Percentage Of Natural Killer Cells Research Articles

Protein phosphatase 2A inhibitor modulates natural killer cell homeostasis in peripheral tissues

Although natural killer (NK) cell responses to tumor and viral infection have been studied, the mechanisms underlying NK cell homeostasis in vivo remain unclear. In this study, we demonstrate the pharmacological action of cytostatin, a protein phosphatase 2A (PP2A) specific inhibitor (PP2Ai), on NK cells in regulating NK cell homeostasis in the peripheral tissues. We found that PP2Ai treatment decreased NK cell percentages in the bone marrow and secondary lymphoid tissues while increasing NK cell percentages in peripheral tissues such as the lung and liver. In the peripheral tissues of PP2Ai-treated mice, Ki-67 expression and BrdU uptake in NK cells were upregulated, and an initial increase in the pre-mature CD11bhiCD27hi NK subset was observed, followed by an increase in the terminally differentiated mature CD11bhiCD27lo NK subset. In addition, bone marrow Ki-67+ NK cells predominantly expressed CX3CR1 in the PP2Ai-treated mice and were further mobilized to the peripheral tissues. Among various target molecules of PP2A, we found that the upregulation of c-Myc pathway and its phosphorylation, along with its downstream cyclin E expression and G1/S cell cycle transition in PP2Ai-treated mice NK cells. Our results suggest that PP2Ai modulates NK cell proliferation through c-Myc and cyclin E, leading to their maturation and trafficking from the bone marrow to the peripheral tissues.

HIV-1 Elite Controllers are Characterised by Elevated Levels of CD69-Expressing Natural Killer Cells.

Human immunodeficiency virus type 1 (HIV-1) elite controllers (ECs) are a rare subset of people living with HIV-1 (PLWH) who control viral replication in the absence of antiretroviral therapy (ART) and may provide a model for a functional cure. We investigated the role of natural killer (NK) cells in HIV-1 ECs from South Africa. Phenotypic (CD69, CD38, CD57, PD-1), functional (CD107a, IFN-γ), and nutrient transporter profiles (glucose transporter 1, CD98) of NK cells from ECs (n=20), viraemic progressors (VPs; n=19), people living with HIV-1 (PLWH) on ART (n=20), and people without HIV-1 (PWOH; n=21) were analysed using flow cytometry. The Kruskal-Wallis test followed by the Mann-Whitney U test were used to determine differences among the study groups. The Spearman's rank correlation coefficient was used to determine significant associations. Compared to the other study groups, the percentage of CD69-expressing NK cells was higher in ECs, whereas the percentage of CD38-expressing NK cells was higher in VPs. Percentages of CD69+CD38- NK cells were elevated in ECs compared to VPs (p = 0.003), but were not different to PLWH on ART and PWOH. Differentiation, exhaustion, and metabolic profiles were not different in ECs compared with PLWH on ART and PWOH, however, NK cell function was lower than in PWOH. These findings demonstrate that NK cells from ECs have an activated, mature profile with low levels of immune exhaustion and a reduced metabolic phenotype suggesting functional competence. This insight could inform the development of novel immunotherapeutic strategies for treating HIV-1.

The incidence and clinical significance of monoclonal and oligoclonal protein bands in multiple myeloma patients after BCMA-CAR-T cell therapy: A retrospective study based on LEGEND-2.

