Frequency Of Natural Killer Cells Research Articles

Active juvenile systemic lupus erythematosus is associated with distinct NK cell transcriptional and phenotypic alterations

While adaptive immune responses have been studied extensively in SLE (systemic lupus erythematosus), there is limited and contradictory evidence regarding the contribution of natural killer (NK) cells to disease pathogenesis. There is even less evidence about the role of NK cells in the more severe phenotype with juvenile-onset (J)SLE. In this study, analysis of the phenotype and function of NK cells in a large cohort of JSLE patients demonstrated that total NK cells, as well as perforin and granzyme A expressing NK cell populations, were significantly diminished in JSLE patients compared to age- and sex-matched healthy controls. The reduction in NK cell frequency was associated with increased disease activity, and transcriptomic analysis of NK populations from active and low disease activity JSLE patients versus healthy controls confirmed that disease activity was the main driver of differential NK cell gene expression. Pathway analysis of differentially expressed genes revealed an upregulation of interferon-α responses and a downregulation of exocytosis in active disease compared to healthy controls. Further gene set enrichment analysis also demonstrated an overrepresentation of the apoptosis pathway in active disease. This points to increased propensity for apoptosis as a potential factor contributing to NK cell deficiency in JSLE.

Immune cell profiles associated with human exposure to perfluorinated compounds (PFAS) suggest changes in natural killer, T helper, and T cytotoxic cell subpopulations

Per- and polyfluoroalkyl substances (PFAS) constitutes a group of highly persistent man-made substances. Recent evidence indicates that PFAS negatively impact the immune system. However, it remains unclear how different PFAS are associated with alterations in circulating leukocyte subpopulations. More detailed knowledge of such potential associations can provide better understanding into mechanisms of PFAS immunotoxicity in humans. In this exploratory study, associations of serum levels of common PFAS (perfluorooctanoic acid (PFOA), perfluorooctane sulfonic acid (PFOS), perfluorononanoic acid (PFNA), and perfluorohexane sulfonic acid (PFHxS)) and immune cell profiles of peripheral blood mononuclear cells, both with and without immunostimulation, were investigated. High-dimensional single cell analysis by mass cytometry was done on blood leukocytes from fifty participants in the Norwegian human biomonitoring EuroMix study.Different PFAS were associated with changes in various subpopulations of natural killer (NK), T helper (Th), and cytotoxic T (Tc) cells. Broadly, PFAS concentrations were related to increased frequencies of NK cells and activated subpopulations of NK cells. Additionally, increased levels of activated T helper memory cell subpopulations point to Th2/Th17 and Treg-like skewed profiles. Finally, PFAS concentrations were associated with decreased frequencies of T cytotoxic cell subpopulations with CXCR3+ effector memory (EM) phenotypes. Several of these observations point to biologically plausible mechanisms that may contribute to explaining the epidemiological reports of immunosuppression by PFAS. Our results suggest that PFAS exposures even at relatively low levels are associated with changes in immune cell subpopulations, a finding which should be explored more thoroughly in a larger cohort. Additionally, causal relationships should be confirmed in experimental studies. Overall, this study demonstrates the strength of profiling by mass cytometry in revealing detailed changes in immune cells at a single cell level.

Myeloid-derived suppressor cells in pleural effusion as a diagnostic marker for early discrimination of pulmonary tuberculosis from pneumonia.

Tuberculous pleural effusion (TPE) stands as one of the primary forms of extrapulmonary tuberculosis (TB) and frequently manifests in regions with a high prevalence of TB, consequently being a notable cause of pleural effusion in such areas. However, the differentiation between TPE and parapneumonic pleural effusion (PPE) presents diagnostic complexities. This study aimed to evaluate the potential of myeloid-derived suppressor cells (MDSCs) in the pleural fluid as a potential diagnostic marker for distinguishing between TPE and PPE. Adult patients, aged 18 years or older, who presented to the emergency room of a tertiary referral hospital and received a first-time diagnosis of pleural effusion, were prospectively enrolled in the study. Various immune cell populations, including T cells, B cells, natural killer (NK) cells, and MDSCs, were analyzed in both pleural fluid and peripheral blood samples. In pleural fluid, the frequency of lymphocytes, including T, B, and NK cells, was notably higher in TPE compared to PPE. Conversely, the frequency of polymorphonuclear (PMN)-MDSCs was significantly higher in PPE. Notably, compared to traditional markers such as the neutrophil-to-lymphocyte ratio and adenosine deaminase level, the frequency of PMN-MDSCs emerged as a more effective discriminator between PPE and TPE. PMN-MDSCs demonstrated superior positive and negative predictive values and exhibited a higher area under the curve in the receiver operating characteristic curve analysis. PMN-MDSCs in pleural effusion increased the levels of reactive oxygen species and suppressed the production of interferon-gamma from T cells following nonspecific stimulation. These findings suggest that MDSC-mediated immune suppression may contribute to the pathology of both TPE and PPE. The frequency of PMN-MDSCs in pleural fluid is a clinically useful indicator for distinguishing between TPE and PPE.

