Frequency Of Natural Killer Cells Research Articles

Phenotypic characterization of NK cells in 5-year-old children exposed to maternal HIV and antiretroviral therapy in early-life

BackgroundHIV-exposed uninfected (HEU) children are at increased risk of morbidity during the first years of life. Although the immune responses of HEU infants in early-life are relatively well described, studies of natural killer (NK) cells in older HEU children are lacking. NK cell subsets were analysed in HEU children and compared to those in HIV unexposed uninfected (HUU) children aged ~ five years.MethodsMulti-parametric flow cytometry was used to characterize peripheral blood-derived NK cell CD56, CD16, CD57, NKG2A and KIR3DL1/KIR2DL2/L3 expression, including intracellular perforin and granzyme B. NK cell subsets were compared between HEU children exposed to prenatal antiretroviral therapy (ART) from conception [long-term (HEULT)]; those exposed to ART during pregnancy [medium-term (HEUMT)] with continued exposure throughout the breastfeeding period and HUU peers. Furthermore, clinical data of the children, including sick clinic visits and hospitalizations documented in morbidity diaries from birth to 5 years were compared between HEU and HUU groups. Frequencies of CD56bright and CD56dim NK cell were correlated with these clinical parameters.Results139 children were enrolled however, 133 comprising 43 HEULT, 38 HEUMT and 52 HUU were included in the main analyses. Total NK cell, CD56bright nor CD56dim NK cell proportions differed between HEU and HUU children. However, HEULT children had lower frequencies of CD56dim NK cells compared to HEUMT children, (p = 0.002) which maintained significance after controlling for preterm birth, p = 0.012. No differences were observed between HEULT and HUU. The expressions of NKG2A, KIR3DL1/KIR2DL2/L3 and CD57 on CD56bright and CD56dim NK cells were similar between the three groups. Furthermore, the frequencies of granzyme B and perforin double positive NK cells were similar between the HUU with HEULT and HEUMT children. CD56dim NK cell counts had a significant moderate negative correlation with recurrent respiratory infections (rho=-0.38; p = 0.010) in HUU children and negatively correlated with total sick clinic visits in HEUMT (rho=-0.40, p = 0.064).ConclusionThe proportions of total NK cell, CD56bright and CD56dim NK cells, NK cells inhibitory and differentiation surface marker expression and cytolytic granule-positive cells were similar between HEU and HUU children. These data suggest that early-life HIV/ART exposure may not result in major changes in NK cell subsets at 5 years of age.

Dynamics of Peripheral Lymphocyte Subsets from Birth until Old Age

The immune system is inexperienced before birth and tends to be tolerogenic, rather than immunogenic. After birth, the adaptive immune system develops while facing microbial challenges, but it can become impaired as old age progresses and persistent inflammation can lead to chronic morbidity, disability and frailty. To investigate the potential contributions of lymphocyte subsets to immunity from birth until old age, we enumerated circulating innate and conventional lymphocytes and measured serum cytokine levels in 10 cord blood samples and in peripheral blood from 10 healthy term neonates, 23 healthy school-age children, 25 young adults and 11 older subjects. Flow cytometric analysis revealed that B cell frequencies increase during childhood and gradually decrease into adulthood, whereas natural killer cell frequencies increase throughout life. T cell frequencies remained relatively constant throughout life, as did their expression of CD4 and CD8. However, all four innate T cell populations studied—invariant natural killer T cells, mucosa-associated invariant T cells and the Vδ1 and the Vδ2 subsets of γδ T cells—were extremely rare in cord blood and in peripheral blood of neonates, but they expanded after birth reaching highest levels in adulthood. Analysis of serum cytokine levels revealed that proinflammatory and T helper type 1 (Th1) cytokine levels increase in adulthood, whereas Th2 and Th17 cytokine levels remain relatively constant. These changes in lymphocyte numbers and cytokine levels across the lifetime are likely to affect immunocompetence, leaving newborn and elderly people susceptible to infection, cancer and immune-mediated disease.

An altered natural killer cell immunophenotype characterizes clinically severe pediatric RSV infection.

Respiratory syncytial virus (RSV) infects nearly all children by 2 years of age and is a leading cause of pediatric hospitalizations. A subset of children with RSV infection (RSV+ children) develop respiratory failure requiring intensive care, but immune mechanisms distinguishing severe pediatric RSV infection are not fully elucidated. Natural killer (NK) cells are key innate immune effectors of viral host defense. In this study of 47 critically ill RSV+ children, we coupled NK cell immunophenotype and cytotoxic function with clinical parameters to identify an NK cell immune signature of severe pediatric RSV disease. Airway NK cells were increased in intubated RSV+ children with severe hypoxemia and prolonged duration of mechanical ventilation and were correlated with clinical severity scores. Peripheral blood NK cells were decreased in RSV+ patients and had altered activating receptor expression, with increased expression of CD69 and decreased expression of NKG2D. Ex vivo, circulating NK cells from RSV+ patients exhibited functional impairment characterized by decreased cytotoxicity as well as aberrant immune synapse assembly and lytic granule trafficking. NK cell frequency and phenotype correlated with clinical measures that defined disease severity. These findings implicate a role for NK cells in mediating RSV immunopathology and suggest that an altered NK cell immunophenotype is associated with severe RSV disease in young children.

