Regulatory Phenotype Research Articles

Impaired immunomodulatory effects of seminal plasma may play a role in unexplained recurrent pregnancy loss: Results of an in vitro study

BackgroundSeminal plasma contains signaling molecules capable of modulating the maternal immune environment to support implantation and pregnancy. Prior studies indicated that seminal plasma induces changes in gene transcription of maternal immune cells. Reduced immune suppressive capacity may lead to pregnancy loss. The aim of this study was to investigate the immunomodulating effects of seminal plasma on T cells and monocytes in the context of recurrent pregnancy loss (RPL). MethodsFemale T cells and monocytes were incubated with seminal plasma of 20 males in unexplained RPL couples (RPL males) and of 11 males whose partners had ongoing pregnancies (control males). The effect of seminal plasma on messenger RNA (mRNA) expression of immune cells was measured. Levels of mRNA expression were related to key signaling molecules present in the seminal plasma. Agglomerative hierarchical cluster analysis was performed on seminal plasma expression profiles and on mRNA expression profiles. ResultsExpression of CD25 and anti-inflammatory IL-10 by female T cells was significantly lower after stimulation with seminal plasma of RPL males compared to control males. Female monocytes treated with seminal plasma of RPL males showed an immune activation signature of relatively elevated HLA-DR expression. Expression of these T cell and monocyte components was particularly correlated with the amounts of TGF-β and VEGF in the seminal plasma. ConclusionOur findings indicate that seminal plasma has immunomodulating properties on female immune cells compatible with the induction of a more regulatory phenotype, which may be impaired in cases of unexplained RPL.

Repertoire analyses reveal T cell antigen receptor sequence features that influence T cell fate.

T cells acquire a regulatory phenotype when their T cell receptors (TCRs) experience an intermediate-to-high affinity interaction with a self-peptide presented via the major histocompatibility complex (MHC). Using TCRβ sequences from flow-sorted human cells, we identified TCR features that promote regulatory T cell (Treg) fate. From these results, we developed a scoring system to quantify TCR-intrinsic regulatory potential (TiRP). When applied to the tumor microenvironment, TiRP scoring helped to explain why only some T cell clones maintained the Tconv phenotype through expansion. To elucidate drivers of these predictive TCR features, we then examined the two elements of the Treg TCR ligand separately: the self-peptide, and the human MHC II molecule. These analyses revealed that hydrophobicity in the third complementarity determining region (CDR3β) of the TCR promotes reactivity to self-peptides, while TCR variable gene (TRBV gene) usage shapes the TCR’s general propensity for human MHC II-restricted activation.

The Ecto-5'nucleotidase/CD73 Mediates Leishmania amazonensis Survival in Macrophages.

Endogenous nucleotides produced by various group of cells under inflammatory conditions act as potential danger signals in vivo. Extracellularly released nucleotides such as ATP are rapidly hydrolyzed to adenosine by the coordinated ectonucleotidase activities of CD39 and CD73. Leishmania is an obligate intracellular parasite of macrophages and capable of modulating host immune response in order to survive and multiply within host cells. In this study, the activity of CD73 induced by Leishmania amazonensis in infected macrophages has been investigated and correlated with parasite survival and infection in vitro. For this, the expression of CD39 and CD73, by flow cytometry, in murine peritoneal macrophages infected with metacyclic promastigotes of L. amazonensis has been analyzed. Our results showed that L. amazonensis-infected macrophages, unlike LPS-treated macrophages, increased CD73 expression. It was also noted that when CD73 enzymatic activity was blocked by α, β-methyleneadenosine 5′-diphosphate sodium salt (APCP), macrophage parasitism was significantly decreased. Interestingly, these effects were not associated with the production of TNF-α, IL-10, or nitric oxide (NO). Together, these data demonstrate that L. amazonensis induces a regulatory phenotype in macrophages, which by activating the CD39/CD73 pathway allows parasite survival through the action of immunomodulatory adenosine receptors.