Natural killer cell subsets and their functional molecules in peripheral blood of the patients with breast cancer.

Natural killer (NK) cells, CD3- lymphocytes, are critical players in cancer immune surveillance. This study aimed to assess two types of CD3- NK cell classifications (subsets), that is, convectional subsets (based on CD56 and CD16 expression) and new subsets (based on CD56, CD27, and CD11b expression), and their functional molecules in the peripheral blood of patients with breast cancer (BC) in comparison with healthy donors (HDs). Thirty untreated females with BC and 20 age-matched healthy women were enrolled. Peripheral blood samples were collected and directly incubated with fluorochrome-conjugated antibodies against CD3, CD56, CD16, CD27, CD11b, CD96, NKG2C, NKG2D, NKp44, CXCR3, perforin, and granzyme B. Red blood cells were then lysed using lysing solution, and the stained cells were acquired on four-color flow cytometer. Our results indicated 15% of lymphocytes in peripheral blood of patients with BC and HDs had NK cells phenotype. However, the frequency of total NK cells (CD3-CD56+), and NK subsets (based on conventional and new classifications) was not significantly different between patients and HDs. We observed mean fluorescent intensity (MFI) of CXCR3 in total NK cells (p = .02) and the conventional cytotoxic (CD3-CD56dim CD16+) NK cells (p = .03) were significantly elevated in the patients with BC compared to HDs. Despite this, the MFI of granzyme B expression in conventional regulatory (CD3-CD56brightCD16- /+) NK cells and CD3-CD56-CD16+ NK cells (p = .03 and p = .004, respectively) in the patients was lower than healthy subjects. The higher expression of chemokine receptor CXCR3 on total NK cells in patients with BC may be associated with increased chemotaxis-related NK cell infiltration. However, lower expression of granzyme B in conventional regulatory NK cells and CD3-CD56-CD16+ NK cells in the patients compared to HDs suggests reduced cytotoxic activity of the NK cells in BC. These results might demonstrate accumulating NK subsets with a dysfunctional phenotype in the peripheral blood of patients with BC.

The ω-3 Polyunsaturated Fatty Acid Docosahexaenoic Acid Enhances NK-Cell Antitumor Effector Functions.

ω-3 polyunsaturated fatty acids (PUFA) are known to directly repress tumor development and progression. In this study, we explored whether docosahexaenoic acid (DHA), a type of ω-3 PUFA, had an immunomodulatory role in inhibiting tumor growth in immunocompetent mice. The number of natural killer (NK) cells but not the number of T or B cells was decreased by DHA supplementation in various tissues under physiologic conditions. Although the frequency and number of NK cells were comparable, IFNγ production by NK cells in both the spleen and lung was increased in DHA-supplemented mice in the mouse B16F10 melanoma tumor model. Single-cell RNA sequencing revealed that DHA promoted effector function and oxidative phosphorylation in NK cells but had no obvious effects on other immune cells. Using Rag2-/- mice and NK-cell depletion by PK136 antibody injection, we demonstrated that the suppression of B16F10 melanoma tumor growth in the lung by DHA supplementation was dependent mainly on NK cells. In vitro experiments showed that DHA directly enhanced IFNγ production, CD107a expression, and mitochondrial oxidative phosphorylation (OXPHOS) activity and slightly increased proliferator-activated receptor gamma coactivator-1α (PGC-1α) protein expression in NK cells. The PGC-1α inhibitor SR-18292 in vitro and NK cell-specific knockout of PGC-1α in mice reversed the antitumor effects of DHA. In summary, our findings broaden the current knowledge on how DHA supplementation protects against cancer growth from the perspective of immunomodulation by upregulating PGC-1α signaling-mediated mitochondrial OXPHOS activity in NK cells.

NK cell receptor profiling of endometrial and decidual NK cells reveals pregnancy-induced adaptations.

Natural killer (NK) cells, with a unique NK cell receptor phenotype, are abundantly present in the non-pregnant (endometrium) and pregnant (decidua) humanuterine mucosa. It is hypothesized that NK cells in the endometrium areprecursors for decidual NK cells present during pregnancy. Microenvironmental changes can alter the phenotype of NK cells, but it is unclear whether decidual NK cell precursors in the endometrium alter their NK cell receptor repertoire under the influence of pregnancy. To examine whether decidual NK cell precursors reveal phenotypic modifications upon pregnancy, we immunophenotyped the NK cell receptor repertoire of both endometrial and early-pregnancy decidual NK cells using flow cytometry. We showed that NK cells in pre-pregnancy endometrium have a different phenotypic composition compared to NK cells in early-pregnancy decidua. The frequency of killer-immunoglobulin-like receptor (KIR expressing NK cells, especially KIR2DS1, KIR2DL2L3S2, and KIR2DL2S2 was significantly lower in decidua, while the frequency of NK cells expressing activating receptors NKG2D, NKp30, NKp46, and CD244 was significantly higher compared to endometrium. Furthermore, co-expression patterns showed a lower frequency of NK cells co-expressing KIR3DL1S1 and KIR2DL2L3S2 in decidua. Our results provide new insights into the adaptations in NK cell receptor repertoire composition that NK cells in the uterine mucosa undergo upon pregnancy.

