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NK cells play important roles in innate defenses against viruses and in the control of tumor growth and metastasis. The regulation/induction of NK cell function is mediated by an array of activating or inhibitory surface receptors. In humans, major activating receptors involved in target cell killing are the natural cytotoxicity receptors (NCRs) and NKG2D. Activating receptors recognize ligands that are overexpressed or expressed de novo upon cell stress, viral infection, or tumor transformation. The HLA-class I-specific inhibitory receptors, including KIRs recognizing HLA-class I allotypic determinants and CD94/NKG2A recognizing the class-Ib HLA-E, constitute a fail-safe mechanism to avoid unwanted NK-mediated damage to healthy cells. Other receptors such as PD-1, primarily expressed by activated T lymphocytes, are important inhibitory checkpoints of immune responses that ensure T-cell tolerance. PD-1 also may be expressed by NK cells in cancer patients. Since PD-1 ligand (PD-L1) may be expressed by different tumors, PD-1/PD-L1 interactions inactivate both T and NK cells. Thus, the reliable evaluation of PD-L1 expression in tumors has become a major issue to select patients who may benefit from therapy with mAbs disrupting PD-1/PD-L1 interactions. Recently, NKG2A was revealed to be an important checkpoint controlling both NK and T-cell activation. Since most tumors express HLA-E, mAbs targeting NKG2A has been used alone or in combination with other therapeutic mAbs targeting PD-1 or tumor antigens (e.g., EGFR), with encouraging results. The translational value of NK cells and their receptors is evidenced by the extraordinary therapeutic success of haploidentical HSCT to cure otherwise fatal high-risk leukemias.

Ultracentrifugationon sucrose density gradientappears to be the best purification protocol for extracellular vesicle (EVs) purification. After this step, to reduce disulfide bridges linking exogenous proteins to the vesicles, the collected samples are routinely washed and treated with dithiothreitol (DTT). Such incubations are performed at temperatures ranging from room temperature up to 95 °C, with either Tris or PBS as buffers. We re-investigated these steps on both exosomes and microvesicles purified from blood (serum) and urine by electrophoretic separation, silver staining and western blots analysis. Data confirm that an extra centrifugation on a sucrose cushion can effectively eliminate contaminants. Tris buffer (50 Mm) and β-mercaptoethanol as a reducing agent at room temperature dramatically improved either sample cleaning. By contrast, especially for exosomes PBS buffer and DTT, above 37 °C, caused massive protein aggregations, yielding blurred SDS-PAGE gels in both samples. Immuno-blot analyses demonstrated that in PBS-DTT contamination with albumin (in serum) or with uromodulin (in urine) occurs. DTT, likely due to its two–SH groups, might form scrambled SS-bonds promoting EVs interaction with environmental macromolecules via disulphide bridges. Therefore, to obtain maximum vesicle purity for biomarker investigations and to maximize both presence of EVs proteins and their accessibility, use of DTT is not recommended.

The NK cell population is characterized by distinct NK cell subsets that respond differently to the various activating stimuli. For this reason, the determination of the optimal cytotoxic activation of the different NK cell subsets can be a crucial aspect to be exploited to counter cancer cells in oncologic patients. To evaluate how the triggering of different combination of activating receptors can affect the cytotoxic responses of different NK cell subsets, we developed a microbead-based degranulation assay. By using this new assay, we were able to detect CD107a+ degranulating NK cells even within the less cytotoxic subsets (i.e., resting CD56bright and unlicensed CD56dim NK cells), thus demonstrating its high sensitivity. Interestingly, signals delivered by the co-engagement of NKp46 with 2B4, but not with CD2 or DNAM-1, strongly cooperate to enhance degranulation on both licensed and unlicensed CD56dim NK cells. Of note, 2B4 is known to bind CD48 hematopoietic antigen, therefore this observation may provide the rationale why CD56dim subset expansion correlates with successful hematopoietic stem cell transplantation mediated by alloreactive NK cells against host T, DC and leukemic cells, while sparing host non-hematopoietic tissues and graft versus host disease. The assay further confirms that activation of LFA-1 on NK cells leads to their granule polarization, even if, in some cases, this also takes to an inhibition of NK cell degranulation, suggesting that LFA-1 engagement by ICAMs on target cells may differently affect NK cell response. Finally, we observed that NK cells undergo a time-dependent spontaneous (cytokine-independent) activation after blood withdrawal, an aspect that may strongly bias the evaluation of the resting NK cell response. Altogether our data may pave the way to develop new NK cell activation and expansion strategies that target the highly cytotoxic CD56dim NK cells and can be feasible and useful for cancer and viral infection treatment.