Glutamine promotes the generation of B10+ cells via the mTOR/GSK3 pathway.

Alterations in cell metabolism can shift the differentiation of immune cells toward a regulatory or inflammatory phenotype, thus, opening up new therapeutic opportunities for immune-related diseases. Indeed, growing knowledge on T- cell metabolism has revealed differences in the metabolic programs of suppressive Tregs as compared to inflammatory Th1 and Th17 cells. In addition to Tregs, IL-10-producing regulatory B cells are crucial for maintaining tolerance, inhibiting inflammation, and autoimmunity. Yet, the metabolic networks regulating diverse B-lymphocyte responses are not well known. Here, we show that glutaminase blockade decreased downstream mTOR activation and attenuated IL-10 secretion. Direct suppression of mTOR activity by rapamycin selectively impaired IL-10 production by B cells whereas secretion was restored upon Glycogen synthase kinase 3 (GSK3) inhibition. Mechanistically, we found mTORC1 activation leads to GSK3 inhibition, identifying a key signalling pathway regulating IL-10 secretion by B lymphocytes. Thus, our results identify glutaminolysis and the mTOR/GSK3 signalling axis, as critical regulators of the generation of IL-10 producing B cells with regulatory functions.

Islet Lymphocytes Maintain a Stable Regulatory Phenotype Under Homeostatic Conditions and Metabolic Stress.

T cells and B cells have been identified in human and murine islets, but the phenotype and role of islet lymphocytes is unknown. Resident immune populations set the stage for responses to inflammation in the islets during homeostasis and diabetes. Thus, we sought to identify the phenotype and effector function of islet lymphocytes to better understand their role in normal islets and in islets under metabolic stress. Lymphocytes were located in the islet parenchyma, and were comprised of a mix of naïve, activated, and memory T cell and B cell subsets, with an enrichment for regulatory B cell subsets. Use of a Nur77 reporter indicated that CD8 T cells and B cells both received local antigen stimulus, indicating that they responded to antigens present in the islets. Analysis of effector function showed that islet T cells and B cells produced the regulatory cytokine IL-10. The regulatory phenotype of islet T cells and B cells and their response to local antigenic stimuli remained stable under conditions of metabolic stress in the diet induced obesity (DIO) model. T cells present in human islets retained a similar activated and memory phenotype in non-diabetic and T2D donors. Under steady-state conditions, islet T cells and B cells have a regulatory phenotype, and thus may play a protective role in maintaining tissue homeostasis.

Metabolite-based dietary supplementation in human type 1 diabetes is associated with microbiota and immune modulation

BackgroundShort-chain fatty acids (SCFAs) produced by the gut microbiota have beneficial anti-inflammatory and gut homeostasis effects and prevent type 1 diabetes (T1D) in mice. Reduced SCFA production indicates a loss of beneficial bacteria, commonly associated with chronic autoimmune and inflammatory diseases, including T1D and type 2 diabetes. Here, we addressed whether a metabolite-based dietary supplement has an impact on humans with T1D. We conducted a single-arm pilot-and-feasibility trial with high-amylose maize-resistant starch modified with acetate and butyrate (HAMSAB) to assess safety, while monitoring changes in the gut microbiota in alignment with modulation of the immune system status.ResultsHAMSAB supplement was administered for 6 weeks with follow-up at 12 weeks in adults with long-standing T1D. Increased concentrations of SCFA acetate, propionate, and butyrate in stools and plasma were in concert with a shift in the composition and function of the gut microbiota. While glucose control and insulin requirements did not change, subjects with the highest SCFA concentrations exhibited the best glycemic control. Bifidobacterium longum, Bifidobacterium adolescentis, and vitamin B7 production correlated with lower HbA1c and basal insulin requirements. Circulating B and T cells developed a more regulatory phenotype post-intervention.ConclusionChanges in gut microbiota composition, function, and immune profile following 6 weeks of HAMSAB supplementation were associated with increased SCFAs in stools and plasma. The persistence of these effects suggests that targeting dietary SCFAs may be a mechanism to alter immune profiles, promote immune tolerance, and improve glycemic control for the treatment of T1D.Trial registrationACTRN12618001391268. Registered 20 August 2018,https://www.anzctr.org.au/Trial/Registration/TrialReview.aspx?id=375792CkvdGnDkuZG7nQTZ5dAk_pVideo