Ozanimod-mediated remission in experimental autoimmune encephalomyelitis is associated with enhanced activity of CNS CD27low/- NK cell subset.

Ozanimod (RPC1063) is an immunomodulator that has been recently approved by the FDA (2020) for the treatment of relapsing-remitting multiple sclerosis (RRMS). It is a selective agonist of the sphingosine-1-phophate receptors 1 and 5, expressed on naïve and central memory T and B cells, as well as natural killer (NK) cells, and is involved in lymphocyte trafficking. Oral administration of ozanimod was reported to result in rapid and reversible reduction in circulating lymphocytes in multiple sclerosis (MS) patients, however, only minimal effect on NK cells was observed. In this study, we sought to investigate the effect of ozanimod on NK cells and assess whether they play any role in ozanimod-induced remission in experimental autoimmune encephalomyelitis (EAE), the animal model of MS. Active EAE induction was done in C57BL/6 female mice, followed by daily oral treatment with ozanimod (0.6mg/kg) starting at disease onset (score 1). Flow cytometry of blood and CNS was performed 24 hours after the last oral dose of ozanimod treatment in diseased mice. Histological analysis of lumbar spinal cord was performed for evaluating the level of inflammation and demyelination. Depletion of peripheral NK cells was done using anti-NK1.1 mouse antibody (mAb) at day 5 post-EAE induction. Ozanimod was effective in reducing the clinical severity of EAE and reducing the percentage of autoreactive CD4+ and CD8+ T cells along with significant inhibition of lymphocyte infiltration into the spinal cord, accompanied by reversed demyelination. Furthermore, ozanimod treatment resulted in a significant increase in the frequency of total NK cells in the blood and CNS along with upregulation of the activating receptor NKG2D on CD27low/- NK cell subset in the CNS. The effectiveness of ozanimod treatment in inhibiting the progression of the disease was reduced when NK cells were depleted using anti-NK1.1 mAb. The current study demonstrated that ozanimod treatment significantly improved clinical symptoms in EAE mice. Ozanimod and anti-NK1.1 mAb appear to function in opposition to one another. Collectively, our data suggest that ozanimod-mediated remission is associated with an increased percentage of total NK cells and CD27low/- NK cells expressing the activating receptor, NKG2D in the CNS.

Age-dependent natural killer cell and interferon γ deficits contribute to severe pertussis in infant mice.

Many respiratory infections are selectively injurious to infants, yet the etiology of age-associated susceptibility is unknown. One such bacterial pathogen is Bordetella pertussis. In adult mice, innate interferon γ (IFN-γ) is produced by natural killer (NK) cells and restricts infection to the respiratory tract. In contrast, infant pertussis resembles disease in NK cell- and IFN-γ-deficient adult mice that experience disseminated lethal infection. We hypothesized that infants exhibit age-associated deficits in NK cell frequency, maturation, and responsiveness to B. pertussis, associated with low IFN-γ levels. To delineate mechanisms behind age-dependent susceptibility, we compared infant and adult mouse models of infection. Infection in infant mice resulted in impaired upregulation of IFN-γ and substantial bacterial dissemination. B. pertussis-infected infant mice displayed fewer pulmonary NK cells than adult mice. Furthermore, the NK cells in the infant mouse lungs had an immature phenotype, and the infant lung showed no upregulation of the IFN-γ-inducing cytokine IL-12p70. Adoptive transfer of adult NK cells into infants, or treatment with exogenous IFN-γ, significantly reduced bacterial dissemination. These data indicate that the lack of NK cell-produced IFN-γ significantly contributes to infant fulminant pertussis and could be the basis for other pathogen-induced, age-dependent respiratory diseases.

Persistence of KIRneg NK cells after haploidentical hematopoietic stem cell transplantation protects from human cytomegalovirus infection/reactivation.

Haploidentical hematopoietic stem cell transplantation (h-HSCT) is a therapeutic option to cure patients affected by hematologic malignancies. The kinetics and the quality of immune-reconstitution (IR) impact the clinical outcome of h-HSCT and limit the onset of life-threatening Human Cytomegalovirus (HCMV) infection/reactivation. Natural Killer (NK) cells are the first lymphocytes that recover after h-HSCT and they can provide rapid innate immune responses against opportunistic pathogens. By performing a longitudinal single-cell analysis of multiparametric flow-cytometry data, we show here that the persistence at high frequencies of CD158b1b2jneg/NKG2Apos/NKG2Cneg/NKp30pos/NKp46pos (KIRneg) NK cells is associated with HCMV infection/reactivation control. These KIRneg NK cells are "unlicensed", and are not terminal-differentiated lymphocytes appearing early during IR and mainly belonging to CD56bright/CD16neg and CD56bright/CD16pos subsets. KIRneg NK cells are enriched in oxidative and glucose metabolism pathways, produce interferon-γ, and are endowed with potent antiviral activity against HCMV ex vivo. Decreased frequencies of KIRneg NK cells early during IR are associated with clinically relevant HCMV replication. Taken together, our findings indicate that the prolonged persistence of KIRneg NK cells after h-HSCT could serve as a biomarker to better predict HCMV infection/reactivation. This phenomenon also paves the way to optimize anti-viral immune responses by enriching post-transplant donor lymphocyte infusions with KIRneg NK cells.

