Fakhri Hassouneh

Aging remodels antiviral immunity, yet its influence on responses to repeated mRNA vaccination is not fully defined. We evaluated humoral and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike–specific T-cell responses in 41 adults—stratified by age (<50 vs. ≥60 years), sex, prior SARS-CoV-2 infection, and cytomegalovirus (CMV) serostatus—before and after a fourth dose of the bivalent BNT162b2 vaccine. Anti-RBD IgG titers increased in nearly all participants, with no measurable impact of age, sex, infection history, or CMV status, and baseline titers predicted post-booster antibody levels. In contrast, cellular immunity showed clear heterogeneity across aging-related variables. Although the booster enhanced IFN-γ production and reduced TNF-α-associated inflammatory activity at the cohort level, older adults and males exhibited significantly lower post-boost frequencies of IFN-γ–producing CD4+ T cells. Prior SARS-CoV-2 infection was associated with attenuated CD4+ recall responses, whereas infection-naïve and female participants showed the strongest functional gains. Immunosenescence markers were associated with reduced cellular responsiveness. CMV-related immune remodeling—including higher anti-CMV IgG levels and expansions of differentiated CD8+ subsets—correlated with diminished IFN-γ responses in CD4+ and CD8+ T cells after boosting, suggesting that chronic CMV imprinting constrains heterologous antiviral immunity even in mid-adult life. Humoral and cellular changes were largely uncoupled, supporting the need to evaluate both arms of adaptive immunity. These findings indicate that while a fourth bivalent BNT162b2 dose reliably reinforces humoral immunity across ages, the magnitude and quality of cellular responses are shaped by age, sex, infection history, and CMV-associated immunosenescence. Incorporating immune-aging markers into vaccination strategies may improve booster efficacy in older populations.

The family of Nectin and Nectin-like molecules (Necls) and their immunoreceptors are essential in the immune response against cancer. While primarily involved in cell adhesion, motility, and proliferation, some Nectin members also serve as ligands for both stimulatory and inhibitory immune receptors, influencing immune responses. The Nectin/Necls and their receptors form a complex regulatory axis crucial for natural killer (NK) and T cell activity. The most relevant ligands in this axis are the Nectin CD112 and the Nectin-like molecule CD155. Both are ligands for the activating receptor DNAX accessory molecule-1 (DNAM-1) and the inhibitory receptor T cell immunoreceptor with Ig and ITIM domains (TIGIT). Additionally, CD155 is a ligand for T cell activation induced late expression (TACTILE), and CD112 for poliovirus receptor related immunoglobulin domain containing (PVRIG), both inhibitory receptors. These ligands bind with higher affinity to their inhibitory receptors than to the activating receptor DNAM-1. Recent advances in cancer immunotherapy have focused on identifying new targets to overcome tumor resistance. Antibody-mediated blockade of inhibitory receptors such as TIGIT, TACTILE, and PVRIG on NK and T cells has shown promising results in animal models and clinical trials, helping to break immune tolerance. Aging significantly increases cancer risk and contributes to the deterioration of the immune response, impairing cancer immune surveillance. Aging is also associated with an altered tumor microenvironment, reducing immune cell infiltration and promoting tumor progression. In elderly cancer patients, both immunosenescence and cancer-related immunosuppression contribute to a defective immune response against tumor cells. Thus, cancer immunotherapy strategies should be personalized to overcome the dysfunctional immune responses in elderly cancer patients. Understanding these interactions, including ligand-receptor binding mechanisms, is vital for developing effective immunotherapies. Since cancer predominantly affects older adults, further research is needed to explore how aging impacts Nectin-receptor interactions and to tailor immunotherapy strategies for elderly patients.

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.

Acute myeloid leukemia (AML) is the most common type of leukemia in adults. Despite advances in treatment, the average survival rate after diagnosis is low. AML also alters the patient's immune response further contributing to disease progression. The aim of this study was to analyze the expression of LAG-3, TIM-3 and PD-1 inhibitory checkpoints on NK cells and T cells from newly diagnosed AML patients and its impact in patient survival. NK cells and T cells from AML patients showed a lower expression of TIM-3 compared with healthy donors, whereas no significant differences were observed in the expression of LAG-3. The percentages of cells co-expressing these receptors showed a decrease in the percentage of LAG-3 + TIM-3 + PD-1 − NK and T cells from AML patients in comparison with healthy donors. Remarkably, the survival analysis of AML patients according to the expression of these receptors showed that higher expression of LAG-3 on NK cells and T cells was associated with better survival. In addition, those AML patients showing higher co-expression of LAG-3 and TIM-3 on NK and T cells had better survival (higher than 6 months) than those with lower co-expression of LAG-3 and TIM-3, therefore supporting that the expansion of NK and T cells with lower expression of these checkpoint receptors reflects a dysfunctional state associated with poor prognosis. This study identifies the expression of LAG-3 and TIM-3 checkpoints on NK and T cells as potential biomarkers of AML prognosis, contributing to define those patients that could benefit from checkpoint blockade therapies.