The emergence of abnormal protein bands (APBs), also known as oligoclonal protein bands, has been documented in patients with multiple myeloma (MM) post hematopoietic stem cell transplantation. However, the incidence rate and clinical significance of APBs remain contentious. Few studies have explored the occurrence and prognostic implications of APBs in patients with MM treated with B-cell maturation antigen (BCMA)-specific chimeric antigen receptor (CAR)-T therapy. In this retrospective study, we examined the frequency, isotypes, and duration of APBs, as well as their correlation with MM disease characteristics, treatment response, clinical outcomes, and immune signature in patients with relapsed/refractory MM who had received LCAR-B38M therapy at the Xi'an site of the phase 1 LEGEND-2 trial. Among 47 patients assessed, 23 (48.9%) developed APBs following CAR-T therapy, with IgG being the most common isotype. The median onset and duration of APBs post-CAR-T infusion were 3.6 and 5.8 months, respectively. Patients with APBs demonstrated significantly improved response to LCAR-B38M therapy, along with longer overall and progression-free survival. Furthermore, those with APBs exhibited enhanced recovery rates of immunoglobulins and higher absolute counts of white blood cells, neutrophils, and lymphocytes post-CAR-T treatment compared to those without APBs. However, no significant differences were observed between the two groups in the percentages of various T-cell subsets and natural killer cells. Overall, the presence of APBs in patients with MM following CAR-T treatment was associated with deeper remission and a more favorable prognosis, suggesting a robust humoral response and subsequent immune reconstitution.

The Impact of Different Anesthetics on the Distribution and Cytotoxic Function of NK Cell Subpopulations: An In Vitro Study

Only some subpopulations of natural killer (NK) cells have cytotoxic functionality, and the effects of anesthetics on these subpopulations are unknown. This study aimed to evaluate the in vitro effects of various anesthetics, both alone and in combination, on the distribution and cytotoxic function of NK cells and their subpopulations. Peripheral blood mononuclear cells (PBMCs) from eight healthy volunteers were treated for 4 h in vitro with dexmedetomidine, remifentanil, lidocaine, propofol, sevoflurane, and combinations in clinically relevant concentrations or left untreated. Flow cytometry was used to quantify the percentage of sampled NK cells and evaluate their distribution (CD56brightCD16neg, CD56brightCD16dim, CD56dimCD16neg, CD56dimCD16bright, and CD56negCD16bright) and cytotoxicity (Granzyme B (GrzB) and perforin) of NK cell subpopulations. Although the percentage of total NK cells did not change following exposure to anesthesia, the most important cytotoxic subpopulation (CD56dimCD16bright NK cells) decreased after exposure to both propofol (−3.58%, p = 0.045) and sevoflurane (−16.10%, p = 0.008) alone, and most combinations, especially in combination with lidocaine (propofol with lidocaine (−9.66%, p = 0.002) and sevoflurane with lidocaine (−21.90%, p < 0.001)). Dexmedetomidine and remifentanil had no effect on CD56dimCD16bright NK cells. Furthermore, no anesthetic regimen or combination altered the expression of GrzB and perforin in NK cells or NK cell subpopulations. In short, propofol and sevoflurane suppressed the highly cytotoxic phenotype (CD56dimCD16bright) of NK cells, with those exposed to sevoflurane combinations showing greater reductions. Immunosuppression was intensified with the inclusion of lidocaine in the anesthetic regimen.

High expression of CD3+T-lymphocytes in cerebrospinal fluid increases the risk of critical cerebral hemorrhage with systemic inflammatory response syndrome (SIRS) after surgery