Distinct peripheral T-cell and NK-cell profiles in HGBL-MYC/BCL2 vs patients with DLBCL NOS

AbstractPatients with high-grade B-cell lymphoma with MYC and BCL2 rearrangements (HGBL-MYC/BCL2) respond poorly to immunochemotherapy compared with patients with diffuse large B-cell lymphoma not otherwise specified (DLBCL NOS) without a MYC rearrangement. This suggests a negative impact of lymphoma-intrinsic MYC on the immune system. To investigate this, we compared circulating T cells and natural killer (NK) cells of patients with HGBL-MYC/BCL2 (n = 66), patients with DLBCL NOS (n = 53), and age-matched healthy donors (HDs; n = 16) by flow cytometry and performed proliferation, cytokine production, and cytotoxicity assays. Compared with HDs, both lymphoma subtypes displayed similar frequencies of CD8+ T cells but decreased CD4+ T cells. Regulatory T-cell (Treg) frequencies were reduced only in patients with DLBCL NOS. Activated (HLA-DR+/CD38+) T cells, PD-1+CD4+ T cells, and PD-1+Tregs were increased in both lymphoma subtypes, but PD-1+CD8+ T cells were increased only in HGBL-MYC/BCL2. Patients with DLBCL NOS, but not patients with HGBL-MYC/BCL2, exhibited higher frequencies of senescent T cells than HDs. Functional assays showed no overt differences between both lymphoma groups and HDs. Deeper analyses revealed that PD-1+ T cells of patients with HGBL-MYC/BCL2 were exhausted with impaired cytokine production and degranulation. Patients with DLBCL NOS, but not patients with HGBL-MYC/BCL2, exhibited higher frequencies of NK cells expressing inhibiting receptor NKG2A. Both lymphoma subtypes exhibited lower TIM-3+– and DNAM-1+–expressing NK cells. Although NK cells of patients with HGBL-MYC/BCL2 showed less degranulation, they were not defective in cytotoxicity. In conclusion, our results demonstrate an increased exhaustion in circulating T cells of patients with HGBL-MYC/BCL2. Nonetheless, the overall intact peripheral T-cell and NK-cell functions in these patients emphasize the importance of investigating potential immune evasion in the microenvironment of MYC-rearranged lymphomas.

A higher number of exhausted local PD1+, but not TIM3+, NK cells in advanced endometriosis

Endometriosis (EMT) is a chronic inflammatory disease characterized by the presence and growth of endometrial-like glandular epithelial and stromal cells outside the uterus. Natural Killer (NK) cell dysfunction/exhaustion has been shown in patients with EMT. In this case-control study, we compared the frequency of exhausted PD-1 or TIM-3 positive NK cells in peripheral blood (PB) and peritoneal fluid (PF) of women with advanced endometriosis to control fertile women. PB and PF were collected from women aged 25–40 who underwent the laparoscopic procedure, including 13 stages III/IV endometriosis and 13 control samples. Multicolor flowcytometry was used to compare the frequency of PD-1 or TIM-3 positive NK (CD3−CD56+) cells in PB and PF of two groups. We demonstrated a higher percentage of PD-1+ NK cells in the peritoneal fluid of patients with endometriosis rather than controls (P-value = 0.039). This significance was related to stage IV of endometriosis (P-value = 0.047). We can not show any significant difference in the number of PD-1 or TIM-3 positive NK cells in peripheral blood. Our results suggest a local exhausted NK cell response in endometriosis that can be a leading factor in the endometriosis pathogenesis.

A Single Bout of Exercise Enhances the Efficacy of Rituximab Against Autologous Chronic Lymphocytic Leukaemia B Cells Ex Vivo By Transiently Increasing Natural Killer Cell Frequency in Blood, and Simultaneously Mobilises CD5 +CD19 +CD20 + B Cells into Blood