CAR-T cell therapy has revolutionized cancer treatment. However, despite the achievements of this approach, there are still clinical challenges to address, such as antigen loss and the design of an optimal CAR structure. Multi-targeted CAR-T therapies, including tandem CAR-T cells, have emerged as a strategy to overcome some of these limitations and improve outcomes. Tandem CAR-T cells are currently being evaluated in preclinical and clinical studies for the treatment of hematological malignancies and solid tumors, showing promising results. These CARs have demonstrated efficacy, safety, and a relatively low relapse rate in these studies. Research suggests that TanCAR-T cells can enhance the outcomes and benefits of CAR-T cell therapy. However, challenges such as identifying the ideal CAR construct, selecting appropriate targets, and improving transduction efficiency remain unresolved, and further research is essential to address these limitations. This review highlights the potential of tandem CAR-T cells as a cancer treatment, summarizing preclinical and clinical studies with this innovative therapy and emphasizing the importance of continued research to overcome its limitations and improve its effectiveness.

Human cytomegalovirus (HCMV) is linked to age-related diseases like cardiovascular disease, neurodegenerative conditions, and cancer. It can also cause congenital defects and severe illness in immunocompromised individuals. Accurate HCMV seroprevalence assessment is essential for public health planning and identifying at-risk individuals. This is the first HCMV seroprevalence study conducted in the general Spanish adult population in 30 years. We studied HCMV seroprevalence and HCMV IgG antibody titres in healthy adult donors (HDs) and HCMV-related disease patients from 2010 to 2013 and 2020 to 2023, categorized by sex and age. We compared our data with 1993 and 1999 studies in Spain. The current HCMV seroprevalence among HDs in Spain is 73.48%. In women of childbearing age, HCMV seroprevalence has increased 1.4-fold in the last decade. HCMV-seropositive individuals comprise 89.83% of CVD patients, 69% of SMI patients, and 70.37% of COVID-19 patients. No differences in HCMV seroprevalence or HCMV IgG antibody titres were observed between patients and HDs. A significant reduction in Spanish HCMV seroprevalence among HDs was observed in 1993. However, women of childbearing age have shown an upturn in the last decade that may denote a health risk in newborns and a change in HCMV seroprevalence trends.

Introduction: Natural 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.Methods: In 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.Results: Our 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.Discussion: In 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.

Cytomegalovirus (CMV) infection is a major driver of accelerated immunosenescence related to CD28^null T cell expansion. CMV infection and these proatherogenic T cells have been independently associated with cardiovascular disease and coronavirus disease 2019 (COVID-19) severity. We investigated the potential contribution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) to immunosenescence and its relationship with CMV. Innate and adaptive immune subpopulations from individuals with mild or asymptomatic SARS-CoV-2 infection (mCOVID-19) and healthy donors were immunophenotyped. A significant increase in CD28^nullCD57⁺CX3CR1⁺ T cell percentages (CD4⁺ [P ≤ .01], CD8⁺ [P ≤ .01], and TcRγδ (CD4⁻CD8⁻) [P ≤ .001]) was found in unnvaccinated CMV-seropositive mCOVID-19 individuals stable up to 12 months after infection. This expansion did not occur in CMV-seronegative mCOVID-19 individuals or in CMV-seropositive individuals infected after SARS-CoV-2 vaccination. There were no significant differences between mCOVID-19 and aortic stenosis groups. Thus, individuals coinfected with SARS-CoV-2 and CMV have accelerated T cell senescence, which might lead to an increased risk of cardiovascular disease.

Aortic stenosis (AS) is a frequent cardiac disease in old individuals, characterized by valvular calcification, fibrosis, and inflammation. Recent studies suggest that AS is an active inflammatory atherosclerotic-like process. Particularly, it has been suggested that several immune cell types, present in the valve infiltrate, contribute to its degeneration and to the progression toward stenosis. Furthermore, the infiltrating T cell subpopulations mainly consist of oligoclonal expansions, probably specific for persistent antigens. Thus, the characterization of the cells implicated in the aortic valve calcification and the analysis of the antigens to which those cells respond to is of utmost importance to develop new therapies alternative to the replacement of the valve itself. However, calcified aortic valves have been only studied so far by histological and immunohistochemical methods, unable to render an in-depth phenotypical and functional cell profiling. Here we present, for the first time, a simple and efficient cytometry-based protocol that allows the identification and quantification of infiltrating inflammatory leukocytes in aortic valve explants. Our cytometry protocol saves time and facilitates the simultaneous analysis of numerous surface and intracellular cell markers and may well be also applied to the study of other cardiac diseases with an inflammatory component.

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.