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The relationship between severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) and host immunity is poorly understood. We performed an extensive analysis of immune responses in 32 patients with severe COVID-19, some of whom succumbed. A control population of healthy subjects was included. Patients with COVID-19 had an altered distribution of peripheral blood lymphocytes, with an increased proportion of mature natural killer (NK) cells and low T-cell numbers. NK cells and CD8+ T cells overexpressed T-cell immunoglobulin and mucin domain-3 (TIM-3) and CD69. NK cell exhaustion was attested by increased frequencies of programmed cell death protein 1 (PD-1) positive cells and reduced frequencies of natural killer group 2 member D (NKG2D)-, DNAX accessory molecule-1 (DNAM-1)- and sialic acid-binding Ig-like lectin 7 (Siglec-7)-expressing NK cells, associated with a reduced ability to secrete interferon (IFN)γ. Patients with poor outcome showed a contraction of immature CD56bright and an expansion of mature CD57+ FcεRIγneg adaptive NK cells compared to survivors. Increased serum levels of IL-6 were also more frequently identified in deceased patients compared to survivors. Of note, monocytes secreted abundant quantities of IL-6, IL-8, and IL-1β which persisted at lower levels several weeks after recovery with concomitant normalization of CD69, PD-1 and TIM-3 expression and restoration of CD8+ T cell numbers. A hyperactivated/exhausted immune response dominate in severe SARS-CoV-2 infection, probably driven by an uncontrolled secretion of inflammatory cytokines by monocytes. These findings unveil a unique immunological profile in COVID-19 patients that will help to design effective stage-specific treatments for this potentially deadly disease.

Hepatocellular carcinoma (HCC) still represents a significant complication of chronic liver disease, particularly when cirrhosis ensues. Current treatment options include surgery, loco-regional procedures and chemotherapy, according to specific clinical practice guidelines. Immunotherapy with check-point inhibitors, aimed at rescuing T-cells from exhaustion, has been applied as second-line therapy with limited and variable success. Natural killer (NK) cells are an essential component of innate immunity against cancer and changes in phenotype and function have been described in patients with HCC, who also show perturbations of NK activating receptor/ligand axes. Here we discuss the current status of NK cell treatment of HCC on the basis of existing evidence and ongoing clinical trials on adoptive transfer of autologous or allogeneic NK cells ex vivo or after activation with cytokines such as IL-15 and use of antibodies to target cell-expressed molecules to promote antibody-dependent cellular cytotoxicity (ADCC). To this end, bi-, tri- and tetra-specific killer cell engagers are being devised to improve NK cell recognition of tumor cells, circumventing tumor immune escape and efficiently targeting NK cells to tumors. Moreover, the exciting technique of chimeric antigen receptor (CAR)-engineered NK cells offers unique opportunities to create CAR-NK with multiple specificities along the experience gained with CAR-T cells with potentially less adverse effects.

Background/Objectives: New SARS-CoV-2 variants are continuously emerging, making it essential to assess the efficacy of vaccine-induced immune protection. Limited information is available regarding T cell responses to BA.2.86 and JN.1 variants, particularly in elderly individuals. Methods: We evaluated T cell and total IgG responses against the receptor-binding domain (RBD) of the ancestral SARS-CoV-2 strain, as well as BA.2.86 and JN.1 omicron subvariants, in two groups of subjects. One group consisted of SARS-CoV-2-exposed elderly individuals who were fully vaccinated with the BNT162B2 mRNA vaccine, with a booster dose of the updated 2023–2024 COVID-19 vaccine (XBB.1.5) at least 15 days after receiving a booster dose of the updated 2023–2024 COVID-19 vaccine. The second group consisted of healthcare workers who were unexposed to SARS-CoV-2 one month after the booster dose of the first-generation BNT162b2 mRNA vaccine. T cell activation-induced markers (AIM) and IFN-γ secretion were evaluated by flow cytometry and ELISpot assays, respectively. Results: Elderly subjects showed reduced IgG levels against JN.1 compared with the ancestral strain. BA.2.86 stimulation resulted in lower IFN-γ levels in the elderly versus the COVID-19-naïve group. AIM analysis showed that among T cells, CD4+ were the most responsive, with a reduced proportion of JN.1-reactive CD4+ T cells compared with the ancestral strain in the SARS-CoV-2-unexposed group. Despite receiving the updated booster, the elderly group showed reduced CD4+ T cell reactivity to BA.2.86. Conclusions: The XBB.1.5-containing vaccine induced lower CD4+ T cell responses against BA.2.86 in the elderly. CD4+ T cells from BNT16b2-vaccinated, COVID-19-naïve subjects recognized ancestral and BA.2.86 RBD strains while showing reduced responses to JN.1. These results emphasize the need for tailored vaccine strategies for emerging variants, particularly in vulnerable populations.