ObjectiveTo assess the frequency of lymphocyte subsets and other laboratory indicators in paired cerebrospinal fluid (CSF) and peripheral blood (PB) samples from critically ill patients with intracerebral hemorrhage (ICH) who developed systemic inflammatory response syndrome (SIRS) following surgery. IntroductionNeuroinflammation and systemic inflammatory responses significantly contribute to secondary brain injury following ICH. Post-surgery SIRS is known to worsen clinical outcomes in ICH patients; however, the immune response in the CSF and PB has not been fully characterized. Understanding immunological changes in ICH patients with SIRS could lead to improved clinical management and prognostic outcomes. MethodsThis study involved a retrospective analysis of data from patients with ICH who underwent surgery in the Neurological Intensive Care Unit (NICU) of Baoding No. 1 Hospital, Hebei Province, China, between January and July 2022. Patients were divided into SIRS and non-SIRS groups based on the clinical criteria. Demographic, clinical, and laboratory data, including lymphocyte subsets in CSF and PB, were collected and analyzed. This study compared lymphocyte subsets and other inflammatory markers between the SIRS and non-SIRS groups. ResultsPatients with SIRS demonstrated higher systolic blood pressure (SBP) at admission, worse 90-day prognoses, elevated inflammatory markers, increased levels of complement proteins C3 and C4, and lower levels of immunoglobulin G (IgG) compared to patients without SIRS. Between 3–6 days post-surgery, SIRS patients showed higher percentages of CD3+T cells, CD4+T cells, and CD4+/CD8+ ratios in the CSF than non-SIRS patients. CD3+T cell percentages in the CSF were consistently higher than those in the PB and were independent of PB levels. In contrast, CD3-CD16+CD56+ natural killer (NK) cell percentages were lower in patients with SIRS. No significant differences in PB lymphocyte subsets were found between the two groups. A high CSF CD3+T cell percentage (≥85.68 %) was identified as the strongest predictor of critical ICH with SIRS after surgery, with an appropriate use criterion (AUC) of 0.7742, sensitivity of 77.42 %, specificity of 76.19 %, and 95 % CI of 0.6655–0.8829 (P < 0.0001). ConclusionElevated levels of CD3+T lymphocytes in CSF are strongly associated with an increased risk of severe cerebral hemorrhage and SIRS following surgery. These findings suggest that monitoring CSF immune markers, particularly CD3+T lymphocytes, could serve as valuable predictors for the development of SIRS in critically ill ICH patients and inform post-surgical treatment strategies.

Evaluation of the natural killer cell subsets and their relationship with serum interferon gamma and vitamin D levels in women with stages III and IV endometriosis: A case-control study.

Natural killer (NK) cells play a critical role in the pathogenesis of endometriosis. Moreover, a normal vitamin D level is remarkably associated with an optimal immune response. So, there may be a probable relationship between these factors and the endometriotic women. This study aimed to evaluate the percentage of NK cells and their subsets and their relationship with serum levels of vitamin D and interferon-gamma (IFN-γ) in women with endometriosis. In this case-control study, 29 women with stage III-IV endometriosis and 30 healthy controls were enrolled. The study was conducted in the Immunology Department of Isfahan University of Medical Sciences, Isfahan, Iran between November 2021 and June 2022. The percentage of NK cells and their subsets, including CD56 CD16 , CD56 CD16 and CD56 CD16 were measured in the peripheral blood samples using flow cytometry. Serum levels of vitamin D and IFN-γ were also measured using the enzyme-linked immunosorbent assay. The mean percentage of NK cells in women with endometriosis increased significantly compared to the control group (p = 0.03). The percentage of CD56 CD16 (p = 0.007) and CD56 CD16 (p = 0.043) increased significantly in women with endometriosis in comparison with the control group, but the percentage of CD56 CD16 subset was not significantly different. No relationship was observed between NK cells and their subsets with vitamin D and IFN-γ in the studied groups. The study of NK cell subsets and their related factors can be useful in assessing and treating women suffering from endometriosis. However, more comprehensive studies are required to draw definitive conclusions about these observations.

In-Depth Analysis of the Peripheral Immune Profile of HER2+ Breast Cancer Patients on Neoadjuvant Treatment with Chemotherapy Plus Trastuzumab Plus Pertuzumab.