Chronic Lymphocytic Leukaemia (CLL) is characterised by the proliferation and accumulation of clonal B cells (B-CLL cells) and is often treated with anti-CD20 monoclonal antibody immunotherapies, including rituximab. One of the mechanism-of-action of rituximab is antibody-dependent cellular cytotoxicity (ADCC) which occurs when natural killer (NK) cells detect rituximab bound to CD20 + target cells. Rituximab often fails to induce stringent disease eradication, due in part to the diffuse distribution of clonal cells across multiple lymphoid and non-lymphoid tissues where NK cell frequency can be low. It is well established that an individual bout of aerobic exercise induces a transient relocation of lymphocytes - including NK cells and B cells - into peripheral blood. We hypothesised that this exercise-induced lymphocytosis could be harnessed to enhance the efficacy of rituximab in CLL by relocating both target and effector cells together with rituximab into blood. In this pilot study n = 20 treatment naïve patients with CLL (mean ± SD: age = 62 ± 10 years; height = 174.0 ± 7.5 cm; body mass = 83.3 ± 16.8 kg; body fat = 31.7 ± 9.8 %; blood leukocytes = 30.70 ± 22.21 ×10 9/L; anaerobic threshold = 14.1 ± 2.9 mL.kg -1.min -1) participated. Participants cycled at a moderate intensity (15% above their anaerobic threshold) for ~30-minutes, with blood samples collected pre-, post-, and 1-hour post-exercise. Given the importance of CD16 + NK cells in evoking ADCC, we enumerated NK cell subsets in blood samples collected pre, post-, and 1-hour post-exercise by flow cytometry. As expected, exercise induced a preferential increase of CD56 +CD16 + (+255%, p < 0.001) and mature, cytotoxic CD56 +CD57 +CD16 + NK cells (+322%, p < 0.001) pre- to post-exercise. Next, using immunomagnetic negative separation, NK cells and primary B-CLL cells were isolated from blood pre- and post-exercise and incubated together with heat inactivated foetal calf serum, with or without the presence of rituximab to determine specific lysis using a calcein-release assay. Rituximab mediated cell lysis increased by +129% following exercise ( p < 0.001), with no change in antibody independent NK cell lysis of B-CLL cells - independent of rituximab - following exercise ( p = 0.25). Blocking CD16 on NK cells - vital for ADCC - blunted the effects of exercise on B-CLL cell lysis ( p = 0.84). In a subset of patients ( n = 9) we also explored the effects of autologous time-point matched plasma (instead of heat inactivated foetal calf serum) on rituximab mediated ADCC, which evoked a +92% increase in lysis pre- to post-exercise ( p = 0.038). Collectively, our results suggest that augmented efficacy of rituximab mediated ADCC was driven by an increase in CD16 + NK cells. We posited that the enhancement to rituximab mediated ADCC would have greater, clinically relevant implications if there was also a concomitant exercise-induced mobilisation of B-CLL cells expressing CD20 into blood from different body tissues. Flow cytometry revealed a +63% increase in CD5 +CD19 +CD20 + B-CLL cells ( p = 0.002) in blood after exercise. Further analyses revealed that CD5 +CD19 +CD20 + B-CLL cells with a phenotype consistent with recent egress from lymphoid tissue (CD5 brightCXCR4 dim; 70%, p = 0.004) and B-CLL cells with a propensity to migrate to peripheral tissues (CD5 dimCXCR4 bright; 67%, p = 0.002) were mobilised, with no change to overall CD20 surface antigen density ( p = 1.0) - determined by median fluorescence intensity. Furthermore, exercise evoked a +69% ( p = 0.022) increase in CD5 +CD19 +CD20 + B-CLL cells expressing CD49d, which is considered one of the strongest predictors of CLL prognosis. Taken together, these data demonstrate that exercise increased the frequency of CD20 + B-CLL cells with lymphoid origins and prognostic relevance into the blood, therefore rendering them susceptible to rituximab mediated ADCC. Our results show that individual bouts of moderate intensity aerobic exercise temporarily increased the number of cytotoxic CD16 + NK cells, and CD20 + B-CLL cells in blood. Additionally, our ex vivo investigations demonstrated enhanced rituximab mediated ADCC following exercise. Thus, exercise could be explored as a means of improving clinical responses in patients receiving rituximab, and/or other anti-CD20 monoclonal antibodies such as, Obinutuzumab.