Angiotensin-(1-7)/MasR axis promotes migration of monocytes/macrophages with a regulatory phenotype to perform phagocytosis and efferocytosis.

Nonphlogistic migration of macrophages contributes to the clearance of pathogens and apoptotic cells, a critical step for the resolution of inflammation and return to homeostasis. Angiotensin-(1-7) [Ang-(1-7)] is a heptapeptide of the renin-angiotensin system that acts through Mas receptor (MasR). Ang-(1-7) has recently emerged as a novel proresolving mediator, yet Ang-(1-7) resolution mechanisms are not fully determined. Herein, Ang-(1-7) stimulated migration of human and murine monocytes/macrophages in a MasR-, CCR2-, and MEK/ERK1/2–dependent manner. Pleural injection of Ang-(1-7) promoted nonphlogistic mononuclear cell influx alongside increased levels of CCL2, IL-10, and macrophage polarization toward a regulatory phenotype. Ang-(1-7) induction of CCL2 and mononuclear cell migration was also dependent on MasR and MEK/ERK. Of note, MasR was upregulated during the resolution phase of inflammation, and its pharmacological inhibition or genetic deficiency impaired mononuclear cell recruitment during self-resolving models of LPS pleurisy and E. coli peritonitis. Inhibition/absence of MasR was associated with reduced CCL2 levels, impaired phagocytosis of bacteria, efferocytosis, and delayed resolution of inflammation. In summary, we have uncovered a potentially novel proresolving feature of Ang-(1-7), namely the recruitment of mononuclear cells favoring efferocytosis, phagocytosis, and resolution of inflammation. Mechanistically, cell migration was dependent on MasR, CCR2, and the MEK/ERK pathway.

CD1dhiPD-L1hiCD27+ Regulatory Natural Killer Subset Suppresses Atopic Dermatitis.

Effector and regulatory functions of various leukocytes in allergic diseases have been well reported. Although the role of conventional natural killer (NK) cells has been established, information on its regulatory phenotype and function are very limited. Therefore, the objective of this study was to investigate the phenotype and inhibitory functions of transforming growth factor (TGF)-β-producing regulatory NK (NKreg) subset in mice with MC903-induced atopic dermatitis (AD). Interestingly, the population of TGF-β-producing NK cells in peripheral blood monocytes (PBMCs) was decreased in AD patients than in healthy subjects. The number of TGF-β+ NK subsets was decreased in the spleen or cervical lymph node (cLN), but increased in ear tissues of mice with AD induced by MC903 than those of normal mice. We further observed that TGF-β+ NK subsets were largely included in CD1dhiPD-L1hiCD27+ NK cell subset. We also found that numbers of ILC2s and TH2 cells were significantly decreased by adoptive transfer of CD1dhiPD-L1hiCD27+ NK subsets. Notably, the ratio of splenic Treg per TH2 was increased by the adoptive transfer of CD1dhiPD-L1hiCD27+ NK cells in mice. Taken together, our findings demonstrate that the TGF-β-producing CD1dhiPD-L1hiCD27+ NK subset has a previously unrecognized role in suppressing TH2 immunity and ILC2 activation in AD mice, suggesting that the function of TGF-β-producing NK subset is closely associated with the severity of AD in humans.

B Cell Receptor Signaling and Protein Kinase D2 Support Regulatory B Cell Function in Pancreatic Cancer.

