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A better understanding of the complex interactions between the immune system and tumour cells from different origins has opened the possibility to design novel procedures of antitumoral immunotherapy. One of these novel approaches is based on the use of autologous or allogeneic natural killer (NK) cells to treat cancer. In the last decade, different strategies to activate NK cells and their use in adoptive NK cell-based therapy have been established. Although NK cells are often considered as a uniform cell population, several phenotypic and functionally distinct NK cells subsets exist in healthy individuals, that are differentially affected by ageing or by apparently innocuous viruses such as cytomegalovirus (CMV). In addition, further alterations in the expression of activating and inhibitory receptors are found in NK cells from cancer patients, likely because of their interaction with tumour cells. Thus, NK cells represent a promising strategy for adoptive immunotherapy of cancer already tested in phase 1/2 clinical trials. However, the existence of NK cell subpopulations expressing different patterns of activating and inhibitory receptors and different functional capacities, that can be found to be altered not only in cancer patients but also in healthy individuals stratified by age or CMV infection, makes necessary a personalized definition of the procedures used in the selection, expansion, and activation of the relevant NK cell subsets to be successfully used in NK cell-based immunotherapy.

NK cells exhibit the highest cytotoxic capacity within the immune system. Alteration of their number or functionality may have a deep impact on overall immunity. This is of particular relevance in aging where the elderly population becomes more susceptible to infection, cancer, autoimmune diseases, and neurodegenerative diseases amongst others. As the fraction of elderly increases worldwide, it becomes urgent to better understand the aging of the immune system to prevent and cure the elderly population. For this, a better understanding of the function and phenotype of the different immune cells and their subsets is necessary. We review here NK cell functions and phenotype in healthy aging as well as in various age-associated diseases.

In recent years, the use of immune checkpoint inhibitors (ICIs) in combination with approved or experimental vaccines has proven to be a promising approach to improve vaccine immunogenicity and efficacy. This strategy seeks to overcome the immunosuppressive mechanisms associated with the vaccine response, thereby achieving increased immunogenicity and efficacy. Most of the information on the use of ICIs combined with vaccines derives from studies on certain anti-tumor vaccines combined with monoclonal antibodies (mAbs) against either cytotoxic T lymphocyte-associated protein 4 (CTLA-4), programmed cell death protein 1 (PD-1), or programmed death-ligand 1 (PD-L1). However, over the past few years, emerging strategies to use new-generation ICIs as molecular adjuvants are paving the way for future advances in vaccine research. Here, we review the current state and future directions of the use of ICIs in experimental and clinical settings, including mAbs and alternative new approaches using antisense oligonucleotides (ASOs), small non-coding RNAs, aptamers, peptides, and other small molecules for improving vaccine efficacy. The scope of this review mainly includes the use of ICIs in therapeutic antitumor vaccines, although recent research on anti-infective vaccines will also be addressed.

Natural killer (NK) cells are lymphocytes of the innate immune response characterized by their role in the destruction of tumor cells. Activation of NK cells depend on a fine balance between activating and inhibitory signals mediated by different receptors. In recent years, a family of paired receptors that interact with ligands of the Nectin/Nectin-like (Necl) family has attracted great interest. Two of these ligands, Necl-5 (usually termed CD155 or PVR) and Nectin-2 (CD112), frequently expressed on different types of tumor cells, are recognized by a group of receptors expressed on T and NK cells that exert opposite functions after interacting with their ligands. These receptors include DNAM-1 (CD226), TIGIT, TACTILE (CD96) and the recently described PVRIG. Whereas activation through DNAM-1 after recognition of CD155 or CD112 enhances NK cell-mediated cytotoxicity against a wide range of tumor cells, TIGIT recognition of these ligands exerts an inhibitory effect on NK cells by diminishing IFN-γ production, as well as NK cell-mediated cytotoxicity. PVRIG has also been identified as an inhibitory receptor that recognizes CD112 but not CD155. However, little is known about the role of TACTILE as modulator of immune responses in humans. TACTILE control of tumor growth and metastases has been reported in murine models, and it has been suggested that it negatively regulates the anti-tumor functions mediated by DNAM-1. In NK cells from patients with solid cancer and leukemia, it has been observed a decreased expression of DNAM-1 that may shift the balance in favor to the inhibitory receptors TIGIT or PVRIG, further contributing to the diminished NK cell-mediated cytotoxic capacity observed in these patients. Analysis of DNAM-1, TIGIT, TACTILE and PVRIG on human NK cells from solid cancer or leukemia patients will clarify the role of these receptors in cancer surveillance. Overall, it can be speculated that in cancer patients the TIGIT/PVRIG pathways are upregulated and represent novel targets for checkpoint blockade immunotherapy.

IntroductionNatural killer (NK) cells are a key component of the innate immune system, involved in defending the host against virus-infected cells and tumor immunosurveillance. Under in vitro culture conditions, IL-12/15/18 can induce a memory-like phenotype in NK cells. These cytokine-induced memory-like (CIML) NK cells possess desirable characteristics for immunotherapies, including a longer lifespan and increased cytotoxicity.MethodsIn this study, NK cells were isolated from peripheral blood of healthy donors and stimulated with IL-12/15/18 to induce a memory-like phenotype or with IL-15 alone as a control. After seven days of culture, multiparametric flow cytometry analysis was performed to evaluate the phenotypic and functional profiles of CIML and control NK cells.ResultsOur results showed a significantly higher expression of CD25, CD69, NKG2D, NKp30, NKp44, NKp46, TACTILE, and Granzyme B in CIML NK cells compared to control NK cells. In contrast, KIR2D expression was significantly lower in CIML NK cells than in control NK cells. Moreover, functional experiments demonstrated that CIML NK cells displayed enhanced degranulation capacity and increased intracellular IFN-γ production against the target cell line K562. Interestingly, the degranulation capacity of CIML NK cells was positively correlated with the expression of the activating receptors NKp46 and NKp30, as well as with the inhibitory receptor TACTILE.DiscussionIn conclusion, this study provides a deep phenotypic characterization of in vitro -expanded CIML NK cells. Moreover, the correlations found between NK cell receptors and degranulation capacity of CIML NK cells allowed the identification of several biomarkers that could be useful in clinical settings.