The Expression pattern of NK cells in systemic lupus erythematosus patients with different disease activities

Data demonstrated the role of natural killer (NK) cells in systemic lupus erythematosus (SLE). We aimed to determine the immunophenotype and frequency of NK cells and their subsets, CXCR3, CD161 expression in blood and renal tissue of SLE patients with and without lupus nephritis and their relationship with disease activity. The study included 31 SLE patients and 11 controls. Study participants underwent full history and thorough clinical examination. SLE patients underwent routine laboratory investigations. Renal tissue biopsies were taken from patients with lupus nephritis. The frequency of NK cell subsets in blood of patients and controls and renal tissue from patients was performed by flow cytometry. An increase in circulatory CD56bright NK cells and its CD56bright CD16dim subtype was associated with the severity of systemic manifestations in SLE patients. Total CD56bright NK cells and its CD56bright CD16bright subtypes in renal tissues were related to renal damage. We detected decreased CD161 expression on NK cells related to renal damage and severity of systemic manifestations in SLE patients. Decreased expression of CXCR3 on NK cell surface in renal tissues causes misdirected trafficking of NK cells that seems to reduce the severity of lupus nephritis. In conclusion, our results paved the way to understanding the role of NK cells in the pathogenesis of SLE which may represent a future target for immune therapy of SLE. NK and CD56dim subset were decreased in the blood and increased in renal tissue of SLE patients, while the CD56bright subset was increased in blood and decreased in renal tissue reflecting their effects on renal damage and severity of manifestations in SLE.

MicroRNA-146a deficiency enhances host protection against murine cytomegalovirus.

Natural killer (NK) cells are innate lymphoid cells that protect a host from viral infections and malignancies. MicroRNA-146a (miR-146a) is an important regulator of immune function that is highly expressed in NK cells and is further upregulated during murine cytomegalovirus (MCMV) infection. Here we utilized mice with a global targeted deletion of miR-146a to understand its impact on the innate immune responses to MCMV infection. MiR-146a-/- mice were protected from lethal MCMV infection, which was intrinsic to the hematopoietic compartment based on bone marrow chimera experiments. NK cell depletion abrogated this protection, implicating NK cells as critical for the miR-146a-/- protection from MCMV. Surprisingly, NK cells from miR-146a-deficient mice were largely similar to control NK cells with respect to development, maturation, trafficking, and effector functions. However, miR-146a-/- mice had increased NK cell numbers and frequency of the most mature Stage IV (CD27-CD11b+) NK cells in the liver at baseline, enhanced STAT1 phosphorylation, and increased selective expansion of Ly49H+ NK cells and T cells during MCMV infection. This study demonstrates a critical role for miR-146a in the host response to MCMV, arising from mechanisms that include increased NK cell numbers and early T-cell expansion.

Polygenic polymorphism is associated with NKG2A repertoire and influences lymphocyte phenotype and function

AbstractCD94/NKG2A is a heterodimeric receptor commonly found on natural killer (NK) and T cells, and its interaction with its ligand HLA-E on adjacent cells leads to inhibitory signaling and cell suppression. We have identified several killer cell lectin–like receptor (KLR)C1 (NKG2A) single nucleotide polymorphisms (SNPs) that are associated with NKG2A expression on NK cells, CD8+ T cells, and Vγ9/Vδ2+ T cells. Additionally, due to strong linkage disequilibrium, polymorphisms in KLRC2 (NKG2C) and KLRK1 (NKG2D) are also associated with NKG2A surface density and frequency. NKG2A surface expression correlates with single-cell NK responsiveness, and NKG2A+ NK cell frequency is associated with total NK repertoire response and inhibitability, making the identification of SNPs responsible for expression and frequency important for predicting the innate immune response. Because HLA-E expression is dependent on HLA class I signal peptides, we analyzed the relationship between peptide abundance and HLA-E expression levels. Our findings revealed a strong association between peptide availability and HLA-E expression. We identified the HLA-C killer immunoglobulin–like receptor ligand epitope as a predictive marker for HLA-ABC expression, with the HLA-C1 epitope associated with high HLA-E expression and the HLA-C2 epitope associated with low HLA-E expression. The relationship between HLA-C epitopes and HLA-E expression was independent of HLA-E allotypes and HLA-B leader peptides. Although HLA-E expression showed no significant influence on NKG2A-mediated NK education, it did affect NK cell inhibition. In summary, these findings underscore the importance of NKG2A SNPs and HLA-C epitopes as predictive markers of NK cell phenotype and function and should be evaluated as prognostic markers for diseases that express high levels of HLA-E.