B cells can act as potent suppressors of anti-tumor T cell immunity, presenting a mechanism of resistance to immunotherapy. In pancreatic ductal adenocarcinoma, B cells can display a T cell-suppressive or regulatory phenotype centered on the expression of the cytokine Interleukin 35 (IL-35). While B cell-mediated immunosuppression presents a barrier to anti-tumorigenic T cell function, it is not clear how regulatory B cell function could be targeted, and the signals that promote this suppressive phenotype in B cells are not well understood. Here we use a novel IL-35 reporter model to understand which signaling pathways are important for immunosuppressive properties in B cells. In vitro analysis of IL-35 reporter B cells revealed a synergy between the BCR and TLR4 signaling pathways is sufficient to induce IL-35 expression. However, in vivo, B cell receptor activation, as opposed to MyD88 signaling in B cells, is central to B cell-mediated suppression and promotion of pancreatic cancer growth. Further analysis identified protein kinase D2 (PKD2) as being a key downstream regulator of IL-35 expression in B cells. Regulatory B cells with an inactivating mutation in PKD2 failed to produce IL-35 or fully suppress effector T cell function in vitro. Furthermore, inhibition of PKD in B cells decreased tumor growth and promoted effector T cell function upon adoptive transfer into B cell-deficient mice. Collectively, these data provide insight into how regulatory B cell function is promoted in pancreatic cancer and identify potential therapeutic targets to restrain this function.

The folate way to T cell fate

The role of folate-dependent one carbon (1C) metabolism in CD4+ Tcell polarization is incompletely understood. In this issue of Immunity, Sugiura etal. (2021) provide evidence that blocking the 1C metabolic enzyme MTHFD2 may curb pro-inflammatory CD4+ Tcells, while redirecting them toward a regulatory Tcell phenotype.

T cell fat catabolism: A novel target for kynurenine?

T cell fat catabolism: A novel target for kynurenine?

Tolerance Induced by Antigen-Loaded PLG Nanoparticles Affects the Phenotype and Trafficking of Transgenic CD4+ and CD8+ T Cells.

We have shown that PLG nanoparticles loaded with peptide antigen can reduce disease in animal models of autoimmunity and in a phase 1/2a clinical trial in celiac patients. Clarifying the mechanisms by which antigen-loaded nanoparticles establish tolerance is key to further adapting them to clinical use. The mechanisms underlying tolerance induction include the expansion of antigen-specific CD4+ regulatory T cells and sequestration of autoreactive cells in the spleen. In this study, we employed nanoparticles loaded with two model peptides, GP33–41 (a CD8 T cell epitope derived from lymphocytic choriomeningitis virus) and OVA323–339 (a CD4 T cell epitope derived from ovalbumin), to modulate the CD8+ and CD4+ T cells from two transgenic mouse strains, P14 and DO11.10, respectively. Firstly, it was found that the injection of P14 mice with particles bearing the MHC I-restricted GP33–41 peptide resulted in the expansion of CD8+ T cells with a regulatory cell phenotype. This correlated with reduced CD4+ T cell viability in ex vivo co-cultures. Secondly, both nanoparticle types were able to sequester transgenic T cells in secondary lymphoid tissue. Flow cytometric analyses showed a reduction in the surface expression of chemokine receptors. Such an effect was more prominently observed in the CD4+ cells rather than the CD8+ cells.

B cells support the repair of injured tissues by adopting MyD88-dependent regulatory functions and phenotype.