Durable natural killer cell response after three doses of SARS-CoV-2 inactivated vaccine in HIV-infected individuals.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccine can induce a potent cellular and humoral immune response to protect against SARS-CoV-2 infection. However, it was unknown whether SARS-CoV-2 vaccination can induce effective natural killer (NK) cell response in people living with human immunodeficiency virus (PLWH) and healthy individuals. Forty-seven PLWH and thirty healthy controls (HCs) inoculated with SARS-CoV-2 inactivated vaccine were enrolled from Beijing Youan Hospital in this study. The effect of SARS-CoV-2 vaccine on NK cell frequency, phenotype, and function in PLWH and HCs was evaluated by flow cytometry, and the response of NK cells to SARS-CoV-2 Omicron Spike (SARS-2-OS) protein stimulation was also evaluated. SARS-CoV-2 vaccine inoculation elicited activation and degranulation of NK cells in PLWH, which peaked at 2 weeks and then decreased to a minimum at 12 weeks after the third dose of vaccine. However, in vitro stimulation of the corresponding peripheral blood monocular cells from PLWH with SARS-2-OS protein did not upregulate the expression of the aforementioned markers. Additionally, the frequencies of NK cells expressing the activation markers CD25 and CD69 in PLWH were significantly lower than those in HCs at 0, 4 and 12weeks, but the percentage of CD16 + NK cells in PLWH was significantly higher than that in HCs at 2, 4 and 12weeks after the third dose of vaccine. Interestingly, the frequency of CD16 + NK cells was significantly negatively correlated with the proportion of CD107a + NK cells in PLWH at each time point after the third dose. Similarly, this phenomenon was also observed in HCs at 0, 2, and 4 weeks after the third dose. Finally, regardless of whether NK cells were stimulated with SARS-2-OS or not, we did not observe any differences in the expression of NK cell degranulation markers between PLWH and HCs. s:SARS-CoV-2 vaccine elicited activation and degranulation of NK cells, indicating that the inoculation of SARS-CoV-2 vaccine enhances NK cell immune response.

Markers of Natural Killer Cell Exhaustion in HIV/HCV Coinfection and Their Dynamics After HCV Clearance Mediated by Direct-Acting Antivirals.

Liver fibrosis is a leading cause of morbimortality in people with HIV/hepatitis C virus (HCV). Natural killer (NK) cells are linked with amelioration of liver fibrosis; however, NK cells from individuals coinfected with HIV/HCV with cirrhosis display impaired functionality and high PD-1 expression. Here, we aimed to study PD-1, TIGIT, and Tim3 as potential exhaustion markers in NK cells from persons coinfected with HIV/HCV with mild and advanced liver fibrosis. We also evaluated the role of PD-1 expression on NK cells after HCV clearance by direct-acting antivirals (DAAs). Peripheral blood mononuclear cells were isolated from individuals coinfected with HIV/HCV (N = 54; METAVIR F0/F1, n = 27; F4, evaluated by transient elastography, n = 27). In 26 participants, samples were collected before, at the end of, and 12 months after successful DAA treatment. The frequency, immunophenotype (PD-1, TIGIT, and Tim3 expression), and degranulation capacity (CD107a assay) of NK cells were determined by flow cytometry. Unlike PD-1, Tim3 and TIGIT were comparably expressed between persons with mild and advanced fibrosis. Degranulation capacity was diminished in NK/TIGIT+ cells in both fibrosis stages, while NK/PD-1+ cells showed a lower CD107a expression in cirrhotic cases. Twelve months after DAA treatment, those with advanced fibrosis showed an improved NK cell frequency and reduced NK/PD-1+ cell frequency but no changes in CD107a expression. In individuals with mild fibrosis, neither PD-1 nor NK cell frequency was modified, although the percentage of NK/CD107a+ cells was improved at 12 months posttreatment. Although DAA improved exhaustion and frequency of NK cells in cirrhotic cases, functionality was reverted only in mild liver fibrosis, remarking the importance of an early DAA treatment.

Gut-microbiota prompt activation of natural killer cell on alcoholic liver disease

ABSTRACT The liver is rich in innate immune cells, such as natural killer (NK) cells, natural killer T cells, and Kupffer cells associated with the gut microbiome. These immune cells are dysfunctional owing to alcohol consumption. However, there is insufficient data on the association between immune cells and gut microbiome in alcoholic liver disease (ALD). Therefore, the purpose of this study was to evaluate the effects of probiotic strains on NK cells in ALD patients. In total, 125 human blood samples [control (n = 22), alcoholic hepatitis (n = 43), and alcoholic cirrhosis (n = 60]) were collected for flow cytometric analysis. C57BL/6J mice were divided into four groups (normal, EtOH-fed, and 2 EtOH+strain groups [Phocaeicola dorei and Lactobacillus helveticus]). Lymphocytes isolated from mouse livers were analyzed using flow cytometry. The frequency of NK cells increased in patients with alcoholic hepatitis and decreased in patients with alcoholic cirrhosis. The expression of NKp46, an NK cell-activating receptor, was decreased in patients with alcoholic hepatitis and increased in patients with alcoholic cirrhosis compared to that in the control group. The number of cytotoxic CD56dimCD16+ NK cells was significantly reduced in patients with alcoholic cirrhosis. We tested the effect of oral administration P. dorei and L. helveticus in EtOH-fed mice. P. dorei and L. helveticus improved liver inflammation and intestinal barrier damage caused by EtOH supply and increased NK cell activity. Therefore, these observations suggest that the gut microbiome may ameliorate ALD by regulating immune cells.

NK cell-induced damage to P.falciparum-infected erythrocytes requires ligand-specific recognition and releases parasitophorous vacuoles that are phagocytosed by monocytes in the presence of immune IgG.

