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 release of soluble ligands of activating Natural Killer (NK) cell receptors may represent a regulatory mechanism of NK cell function both in physiologic and in pathologic conditions. Here, we ...identified the extracellular matrix protein Nidogen-1 (NID1) as a ligand of NKp44, an important activating receptor expressed by activated NK cells. When released as soluble molecule, NID1 regulates NK cell function by modulating NKp44-induced IFN-γ production or cytotoxicity. In particular, it also modulates IFN-γ production induced by Platelet-Derived Growth Factor (PDGF)-DD following NKp44 engagement. We also show that NID1 may be present at the cell surface. In this form or when bound to a solid support (bNID1), NID1 fails to induce NK cell cytotoxicity or cytokine release. However, analysis by mass spectrometry revealed that exposure to bNID1 can induce in human NK cells relevant changes in the proteomic profiles suggesting an effect on different biological processes.
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.
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.
Innate lymphoid cells (ILCs) comprise cytotoxic NK cells and helper‐ILCs, which are further divided in ILC1, ILC2, and ILC3. Helper‐ILCs mirror the effector functions of helper T‐cell subsets and ...contribute to host immune defense, tissue homeostasis and repair through cytokine secretion. Although they are mainly tissue‐resident, helper ILCs are also found in the peripheral blood (PB). In the human setting, it may be needed to analyze circulating helper ILCs to compare pathological to physiological conditions. In this review, we provide simple guidelines and a list of markers useful to study human PB helper ILCs phenotype and function by flow cytometry.
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