Mariella Della Chiesa

Despite growing enthusiasm for immunotherapy in cancer treatment, only a few colorectal cancer (CRC) patients experience lasting therapeutic effect. This limited efficacy is likely driven by different tumor-intrinsic resistance mechanisms, including loss or downregulation of HLA-I molecules, which may impair antigen presentation and cytotoxic T cell recognition. Interestingly, the downregulation of HLA-I can augment tumor susceptibility to natural killer (NK) cell attack. However, NK cell activity may be inhibited by immunosuppressive signals within the tumor microenvironment. This study describes two patients with metastatic CRC and microsatellite instability who showed durable responses to pembrolizumab despite unfavorable prognostic factors (tumor recurrence, BRAF V600E mutation and markedly reduced HLA-I on tumor cells). Tumor samples obtained at surgical resection also revealed PD-L1 expression on tumor cells and a substantial fraction of tumor-infiltrating PD-1 ⁺ NK cells. These findings raise the hypothesis that PD-1/PD-L1 blockade may have contributed to NK-cell-mediated antitumor activity in the context of markedly reduced HLA-I expression. Furthermore, assessing PD-L1 and HLA-I expression on tumor cells at diagnosis could help identify patients who could benefit most from pembrolizumab treatment, alone or combined with inhibitors of HLA-I-specific inhibitory NK receptors. These observations provide a rationale for further investigation in larger and multicenter patient cohorts.

Natural killer (NK) cells are uniquely equipped to eliminate transformed cells without prior antigen sensitization, yet their therapeutic potential in solid tumors remains only partially realized. At the center of this paradox lies a complex network of inhibitory pathways, dominated by killer cell immunoglobulin-like receptors (KIRs) and the CD94/NKG2A axis, which continuously calibrate NK-cell self-tolerance and effector competence. Tumors exploit these regulatory circuits through dynamic remodeling of HLA-I expression: while loss of classical HLA-I impairs CD8 ⁺ T-cell recognition, preservation or upregulation of non-classical HLA, particularly HLA-E, sustains inhibitory signaling and promotes immune escape. These mechanisms are further amplified by the tumor microenvironment (TME), where stromal barriers, hypoxia, metabolic stress, and immunosuppressive networks collectively restrict NK-cell infiltration, persistence, and cytotoxicity. Such multilayered suppression helps explain why therapeutic blockade of KIR or NKG2A alone has yielded only modest clinical benefit in most solid tumors, despite compelling biological rationale. Emerging evidence suggests that the KIR- and CD94/NKG2-centered network should be viewed not only as a therapeutic target but also as a framework for the next-generation of NK-cell-based immunotherapies. Future strategies will likely combine checkpoint modulation with donor- and patient-tailored NK-cell selection, engineered NK-cell products with enhanced metabolic resilience and reduced checkpoint sensitivity, and interventions aimed at remodeling the tumor niche to restore trafficking, persistence, and functional fitness. In this mini-review, we discuss how KIR- and CD94/NKG2-mediated signaling is shaped by the TME and examine emerging combinatorial and personalized approaches designed to unlock the full therapeutic potential of NK cells in solid tumors.

Background: Natural killer (NK) cells are innate lymphocytes endowed with potent cytotoxic activity. The presence of tumor-associated NK cells has been correlated with better prognosis in several solid tumors including colorectal cancer (CRC). This malignant disease is the second cause of cancer death worldwide and is in urgent need for novel approaches to improve current immunotherapies. Since CRC microenvironment can induce NK cell dysfunction and hinder cancer control, understanding tumor-associated NK cell features is mandatory to fully unlock their immunotherapeutic potential.Purpose: Our study aims at elucidating the molecular and functional characteristics of tumor-associated NK cells in CRC focusing on the expression of immune checkpoints that critically regulate NK cell function. We performed an in-depth cytofluorimetric analysis of tumor-associated NK cells obtained by tissue dissociation of samples derived from 80 CRC patients comparing tumor with matched tumor-free tissue and peripheral blood, stratifying patients by tumor stage or MSI/MSS condition. Tumor tissue was also analyzed by immunohistochemistry.Results: NK cells expressing immune checkpoints (i.e., KIR, NKG2A and TIM-3) were significantly enriched in tumor compared to tumor-free tissue, and an increase in PD-1⁺ NK cells was observed in tumors compared to peripheral blood and tumor-free tissue, indicating TME-induced modulation. Notably, tumor-associated PD-1⁺ NK cells characterized MSI rather than MSS CRC. In addition, tumor-associated NK cells also expressed tissue residency markers (CD103 and/or CD49a) and displayed a distinct profile also including the PD-1⁺ NK cell subset in MSI CRC, possibly representing NK cells recruited from circulation, retained in tumors, and reconfigured by TME signals. Importantly, tissue resident NK cells adequately expressed activating NK receptors and cytotoxic molecules.Conclusions: These results suggest, together with an increased PD-L1 expression on MSI tumor cells, that the efficacy of immunotherapies in MSI CRC based on PD-1/PD-L1 blockade could also rely on a superior anti-tumor potential of PD-1⁺ NK cells. Conversely, MSS CRC, in which tumor-associated PD-1⁺ NK cells are scarce, could benefit more from immunotherapies blocking NKG2A and/or KIRs. Thus, novel approaches based on NK cell features related to CRC type, fully exploiting circulating and resident NK cell anti-tumor activity, could be key to next-generation therapies.