Exogenously applied mature naïve B220+ /CD19+ /IgM+ /IgD+ B cells are strongly protective in the context of tissue injury. However, the mechanisms by which B cells detect tissue injury and aid repair remain elusive. Here, we show in distinct models of skin and brain injury that MyD88-dependent toll-like receptor (TLR) signaling through TLR2/6 and TLR4 is essential for the protective benefit of B cells in vivo, while B cell-specific deletion of MyD88 abrogated this effect. The B cell response to injury was multi-modal with simultaneous production of both regulatory cytokines, such as IL-10, IL-35, and transforming growth factor beta (TGFβ), and inflammatory cytokines, such as tumor necrosis factor alpha (TNFα), IL-6, and interferon gamma. Cytometry analysis showed that this response was time and environment-dependent in vivo, with 20%-30% of applied B cells adopting an immune modulatory phenotype with high co-expression of anti- and pro-inflammatory cytokines after 18-48 h at the injury site. B cell treatment reduced the expression of TNFα and increased IL-10 and TGFβ in infiltrating immune cells and fibroblasts at the injury site. Proteomic analysis further showed that B cells have a complex time-dependent homeostatic effect on the injured microenvironment, reducing the expression of inflammation-associated proteins, and increasing proteins associated with proliferation, tissue remodeling, and protection from oxidative stress. These findings chart and validate a first mechanistic understanding of the effects of B cells as an immunomodulatory cell therapy in the context of tissue injury.

Targeting HGF/c-Met Axis Decreases Circulating Regulatory T Cells Accumulation in Gastric Cancer Patients.

Simple SummaryRestoring an effective immune response is the key goal of immunotherapy. One of the major mechanisms of tumor-induced immunosuppression is regulatory T cells (Treg) accumulation. In this study, using in vitro and in vivo analysis, we assessed the impact of the HGF/c-Met pathway, involved notably in tumor angiogenesis, on Treg accumulation in patients with gastric cancer. First, we reported that c-Met is expressed on circulating monocytes of gastric cancer patients and this expression seems to be associated with the worst outcome. Secondly, during in vitro cultures, c-Met+ monocytes differentiate into dendritic cells with tolerogenic properties able to induce the proliferation of Treg. Finally, rilotumumab, an anti-HGF antibody, decreases the percentage of circulating Treg in gastric cancer patients. Using HGF/c-Met inhibitors to partially reverse immunosuppression could lead to the development of new treatment associations, for example with immune checkpoint blockers.Elucidating mechanisms involved in tumor-induced immunosuppression is of great interest since it could help to improve cancer immunotherapy efficacy. Here we show that Hepatocyte Growth Factor (HGF), a pro-tumoral and proangiogenic factor, and its receptor c-Met are involved in regulatory T cells (Treg) accumulation in the peripheral blood of gastric cancer (GC) patients. We observed that c-Met is expressed on circulating monocytes from GC patients. The elevated expression on monocytes is associated with clinical parameters linked to an aggressive disease phenotype and correlates with a worse prognosis. Monocyte-derived dendritic cells from GC patients differentiated in the presence of HGF adopt a regulatory phenotype with a lower expression of co-stimulatory molecules, impaired maturation capacities, and an increased ability to produce interleukin-10 and to induce Treg differentiation in vitro. In the MEGA-ACCORD20-PRODIGE17 trial, GC patients received an anti-HGF antibody treatment (rilotumumab), which had been described to have an anti-angiogenic activity by decreasing proliferation of endothelial cells and tube formation. Rilotumumab decreased circulating Treg in GC patients. Thus, we identified that HGF indirectly triggers Treg accumulation via c-Met-expressing monocytes in the peripheral blood of GC patients. Our study provides arguments for potential alternative use of HGF/c-Met targeted therapies based on their immunomodulatory properties which could lead to the development of new therapeutic associations in cancer patients, for example with immune checkpoint inhibitors.

Inhibitory effects of tolerogenic probiotics on migratory potential of lupus patient-derived DCs.