Extracellular vesicles (EVs) are emerging as critical mediators of intercellular communication in the tumor microenvironment (TME), profoundly influencing cancer progression. These nano-sized vesicles, released by both tumor and stromal cells, carry a diverse cargo of proteins, nucleic acids, and lipids, reflecting the dynamic cellular landscape and mediating intricate interactions between cells. This review provides a comprehensive overview of the biogenesis, composition, and functional roles of EVs in cancer, highlighting their significance in both basic research and clinical applications. We discuss how cancer cells manipulate EV biogenesis pathways to produce vesicles enriched with pro-tumorigenic molecules, explore the specific contributions of EVs to key hallmarks of cancer, such as angiogenesis, metastasis, and immune evasion, emphasizing their role in shaping TME and driving therapeutic resistance. Concurrently, we submit recent knowledge on how the cargo of EVs can serve as a valuable source of biomarkers for minimally invasive liquid biopsies, and its therapeutic potential, particularly as targeted drug delivery vehicles and immunomodulatory agents, showcasing their promise for enhancing the efficacy and safety of cancer treatments. By deciphering the intricate messages carried by EVs, we can gain a deeper understanding of cancer biology and develop more effective strategies for early detection, targeted therapy, and immunotherapy, paving the way for a new era of personalized and precise cancer medicine with the potential to significantly improve patient outcomes.

Natural killer (NK) cells contribute to the first line of defense against viruses and to the control of tumor growth and metastasis spread. The discovery of HLA class I specific inhibitory receptors, primarily of killer Ig-like receptors (KIRs), and of activating receptors has been fundamental to unravel NK cell function and the molecular mechanisms of tumor cell killing. Stemmed from the seminal discoveries in early '90s, in which Alessandro Moretta was the major actor, an extraordinary amount of research on KIR specificity, genetics, polymorphism, and repertoire has followed. These basic notions on NK cells and their receptors have been successfully translated to clinical applications, primarily to the haploidentical hematopoietic stem cell transplantation to cure otherwise fatal leukemia in patients with no HLA compatible donors. The finding that NK cells may express the PD-1 inhibitory checkpoint, particularly in cancer patients, may allow understanding how anti-PD-1 therapy could function also in case of HLA class I tumors, usually susceptible to NK-mediated killing. This, together with the synergy of therapeutic anti-checkpoint monoclonal antibodies, including those directed against NKG2A or KIRs, emerging in recent or ongoing studies, opened new solid perspectives in cancer therapy.

The identification of inhibitory NK cell receptors specific for HLA-I molecules (KIRs and NKG2A) provided the molecular basis for clarifying the mechanism by which NK cells kill transformed cells while sparing normal cells. The direct interactions between inhibitory NK cell receptors and their HLA-I ligands enable NK cells to distinguish healthy from transformed cells, which frequently show an altered expression of HLA-I molecules. Indeed, NK cells can kill cancer cells that have lost, or under express, HLA-I molecules, but not cells maintaining their expression. In this last case, it is possible to use anti-KIR or anti-NKG2A monoclonal antibodies to block the inhibitory signals generated by these receptors and to restore the anti-tumor NK cell activity. These treatments fall within the context of the new immunotherapeutic strategies known as \"immune checkpoint blockade.\" These antibodies are currently used in clinical trials in the treatment of both hematological and solid tumors. However, a more complex scenario has recently emerged. For example, NK cells can also express additional immune checkpoints, including PD-1, that was originally described on T lymphocytes, and whose ligands (PD-Ls) are usually overexpressed on tumor cells. Thus, it appears that the activation of NK cells and their potentially harmful effector functions are under the control of different immune checkpoints and their simultaneous expression could provide additional levels of suppression to anti-tumor NK cell responses. This review is focused on PD-1 immune checkpoint in NK cells, its potential role in immunosuppression, and the therapeutic strategies to recover NK cell cytotoxicity and anti-tumor effect.

The characterization of frequency and phenotypes of natural killer (NK) cells and T cells in BAL and peripheral blood of patients with sarcoidosis was evaluated, to discriminate the differential status of these cells in these two compartments. The analysis revealed that CD56 bright CD16 neg resulted higher in BAL than PB of sarcoidosis and healthy subjects, while CD56 dim CD16 + showed a different proportion between BAL and PB of both Sarcoidosis patients and HC. Moreover, in comparison with autologous PB, BAL was characterized by a higher expression of activated NK cell markers NKp44, CD69 and CD25. Significantly increased levels of PD-1 + NK cells in the BAL of patients were detected. Regarding the maturation of CD4 and CD8, an increase of Effector Memory T cells (T EM ) was reported in BAL compared to PB. A better characterization of NK and T cells may lead to an improvement of the pathogenetic mechanisms in sarcoidosis.