Background: Adaptive human natural killer (NK) cells are an NK cell subpopulation arising upon cytomegalovirus (CMV) infection. They are characterized by CD94/NKG2C expression, a mature CD57⁺KIR⁺NKG2A^– phenotype, a prolonged lifespan, and remarkable antitumor functions. In light of these features, adaptive NK cells represent suitable candidate to design next-generation therapies, based on their enhanced effector function which could be further boosted by Chimeric Antigen Receptors-engineering, or the combination with cell engagers. For therapeutic approaches, however, it is key to generate large numbers of functional cells.Purpose: We developed a method to efficiently expand adaptive NK cells from NK-enriched cell preparations derived from the peripheral blood of selected CMV-seropositive healthy donors. The method is based on the use of an anti-CD94 monoclonal antibody (mAb) combined with IL-2 or IL-15.Results: By setting this method we were able to expand high numbers of NK cells showing the typical adaptive phenotype, CD94/NKG2C⁺ CD94/NKG2A^- CD57⁺, and expressing a single self-inhibitory KIR. Expanded cells maintained the CMV-induced molecular signature, exhibited high ADCC capabilities and degranulation against a HLA-E⁺ target. Importantly, mAb-expanded adaptive NK cells did not upregulate PD-1 or other regulatory immune checkpoints that could dampen their function.Conclusions: By this study we provide hints to improve previous expansion methods, by eliminating the use of genetically modified cells as stimulators, and obtaining effectors not expressing unwanted inhibitory receptors. This new protocol for expanding functional adaptive NK cells is safe, cost-effective and easily implementable in a GMP context, suitable for innovative immunotherapeutic purposes.

High-risk neuroblastomas (HR-NB) still have an unacceptable 5-year overall survival despite the aggressive therapy. This includes standardized immunotherapy combining autologous hemopoietic stem cell transplantation (HSCT) and the anti-GD2 mAb. The treatment did not significantly change for more than one decade, apart from the abandonment of IL-2, which demonstrated unacceptable toxicity. Of note, immunotherapy is a promising therapeutic option in cancer and could be optimized by several strategies. These include the HLA-haploidentical αβT/B-depleted HSCT, and the antibody targeting of novel NB-associated antigens such as B7-H3, and PD1. Other approaches could limit the immunoregulatory role of tumor-derived exosomes and potentiate the low antibody-dependent cell cytotoxicity of CD16 dim/neg NK cells, abundant in the early phase post-transplant. The latter effect could be obtained using multi-specific tools engaging activating NK receptors and tumor antigens, and possibly holding immunostimulatory cytokines in their construct. Finally, treatments also consider the infusion of novel engineered cytokines with scarce side effects, and cell effectors engineered with chimeric antigen receptors (CARs). Our review aims to discuss several promising strategies that could be successfully exploited to potentiate the NK-mediated surveillance of neuroblastoma, particularly in the HSCT setting. Many of these approaches are safe, feasible, and effective at pre-clinical and clinical levels.

Human Natural Killer (NK) cells are all round players in immunity thanks to their powerful and immediate response against transformed cells and the ability to modulate the subsequent adaptive immune response. The potential of immunotherapies based on NK cell involvement has been initially revealed in the hematological setting but has inspired the design of different immune tools to also be applied against solid tumors, including colorectal cancer (CRC). Indeed, despite cancer prevention screening plans, surgery, and chemotherapy strategies, CRC is one of the most widespread cancers and with the highest mortality rate. Therefore, further efficient and complementary immune-based therapies are in urgent need. In this review, we gathered the most recent advances in NK cell-based immunotherapies aimed at fighting CRC, in particular, the use of monoclonal antibodies targeting tumor-associated antigens (TAAs), immune checkpoint blockade, and adoptive NK cell therapy, including NK cells modified with chimeric antigen receptor (CAR-NK).

Human NK cells can control tumor growth and metastatic spread thanks to their powerful cytolytic activity which relies on the expression of an array of activating receptors. Natural cytotoxicity receptors (NCRs) NKG2D and DNAM-1 are those non-HLA-specific activating NK receptors that are mainly involved in sensing tumor transformation by the recognition of different ligands, often stress-induced molecules, on the surface of cancer cells. Tumors display several mechanisms aimed at dampening/evading NK-mediated responses, a relevant fraction of which is based on the downregulation of the expression of activating receptors and/or their ligands. In this review, we summarize the role of the main non-HLA-specific activating NK receptors, NCRs, NKG2D and DNAM-1, in controlling tumor growth and metastatic spread in solid malignancies affecting the gastrointestinal tract with high incidence in the world population, i.e., pancreatic ductal adenocarcinoma (PDAC), colorectal cancer (CRC), and gastric cancer (GC), also describing the phenotypic and functional alterations induced on NK cells by their tumor microenvironment.

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.

Natural killer (NK) lymphocytes are an integral component of the innate immune system and represent important effector cells in cancer immunotherapy, particularly in the control of hematological malignancies. Refined knowledge of NK cellular and molecular biology has fueled the interest in NK cell-based antitumor therapies, and recent efforts have been made to exploit the high potential of these cells in clinical practice. Infusion of high numbers of mature NK cells through the novel graft manipulation based on the selective depletion of T cells and CD19⁺ B cells has resulted into an improved outcome in children with acute leukemia given human leucocyte antigen (HLA)-haploidentical hematopoietic transplantation. Likewise, adoptive transfer of purified third-party NK cells showed promising results in patients with myeloid malignancies. Strategies based on the use of cytokines or monoclonal antibodies able to induce and optimize NK cell activation, persistence, and expansion also represent a novel field of investigation with remarkable perspectives of favorably impacting on outcome of patients with hematological neoplasia. In addition, preliminary results suggest that engineering of mature NK cells through chimeric antigen receptor (CAR) constructs deserve further investigation, with the goal of obtaining an “off-the-shelf” NK cell bank that may serve many different recipients for granting an efficient antileukemia activity.