The impact of CLDN18.2 expression on effector cells mediating antibody-dependent cellular cytotoxicity in gastric cancer

Activating antibody-dependent cellular cytotoxicity (ADCC) by targeting claudin-18 isoform 2 (CLDN18.2) using zolbetuximab, a monoclonal antibody against CLDN18.2, has been considered a promising novel therapeutic strategy for gastric cancer (GC). However, the impact of CLDN18.2 expression on natural killer (NK) cells and monocytes/macrophages—crucial effector cells of ADCC—in GC has not been fully investigated. In the present study, we assessed the impact of CLDN18.2 expression on clinical outcomes, molecular features, and the frequencies of tumor-infiltrating NK cells and macrophages, as well as peripheral blood NK cells and monocytes, in GC by analyzing our own GC cohorts. The expression of CLDN18.2 did not significantly impact clinical outcomes of GC patients, while it was significantly and positively associated with Epstein–Barr virus (EBV) status and PD-L1 expression. The frequencies of tumor-infiltrating NK cells and macrophages, as well as peripheral blood NK cells and monocytes, were comparable between CLDN18.2-positive and CLDN18.2-negative GCs. Importantly, both CLDN18.2 expression and the number of tumor-infiltrating NK cells were significantly higher in EBV-associated GC compared to other molecular subtypes. Our findings support the effectiveness of zolbetuximab in CLDN18.2-positive GC, and offer a novel insight into the treatment of this cancer type, highlighting its potential effectiveness for CLDN18.2-positive/EBV-associated GC.

NK cells with adhesion defects and reduced cytotoxic functions are associated with a poor prognosis in multiple myeloma

AbstractThe promising results obtained with immunotherapeutic approaches for multiple myeloma (MM) call for a better stratification of patients based on immune components. The most pressing being cytotoxic lymphocytes such as natural killer (NK) cells that are mandatory for MM surveillance and therapy. Here, we performed a single-cell RNA sequencing analysis of NK cells from 10 patients with MM and 10 age/sex–matched healthy donors that revealed important transcriptomic changes in the NK cell landscape affecting both the bone marrow (BM) and peripheral blood compartment. The frequency of mature cytotoxic CD56dim NK cell subsets was reduced in patients with MM at the advantage of late-stage NK cell subsets expressing NF-κB and interferon-I inflammatory signatures. These NK cell subsets accumulating in patients with MM were characterized by low CD16 and CD226 expression and poor cytotoxic functions. MM CD16/CD226Lo NK cells also had adhesion defects with reduced lymphocyte function-associated antigen 1 (LFA-1) integrin activation and actin polymerization that may account for their limited effector functions in vitro. Finally, analysis of BM-infiltrating NK cells in a retrospective cohort of 177 patients with MM from the Intergroupe Francophone du Myélome (IFM) 2009 trial demonstrated that a high frequency of NK cells and their low CD16 and CD226 expression were associated with a shorter overall survival. Thus, CD16/CD226Lo NK cells with reduced effector functions accumulate along MM development and negatively affect patients’ clinical outcomes. Given the growing interest in harnessing NK cells to treat myeloma, this improved knowledge around MM–associated NK cell dysfunction will stimulate the development of more efficient immunotherapeutic drugs against MM.

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.

Clinical, genomic and immune microenvironmental determinants of nivolumab response in head and neck squamous cell carcinoma.

In view of improving biomarkers predicting the efficacy of immunotherapy for head and neck squamous cell carcinoma (R/M HNSCC), this multicenter retrospective study aimed to identify clinical, tumor microenvironmental, and genomic factors that are related to therapeutic response to the anti- Programmed cell death protein 1 (PD-1) antibody, nivolumab, in patients with R/M HNSCC. The study compared 53 responders and 47 non-responders, analyzing formalin-fixed paraffin-embedded samples using 14-marker multiplex immunohistochemistry and targeted gene sequencing. Of 100 patients included, responders had significantly lower smoking and alcohol index, higher incidence of immune related adverse events, and higher PD-1 ligand (PD-L1) expression in immune cells as well as PD-L1 combined positive score (CPS) than non-responders. The frequency of natural killer cells was associated with nivolumab response in patients with prior cetuximab use, but not in cetuximab-naïve status. Age-stratified analysis showed nivolumab response was linked to high CPS and lymphoid-inflamed profiles in patients aged ≥ 65. In contrast, lower NLR in peripheral blood counts was associated with response in patients aged < 65. Notably, TP53 mutation-positive group had lower CPS and T cell densities, suggesting an immune-excluded microenvironment. Patients with altered tumor suppressor gene pathways, including TP53, CDKN2A, and SMAD4 mutations, had lower CPS, higher smoking index, and were associated with poor responses. Nivolumab treatment efficacy in HNSCC is influenced by a combination of clinical factors, age, prior treatment, immune environmental characteristics, and gene mutation profiles.

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.