Natural killer (NK) cells are emerging as unique players in the immune response against cancer; however, only limited data are available on tumor infiltrating NK cells in head and neck squamous cell carcinoma (HNSCC), one of the most common cancer. Occurrence of HNSCC is closely related to the immune microenvironment, and immunotherapy is increasingly being applied to this setting. However, the limited success of this type of treatment in this tumor calls for further investigation in the field. Surgical HNSSC specimens of 32 consecutive patients were mechanically and enzymatically dissociated. Tumor cells were separated from infiltrating cells by short centrifugation and infiltrating NK cells were phenotypically and functionally characterized by multiple antibody staining and flow cytometry. Tumor infiltrating NK cells in HNSCC showed a peculiar phenotype predominantly characterized by increased NKG2A and reduced Siglec-7, NKG2D, NKp30 and CD16 expression. This phenotype was associated with a decreased ability to perform antibody-dependent cellular cytotoxicity (ADCC). However, NK, CD4 and CD8 shared an increment of glucocorticoid-induced tumor necrosis factor-related (GITR) costimulatory receptor which could be exploited for immunotherapy with agonistic anti-GITR antibodies combined with checkpoint inhibitors.

Immune control of human cytomegalovirus (HCMV) replication is critical in bone marrow and solid organ transplant recipients, where uncontrolled replication can lead to high mortality. Current commercial immune monitoring tools have several limitations, such as a lack of appropriate test cutoff values and the inability to characterise antigen-specific T cells. The main aim of our study was to develop a new interferon-γ (IFN-γ) release assay (IGRA), easy to use, to quantify and characterise the HCMV-specific T-cell response (pp65-IGRA). Secondary analyses included an evaluation of the performance of pp65-IGRA to assess whether its specificity and sensitivity were equal to or greater than those of the intracellular cytokine staining (ICS) and enzyme-linked immunospot (ELISpot) assays. In the study, 76 immunocompetent donors and nine solid organ transplant recipients were enrolled. Blood samples or peripheral blood mononuclear cells were stimulated with HCMV pp65-recombinant protein or with a complete pool of overlapping pp65 peptides. IFN-γ production was analysed by enzyme-linked immunoassay, ELISpot assays, and flow cytometry. For each assay, appropriate cutoff values were calculated. Our data demonstrate the suitability of pp65-IGRA for the quantification of HCMV-specific CD4 + T-cell responses and may support its use in routine clinical practice to improve the management of immunocompromised patients.

Cholangiocarcinoma (CCA) is a rare cancer characterized by a global increasing incidence. Extracellular vesicles (EV) contribute to many of the hallmarks of cancer through transfer of their cargo molecules. The sphingolipid (SPL) profile of intrahepatic CCA (iCCA)-derived EVs was characterized by liquid chromatography-tandem mass spectrometry analysis. The effect of iCCA-derived EVs as mediators of inflammation was assessed on monocytes by flow cytometry. iCCA-derived EVs showed downregulation of all SPL species. Of note, poorly-differentiated iCCA-derived EVs showed a higher ceramide and dihydroceramide content compared with moderately-differentiated iCCA-derived EVs. Of note, higher dihydroceramide content was associated with vascular invasion. Cancer-derived EVs induced the release of pro-inflammatory cytokines in monocytes. Inhibition of synthesis of ceramide with Myriocin, a specific inhibitor of the serine palmitoyl transferase, reduced the pro-inflammatory activity of iCCA-derived EVs, demonstrating a role for ceramide as mediator of inflammation in iCCA. In conclusion, iCCA-derived EVs may promote iCCA progression by exporting the excess of pro-apoptotic and pro-inflammatory ceramides.