Objective(s):The present in vitro study aimed to evaluate whether Lactobacillus delbrueckii and Lactobacillus rhamnosus treatments can induce regulatory phenotype, together with modulating the expression of chemokine receptors (CRs) in dendritic cells (DCs). The CRs of DCs have been found to be involved in the pathogenesis of Systemic lupus erythematosus (SLE) through directing recruitment and migration of immune cells.Materials and Methods: In brief, monocytes of patients with SLE and healthy donors were isolated and differentiated to regulatory or inflammatory mature DCs through treatment with L. delbrueckii, L. rhamnosus, mixed probiotics, and LPS. Results:FACScan analysis showed that the expression of CRs including CXCR3, CCR5, CCR4, and CCR3, was significantly reduced in all probiotic-treated groups of SLE and healthy (control) donors when compared with the LPS treated group. Conclusion:The results demonstrated that tolerogenic probiotics could prevent or decrease the expression of inflammatory CRs on the surface of tolerogenic DCs during the maturation process.

Immune Regulatory Cell Bias Following Alemtuzumab Treatment in Relapsing-Remitting Multiple Sclerosis.

Alemtuzumab is a highly effective treatment for relapsing-remitting multiple sclerosis. It selectively targets the CD52 antigen to induce profound lymphocyte depletion, followed by recovery of T and B cells with regulatory phenotypes. We previously showed that regulatory T cell function is restored with cellular repletion, but little is known about the functional capacity of regulatory B-cells and peripheral blood monocytes during the repletion phase. In this study (ClinicalTrials.gov ID# NCT03647722) we simultaneously analyzed the change in composition and function of both regulatory lymphocyte populations and distinct monocyte subsets in cross-sectional cohorts of MS patients prior to or 6, 12, 18, 24 or 36 months after their first course of alemtuzumab treatment. We found that the absolute number and percentage of cells with a regulatory B cell phenotype were significantly higher after treatment and were positivity correlated with regulatory T cells. In addition, B cells from treated patients secreted higher levels of IL-10 and BDNF, and inhibited the proliferation of autologous CD4+CD25- T cell targets. Though there was little change in monocytes populations overall, following the second annual course of treatment, CD14+ monocytes had a significantly increased anti-inflammatory bias in cytokine secretion patterns. These results confirmed that the immune system in alemtuzumab-treated patients is altered in favor of a regulatory milieu that involves expansion and increased functionality of multiple regulatory populations including B cells, T cells and monocytes. Here, we showed for the first time that functionally competent regulatory B cells re-appear with similar kinetics to that of regulatory T-cells, whereas the change in anti-inflammatory bias of monocytes does not occur until after the second treatment course. These findings justify future studies of all regulatory cell types following alemtuzumab treatment to reveal further insights into mechanisms of drug action, and to identify key immunological predictors of durable clinical efficacy in alemtuzumab-treated patients.

The molecular and phenotypic makeup of fetal human skin T lymphocytes.

ABSTRACTThe adult human skin contains a vast number of T cells that are essential for skin homeostasis and pathogen defense. T cells are first observed in the skin at the early stages of gestation; however, our understanding of their contribution to early immunity has been limited by their low abundance and lack of comprehensive methodologies for their assessment. Here, we describe a new workflow for isolating and expanding significant amounts of T cells from fetal human skin. Using multiparametric flow cytometry and in situ immunofluorescence, we found a large population with a naive phenotype and small populations with a memory and regulatory phenotype. Their molecular state was characterized using single-cell transcriptomics and TCR repertoire profiling. Importantly, culture of total fetal skin biopsies facilitated T cell expansion without a substantial impact on their phenotype, a major prerequisite for subsequent functional assays. Collectively, our experimental approaches and data advance the understanding of fetal skin immunity and potential use in future therapeutic interventions.

An autologous culture model of nodal B-cell lymphoma identifies ex vivo determinants of response to bispecific antibodies