Natural killer (NK) cells lyse virus-infected cells and transformed cells through polarized delivery of lytic effector molecules into target cells. We have shown that NK cells lyse Plasmodium falciparum-infected red blood cells (iRBC) via antibody-dependent cellular cytotoxicity (ADCC). A high frequency of adaptive NK cells, with elevated intrinsic ADCC activity, in people chronically exposed to malaria transmission is associated with reduced parasitemia and resistance to disease. How NK cells bind to iRBC and the outcome of iRBC lysis by NK cells has not been investigated. We applied gene ablation in inducible erythrocyte precursors and antibody-blocking experiments with iRBC to demonstrate a central role of CD58 and ICAM-4 as ligands for adhesion by NK cells via CD2 and integrin αMβ2, respectively. Adhesion was dependent on opsonization of iRBC by IgG. Live imaging and quantitative flow cytometry of NK-mediated ADCC toward iRBC revealed that damage to the iRBC plasma membrane preceded damage to P. falciparum within parasitophorous vacuoles (PV). PV were identified and tracked with a P.falciparum strain that expresses the PV membrane-associated protein EXP2 tagged with GFP. After NK-mediated ADCC, PV were either found inside iRBC ghosts or released intact and devoid of RBC plasma membrane. Electron microscopy images of ADCC cultures revealed tight NK-iRBC synapses and free vesicles similar in size to GFP+ PV isolated from iRBC lysates by cell sorting. The titer of IgG in plasma of malaria-exposed individuals that bound PV was two orders of magnitude higher than IgG that bound iRBC. This immune IgG stimulated efficient phagocytosis of PV by primary monocytes. The selective NK-mediated damage to iRBC, resulting in release of PV, and subsequent phagocytosis of PV by monocytes may combine for efficient killing and removal of intra-erythrocytic P.falciparum parasite. This mechanism may mitigate the inflammation and malaria symptoms during blood-stage P. falciparum infection.

Effect of high dose vitamin D3 on the HIV-1 reservoir: A pilot randomised controlled trial

IntroductionAntiretroviral therapy for people living with HIV-1 must be taken lifelong due to the persistence of latent virus in long-lived and proliferating CD4+ T cells. Vitamin D3 is a steroidal gene transcription regulator which exerts diverse effects on immune and epithelial cells including reductions in CD4+ T cell proliferation and improvement in gut barrier integrity. We hypothesised that a high dose of vitamin D3 would reduce the size of the HIV-1 reservoir by reducing CD4+ T cell proliferation. MethodsWe performed a randomised placebo-controlled trial evaluating the effect of 24 weeks of vitamin D3 (10,000 international units per day) on the HIV-1 reservoir and immunologic parameters in 30 adults on antiretroviral therapy; participants were followed for 12 weeks post-treatment. The primary endpoint was the effect on total HIV-1 DNA at week 24. Parameters were assessed using mixed-effects models. ResultsWe found no effect of vitamin D3 on the change in total HIV-1 DNA from week 0 to week 24 relative to placebo. There were also no changes in integrated HIV-1 DNA, 2-long-terminal repeat (2-LTR) circles or cell-associated HIV-1 RNA. Vitamin D3 induced a significant increase in the proportion of central memory CD4+ and CD8+ T cells, a reduction in the proportion of senescent CD8+ T cells and a reduction in the natural killer cell frequency at all time points including week 36, 12 weeks after the study drug cessation. At week 36, there was a significant reduction in total HIV-1 DNA relative to placebo and persistently elevated 25-hydroxyvitamin D levels. No significant safety issues were identified. ConclusionsVitamin D3 administration had a significant impact on the T cell differentiation but overall effects on the HIV-1 reservoir were limited and a reduction in HIV-1 DNA was only seen following cessation of the study drug. Additional studies are required to determine whether the dose and duration of vitamin D3 can be optimised to promote a continued depletion of the HIV-1 reservoir over time. Trial registrationClinicalTrials.gov NCT03426592.

NK cells in peripheral blood carry trogocytosed tumor antigens from solid cancer cells.

The innate immune lymphocyte lineage natural killer (NK) cell infiltrates tumor environment where it can recognize and eliminate tumor cells. NK cell tumor infiltration is linked to patient prognosis. However, it is unknown if some of these antitumor NK cells leave the tumor environment. In blood-borne cancers, NK cells that have interacted with leukemic cells are recognized by the co-expression of two CD45 isoforms (CD45RARO cells) and/or the plasma membrane presence of tumor antigens (Ag), which NK cells acquire by trogocytosis. We evaluated solid tumor Ag uptake by trogocytosis on NK cells by performing co-cultures in vitro. We analyzed NK population subsets by unsupervised dimensional reduction techniques in blood samples from breast tumor (BC) patients and healthy donors (HD). We confirmed that NK cells perform trogocytosis from solid cancer cells in vitro. The extent of trogocytosis depends on the target cell and the antigen, but not on the amount of Ag expressed by the target cell or the sensitivity to NK cell killing. We identified by FlowSOM (Self-Organizing Maps) several NK cell clusters differentially abundant between BC patients and HD, including anti-tumor NK subsets with phenotype CD45RARO+CD107a+. These analyses showed that bona-fide NK cells that have degranulated were increased in patients and, additionally, these NK cells exhibit trogocytosis of solid tumor Ag on their surface. However, the frequency of NK cells that have trogocytosed is very low and much lower than that found in hematological cancer patients, suggesting that the number of NK cells that exit the tumor environment is scarce. To our knowledge, this is the first report describing the presence of solid tumor markers on circulating NK subsets from breast tumor patients. This NK cell immune profiling could lead to generate novel strategies to complement established therapies for BC patients or to the use of peripheral blood NK cells in the theranostic of solid cancer patients after treatment.