NK cells are innate lymphoid cells that can acquire a memory-like phenotype in vitro when stimulated with IL-12, IL-15, and IL-18. These cytokine-induced memory-like (CIML) NK cells exhibit prolonged lifespan and increased cytotoxicity, making them ideal for immunotherapy. This study characterizes two CIML NK cell subsets based on CD16 expression. NK cells were isolated from the peripheral blood of healthy donors and stimulated overnight to induce a memory-like phenotype. After seven days, we analyzed the phenotype and degranulation potential of CD16−/CD56 + and CD16+/CD56 + cells. The subsets were purified by fluorescence-activated cell sorting (FACS) and examined using high-throughput multiplexed quantitative proteomics. CD16 − cells showed higher levels of activating receptors, increased Granulysin expression, and lower inhibitory receptor expression compared to CD16 + cells. Functionally, CD16 − cells exhibited greater degranulation capacity, as determined by CD107a/b expression, when co-incubated with K562 and melanoma cells. Proteomic profiling identified 35 differentially expressed proteins out of 4,750, with 22 downregulated and 13 upregulated in the CD16 − subset. Key proteins included Granzyme family proteins, NCAM1, CALM1, CD247, and Fc receptors. This study provides a detailed characterization of CIML NK cells based on CD16 expression. Our findings highlight the molecular and functional diversity of CIML NK cells and may guide improved cancer immunotherapy strategies.

Cytomegalovirus (CMV) latent infection has a deleterious effect on the efficacy of influenza vaccination in the elderly, suggesting that CMV restricts immunological diversity impairing the immune system functionality in old age. Polyfunctional T cells produce multiple cytokines and higher amounts than mono-functional T cells. High number of polyfunctional T cells correlates with better prognosis during infection. Thus, the efficiency of T cell response associates with quality (polyfunctionality) rather than with quantity (percentage of T cells). We analyze the effect of CMV infection on CD8+ T cells polyfunctionality --degranulation (CD107a), IFN-gamma and TNF-alpha production--, from young CMV-seropositive and CMV-seronegative individuals and in middle age CMV-seropositive donors, in response to Staphylococcal Enterotoxin B (SEB). Our results show a higher percentage of polyfunctional CD8+ T cells in young CMV-seropositive individuals compared to CMV-seronegative. Also, we find an expansion of CD8+CD57+ T cells in CMV-seropositive individuals, which are more polyfunctional than CD8+CD57- cells. In middle age individuals there is a higher frequency of SEB-responding CD8+ T cells, mainly TNF-alpha or TNF-alpha/IFN-gamma producers, whereas the percentage of polyfunctional cells (IFN-gamma/TNF-alpha/CD107a) is similar to the percentages found in young CMV-seropositive. Therefore, whereas it has been shown that CMV latent infection can be detrimental for immune response in old individuals, our results indicate that CMV-seropositivity is associated to higher levels of polyfunctional CD8+ T cells in young and middle age donors. This increase in polyfunctionality, which can provide an immunological advantage in the response to other pathogens, is due to a CD8+CD57+ T cell expansion in CMV-seropositive individuals and it is independent of age. Conversely, age could contribute to the inflammation found in old individuals by increasing the percentage of cells producing pro-inflammatory cytokines. These findings highlight the necessity of further studies on the benefits/detrimental effects of CMV infection in the response to vaccination and other infections.

The impact of biological sex on T-cell immunity to Cytomegalovirus (CMV) has not been investigated in detail with only one published study comparing CMV-specific T-cell responses in men and women. Many studies, however, have shown an association between CMV infection and immunosenescence, with broad effects on peripheral blood lymphocyte subsets as well as the T and B-cell repertoires. Here, we provide a detailed analysis of CMV-specific T-cell responses in (n=94) CMV+ older people, including 47 women and 47 men aged between 60 and 93 years. We explore sex differences with respect to 16 different CMV proteins arranged in 14 peptide pools (overlapping peptides). Following ex vivo stimulation, CD4 and CD8 T-cells producing IFN-γ, TNF, and IL-2 were enumerated by flow-cytometry (intracellular cytokine staining). T-cell responses were evaluated in terms of each cytokine separately or in terms of cytokines produced simultaneously (polyfunctionality). Surface memory phenotype and CD3 downmodulation were assessed in parallel. The polyfunctionality index and a memory subset differentiation score were used to identify associations between response size, cytokine production, polyfunctionality, and memory subset distribution. While no significant sex differences were found with respect to overall CMV target protein selection, the T-cell response in men appeared more focused and accompanied by a more prominent accumulation of CMV-specific memory CD4 and CD8 T-cells. T-cell polyfunctionality and differentiation were similar in the sexes, however, CMV-specific T-cells in men produced more pro-inflammatory cytokines. Particularly, TNF production by CD4 T-cells was stronger in men than in women. Also, compared with women, men had larger responses to CMV proteins with immediate-early/early kinetics than women, which might have been driven by CMV reactivation. In conclusion, the CMV-specific T-cell response in men was larger and more pro-inflammatory than in women. Our findings may help explain sex differences in CMV-associated pathologies.