immune checkpoint blockade have been developed, including miRNA exploiting systems. Herein, we provide an overview of the current knowledge on inhibitory immune checkpoints on NK cells and ILCs and we discuss how to target these innate lymphocytes by ICI in both solid tumors and hematological malignancies.