Bi-Specific Killer Cell Engager Enhances NK Cell Activity against Interleukin-13 Receptor Alpha-2 Positive Gliomas.

Glioblastoma (GBM) is a lethal brain tumor with limited therapeutic options. Bi-specific killer cell engagers (BiKEs) are novel immunotherapies designed to engage natural killer (NK) cells against cancer. We designed a BiKE molecule consisting of a single-domain CD16 antibody, an interleukin-15 linker, and a single-chain variable antibody against the glioma-associated antigen interleukin 13 receptor alpha 2 (IL13Rα2). Recombinant BiKE protein was expressed in HEK cells and purified. Flow cytometric analysis of co-cultures of peripheral blood-derived NK cells with GBM6 and GBM39 patient-derived xenograft lines revealed significantly increased activation of NK cells (CD25+CD69+) and increased glioma cell killing following BiKE treatment compared to controls (n = 4, p < 0.01). Glioma cell killing was also confirmed via immunofluorescence staining for cleaved caspase-3 (p < 0.05). In vivo, intracranial delivery of NK cells with BiKE extended median survival in mice bearing GBM6 (p < 0.01) and GBM12 (p < 0.01) tumors compared to controls. Finally, histological analysis of brain tissues revealed a higher frequency of peritumoral NK cells in mice treated with BiKE than with NK cells alone (p < 0.05). In conclusion, we demonstrate that a BiKE generated in a mammalian expression system is functional in augmenting NK cell targeting of IL13Rα2-positive gliomas.

Mass cytometry immune profiling to detect signatures of clinical activity of bintrafusp alfa (BA) in patients with advanced non-small cell lung cancer (NSCLC).

e21016 Background: BA, a bifunctional fusion protein targeting programed death-ligand 1 and transforming growth factor-β, is clinically active in a subset of patients (pts) with advanced NSCLC. In this study, mass cytometry was used to identify immune features associated with response and survival in pts with NSCLC treated with BA. Methods: Peripheral blood mononuclear cells were collected at baseline and after 1 and 3 doses of BA from 12 NSCLC pts treated every 2 weeks in a dose expansion biomarker cohort of a phase I clinical trial (NCT02517398). Samples were analyzed using a 44-marker mass cytometry panel designed to detect immune cell subsets and functional states of the innate and adaptive immune system. Manually gated immune cell subpopulations and unsupervised clusters were analyzed with Kruskal Wallis and Wilcoxon Rank Sum testing for multigroup comparison; and with linear correlation, univariate Cox regression and Kaplan Meier methods for progression free survival (PFS) analysis. Results: Among 12 pts, 3 had an objective response (OR), 6 had stable disease (SD) and 3 developed progressive disease (PD). Immune cell subsets associated with clinical activity by multigroup analyses included memory B cells, CD16+ natural killer (NK) cells, and double negative T cells (DNT). Pts with OR had a 2-fold higher frequency of memory B cells at baseline compared to pts who developed SD or PD. After 1 dose of BA, pts who subsequently developed an OR or SD had a 5-fold increase of CD16+ NK cells compared to pts with PD. After 3 doses of BA, pts who subsequently developed an OR had 4-fold more memory B cells and 3-fold more T-bet+ DNTs compared to pts with PD or SD. Immune cell subpopulations associated with survival included 3 unsupervised clusters annotated by the clustering algorithm as CD16+ NK cells, DNTs and classical monocytes. Fewer CD16+ NK cells at baseline were associated with longer PFS (p = 0.0053). Longer PFS was also associated with higher CD161+ CD127+ DNT cells after 1 dose of BA relative to baseline (p = 0.029), while an expansion of IgG4+ PD-1+ classical monocytes after 3 doses of BA was associated with shorter PFS (p = 0.015). Linear correlation analysis did not find strong associations between any one immune state and survival. Conclusions: Our study demonstrates the ability of mass cytometry immune profiling to detect potential immunological markers of response and survival in NSCLC pts treated with BA. The association between CD16+ NK cell frequencies and clinical benefit could reflect the ability of BA to facilitate antibody-dependent cellular cytotoxicity by NK cells in NSCLC. A high fraction of memory B cells at baseline and after 3 doses identifies pts likely to respond to BA. If validated in larger studies, these novel immune signatures can be used to improve clinical outcomes in pts with NSCLC considering immunotherapy.