Olfactory and taste disorders (OTD) are commonly found as presenting symptoms of SARS-CoV-2 infection in patients with clinically mild COVID-19. Virus-specific T cells are thought to play an important role in the clearance of SARS-CoV-2; therefore the study of T cell specific immune responses in patients with mild symptoms may help to understand their possible role in protection from severe disease. We evaluated SARS-CoV-2-specific T cell responses to four different peptide megapools covering all SARS-CoV-2 proteins during the acute phase of the disease in 33 individuals with mild or no other symptom beside OTD and in 22 age-matched patients with severe infection. A control group of 15 outpatients with OTD and consistently negative nasopharyngeal SARS-CoV-2 RNA swabs and virus-specific IgG serology was included in the study. Increased frequencies of virus-specific CD4 + and CD8 + T cells were found in SARS-CoV-2 positive patients with OTD compared with those with severe COVID-19 and with SARS-CoV-2 negative OTD individuals. Moreover, enhanced CD4 + and CD8 + T-cell activation induced by SARS-CoV-2 peptides was associated with higher interferon (IFN)γ production. Increased frequencies of Spike (S1/S2)-specific CD4 + T cells showing enhanced IFNγ secretion and granzyme B content were associated with serum spike-specific IgG in the OTD group. In conclusion, patients with SARS-CoV-2 induced OTD develop highly functional virus-specific CD4 + and CD8 + T cells during the symptomatic phase of the disease, suggesting that robust and coordinated T-cell responses provide protection against extension of COVID-19 to the lower respiratory tract.

Despite the availability of an effective prophylactic vaccine leading to sterilizing immunity, hepatitis B virus (HBV) is responsible for chronic liver disease in more than 250 million individuals, potentially leading to cirrhosis and hepatocellular carcinoma. Antiviral drugs able to completely suppress virus replication are indeed available but they are, by and large, unable to eradicate the virus. Several alternative new treatment approaches are currently being developed but none have so far captured the interest of clinicians for possible clinical development. A constant feature of chronic HBV infection is T-cell exhaustion resulting from persistent exposure to high antigen concentrations as shown by the high expression of programmed cell death protein 1 (PD-1) by HBV-specific CD8 T cells. One way of tackling this problem is to develop HBV-specific neutralizing antibodies that would clear excess envelope proteins from the circulation, allowing for nucleos(t)ide analogs or other antiviral drugs now in preclinical and early clinical development to take advantage of a reconstituted adaptive immunity. Several fully human monoclonal antibodies (mAb) have been developed from HBV-vaccinated and subjects convalescent from acute hepatitis B that show different properties and specificities. It is envisaged that such neutralizing mAb may be used as adjuvant treatment to reduce viral protein load, thus rescuing adaptive immunity in an effort to optimize the effect of antiviral drugs.

Natural killer (NK) cells are involved in innate immune responses to viral infections either via direct cytotoxicity which destroys virus-infected cells or production of immunoregulatory cytokines which modulate adaptive immunity and directly inhibit virus replication. These functions are mediated by different NK subpopulations, with cytotoxicity being generally performed by CD56(dim) NK cells, whereas CD56(bright) NK cells are mainly involved in cytokine secretion. NK functional defects are usually combined so that impaired degranulation is often associated with deficient cytokine production. Innate immunity is thought to be relevant in the control of hepatitis virus infections such as hepatitis B virus (HBV) and hepatitis C virus (HCV), and recent findings reproducibly indicate that NK cells in chronic viral hepatitis are characterized by a functional dichotomy, featuring a conserved or enhanced cytotoxicity and a reduced production of interferon (IFN)-γ and tumor necrosis factor-α. In chronic HCV infection this appears to be caused by altered IFN-α signaling resulting from increased signal transducer and activator of transcription 1 (STAT1) phosphorylation, which polarizes NK cells toward cytotoxicity, and a concomitantly reduced IFN-α induced STAT4 phosphorylation yielding reduced IFN-γ mRNA levels. These previously unappreciated findings are compatible on the one hand with the inability to clear HCV and HBV from the liver and on the other they may contribute to understand why these patients are often resistant to IFN-α-based therapies.