Milk is a primary source of vital nutrients and bioactive components fundamental to the growth and development of both newborn animals and humans. Produced by economically significant livestock species (including cattle, buffaloes, goats, sheep and camels), milk is a complex matrix rich in caseins, vitamins, fats, and proteins. Beyond its classical nutritional profile, milk serves as a pivotal vehicle for milk-derived extracellular vesicles (mEVs). These specialized food-derived EVs (fEVs) exert pleiotropic effects that resonate with the One Health paradigm, linking animal well-being and human nutrition to broader ecosystem stability. mEVs offer unique advantages, such as high biocompatibility and gastrointestinal stability, also rendering them potential therapeutic tools as drug delivery systems. However, challenges remain regarding the standardization of mEVs and the variability of their molecular cargo. This review provides a comprehensive comparative analysis of mEVs across a diverse taxonomic range, including bovines, water buffaloes, yaks, camels, goats, pigs, horses, donkeys, and humans, highlighting their distinct functional signatures. Indeed, a critical issue in mEV research is the isolation process: recommendations to minimize contamination from milk fat globules and casein micelles (which can cover EV signals) are given. Finally, current detection methods and instrumentation, with a specific focus on advancing flow cytometry (FC) approaches are discussed. Key insights include the use of conventional FC (with fluorescence triggering, the necessity of rigorous controls and calibration, and the utility of bead-based assays to overcome resolution limits) and imaging flow cytometry (IFC). In both technical approaches, the application of different EV generic fluorescent markers and the strategic selection of tetraspanins (i.e., CD9, CD63, CD81), is mandatory: we emphasize that selecting the correct antibody clones and accounting for inter-species cross-reactivity are essential steps for ensuring the accuracy and reproducibility of mEV research across mammalian species.

Natural killer (NK) cells are important effectors playing a relevant role in innate immunity, primarily in tumor surveillance and in defenses against viruses. Human NK cells recognize HLA class I molecules through surface receptors (KIR and NKG2A) that inhibit NK cell function and kill target cells that have lost (or underexpress) HLA class I molecules as it occurs in tumors or virus-infected cells. NK cell activation is mediated by an array of activating receptors and co-receptors that recognize ligands expressed primarily on tumors or virus-infected cells. In vivo anti-tumor NK cell activity may be suppressed by tumor or tumor-associated cells. Alloreactive NK cells (i.e. those that are not inhibited by the HLA class I alleles of the patient) derived from HSC of haploidentical donors play a major role in the cure of high-risk leukemia, by killing leukemia blasts and patient's DC, thus preventing tumor relapses and graft-versus-host disease. The expression of the HLA-C2-specific activating KIR2DS1 may also contribute to NK alloreactivity in patients expressing C2 alleles. A clear correlation has been proven between the size of the alloreactive NK cell population and the clinical outcome. Recently, haplo-HSCT has been further improved with the direct infusion, together with HSC, of donor-derived, mature alloreactive NK cells and TCRγδ + T cells - both contributing to a prompt anti-leukemia effect together with an efficient defense against pathogens during the 6- to 8-week interval required for the generation of alloreactive NK cells from HSC.

Suppressor of cytokine signaling 1 (SOCS1) haploinsufficiency is a recently described inborn error of immunity characterized by autoimmunity, inflammation, lymphoproliferation, and increased infection susceptibility. SOCS1, a negative regulator of cytokine signaling via the JAK/STAT pathway, explains the condition’s broad phenotypic variability. Single nucleotide polymorphisms in SOCS1 have been linked to multiple sclerosis (MS), and SOCS1 mimetics have shown efficacy in MS animal models. However, neurological involvement has not been previously reported in patients with SOCS1 insufficiency. We describe a family with a heterozygous SOCS1 variant, highlighting neurological manifestations such as MS, autoimmune encephalitis, and recurrent complex regional pain syndrome as novel features. Next-Generation Sequencing and segregation analysis were performed on PBMCs from patients and healthy donors. Functional studies included luciferase reporter assays in HeLa cells expressing the SOCS1 mutant, flow cytometry for phenotypic analysis, and gene expression profiling of the type-I interferon (IFN) signature. Intraepidermal nerve fiber density was evaluated via immunohistochemistry on skin biopsy. Genetic analysis confirmed the variant’s inheritance. Transfected cells carrying the SOCS1 variant showed increased STAT1 transcriptional activity after IFN-γ stimulation. Elevated STAT5 phosphorylation and T-cell proliferation were observed in response to IL-2. Peripheral blood revealed an elevated IFN signature during relapse. Skin biopsy showed reduced intraepidermal nerve fiber density. This report expands the clinical spectrum of SOCS1-related disorders to include neurological symptoms, emphasizing SOCS1’s critical role in regulating inflammation in the central and peripheral nervous systems.