AbstractBispecific antibodies (BsAbs) can induce long-term responses in patients with refractory and relapsed B-cell lymphoma. Nevertheless, response rates across patients are heterogeneous, and the factors determining quality and duration of responses are poorly understood. To identify key determinants of response to BsAbs, we established a primary, autologous culture model allowing us to mimic treatment with CD3xCD19 and CD3xCD20 BsAbs within the lymph node microenvironment ex vivo. T cell–mediated killing of lymphoma cells and proliferation of T cells varied significantly among patients but highly correlated between BsAbs targeting CD20 or CD19. Ex vivo response to BsAbs was significantly associated with expansion of T cells and secretion of effector molecules (eg, granzyme B, perforin) but not with expression of T-cell exhaustion (eg, PD1, TIM3) or activation markers (eg, CD25, CD69) or formation of intercellular contacts. In addition, we identified a distinct phenotype of regulatory T cells that was linked to ex vivo response independently from T-cell frequency at baseline. High expression levels of Aiolos (IKZF1), ICOS, and CXCR5 were positively associated with ex vivo response, whereas strong expression of Helios (IKZF2) had an unfavorable impact on ex vivo response to BsAbs. We further showed that lenalidomide, nivolumab, and atezolizumab improved ex vivo response to BsAbs by potentiating T-cell effector functions. In summary, our ex vivo study identified a distinct regulatory T-cell phenotype as a potential contributor to treatment failure of BsAbs and suggests drug combinations of high clinical relevance that could improve the efficacy of BsAbs.

TIGER: The gene expression regulatory variation landscape of human pancreatic islets.

SUMMARYGenome-wide association studies (GWASs) identified hundreds of signals associated with type 2 diabetes (T2D). To gain insight into their underlying molecular mechanisms, we have created the translational human pancreatic islet genotype tissue-expression resource (TIGER), aggregating >500 human islet genomic datasets from five cohorts in the Horizon 2020 consortium T2DSystems. We impute genotypes using four reference panels and meta-analyze cohorts to improve the coverage of expression quantitative trait loci (eQTL) and develop a method to combine allele-specific expression across samples (cASE). We identify >1 million islet eQTLs, 53 of which colocalize with T2D signals. Among them, a low-frequency allele that reduces T2D risk by half increases CCND2 expression. We identify eight cASE colocalizations, among which we found a T2D-associated SLC30A8 variant. We make all data available through the TIGER portal (http://tiger.bsc.es), which represents a comprehensive human islet genomic data resource to elucidate how genetic variation affects islet function and translates into therapeutic insight and precision medicine for T2D.

PD-1, CTLA-4, LAG-3, and TIGIT: The roles of immune checkpoint receptors on the regulation of human NK cell phenotype and functions

The roles of immune checkpoint receptors were defined in many cancers and autoimmune diseases, while there is limited information on their functional roles in the NK cells of healthy individuals. Immune checkpoint receptor expression of NK cell subsets and their association with NK cell functions (cytotoxic capacity and cytokine production) in healthy population were investigated. PD-1, CTLA-4, LAG-3 and TIGIT expression of peripheral blood NK cells, cytokine levels (TNF-α, IFN-γ, IL-10) and cytotoxic functions (granzyme A, perforin, CD107a; with/without K562 target cell stimulation) were evaluated by flow cytometry. CD56dimCD16dim NK cells had the highest expression of TIGIT, while CD56dimCD16− NK cells had highest expression of PD-1, CTLA-4 and LAG-3. PD-1+ NK cells, CTLA-4+ NK cells and LAG-3+ NK cells had increased amount of IL-10 however, reduced IFN-γ and TNF-α levels. Cytotoxic granule expressions (perforin and granzyme A) were reduced in PD-1+ NK cells, CTLA-4+ NK cells and LAG-3+ NK cells. However, TIGIT expression did not alter perforin and granzyme A expressions. Degranulation capacity was reduced in three groups of NK cells (PD-1+ or LAG-3+ or TIGIT+). TIGIT+ NK cells responded strongly to target cell stimulation, while NK cells in the other groups (PD-1+ or CTLA-4+ or LAG-3+) were resistant. PD-1+ NK cells, CTLA-4+ NK cells and LAG-3+ NK cells had a regulatory phenotype, impaired cytotoxic functions, and response to target cell stimulation. In contrast, TIGIT+ NK cells had strong baseline cytotoxic activity that further increased in response to target cell stimulation.