Currently, therapy for early-stage human epidermal growth factor receptor 2-positive (HER2+) breast cancer (BC) is based on the combination of trastuzumab and pertuzumab plus chemotherapy in a neoadjuvant regimen. The INMUNOHER study aimed to detect immunological markers in peripheral blood and their association with treatment response. Sixty-two HER2+ BC patients were recruited. Pre-treatment samples were obtained before the start of treatment, while post-treatment samples were obtained after completing therapy and before surgery and were analyzed by flow cytometry. The pathologic complete response (pCR) rate achieved was 82.3%. The expression of the NKp30, PD-1, and TIM-3 receptors was reduced in the Natural Killer (NK)-CD56dim subset of patients who did not achieve pCR. Following therapy, many changes were found in leukocytes, including alterations in T cell lymphocyte proportions. Also, the percentage of NK cells decreased, and several phenotypic changes were observed in this population. After treatment, IFN-γ production by NK cells against HER2+-cells with or without trastuzumab was significantly reduced. HER2-targeted therapy plus chemotherapy demonstrated high efficacy in most patients, reducing the statistical power for finding immunological markers. However, NK subset phenotypes correlated better with response groups, and numerous changes in the percentage of leukocytes and T and NK cells, as well as changes in the functionality of NK cells, were observed in most patients after treatment, encouraging further research into these immune populations.

Accumulation of CD56+ CD16- Natural Killer Cells in Response to Preoperative Chemotherapy for Breast Cancer.

The activation of the antitumor immune responses of T cells and natural killer (NK) cells is important to induce breast tumor shrinkage via preoperative chemotherapy. We evaluated how antitumor immune responses contribute to the effects of such therapy. Forty-three patients with stages I - IV breast cancer who underwent surgery between August 2018 and Jun 2023 after preoperative chemotherapy were enrolled. Peripheral natural killer (pNK) cell activity was assessed by 51Cr-release assay, and the counts and percentages of CD4+, CD8+, and NK cells and their subsets in peripheral blood were measured before and after chemotherapy by two-color flow cytometry. Associations of cell population changes with chemotherapy responses were analyzed. On univariate analysis, relative to grade (G) ≤ 1 effects, G ≥ 2 therapeutic effects were associated significantly with human epidermal growth factor receptor 2 (HER-2)+ breast cancer (P = 0.024) and post-chemotherapy CD56+ CD16- NK cell accumulation (8.4% vs. 5.5%, P = 0.042), and tended to be associated with increased pre-chemotherapy CD56+ CD16- NK cell percentages (5.4% vs. 3.3%, P = 0.054) and pNK cell activity (42.0% vs. 34.5%, P = 0.057). The accumulation and increased percentage of CD56+ CD16- NK cells in patients with G ≥ 2 effects were not associated with changes in pNK cell activity or the disappearance of axillary lymph-node metastases. On multivariate analysis, G ≥ 2 therapeutic effects tended to be associated with higher pre-chemotherapy pNK levels (odds ratio = 0.96; 95% confidence interval: 0.921 - 1.002; P = 0.067). The accumulation of the immunoregulatory CD56+ CD16- NK cell subset in the peripheral blood before and after chemotherapy may lead to the production of cytokines that induce an antitumor immune response. Activation of the immune response mediated by CD56+ CD16- pNK cells after chemotherapy and their high counts before chemotherapy may contribute to the improvement of therapeutic effects against breast cancer.

Addition of CD14 improves discrimination of lymphocytes in the TBNK phenotyping panel.

Peripheral blood lymphocyte phenotyping panels typically include CD45 for discrimination of the lymphocyte population, and fluorophore-conjugated monoclonal antibodies to identify T, B, and Natural Killer (NK) cells. While CD45 combined with side scatter is generally sufficient to clearly distinguish lymphocytes from monocytes in the majority of peripheral blood samples, it is challenging to accurately gate lymphocytes in samples from patients with monocytosis or significant lymphopenia, or from very young infants. Addition of a monocyte marker to lymphocyte phenotyping panels for monocyte exclusion has previously been evaluated for improved discrimination of lymphocytes, albeit largely in healthy donor adult samples. Here we evaluate the effect of the addition of CD14 to a standard lymphocyte phenotyping panel on total lymphocyte, T, B, and NK cell percentages in a predominantly pediatric population of patients under evaluation chiefly for immunodeficiency, immune-depletion, or immune reconstitution. Addition of CD14 to the standard lymphocyte phenotyping improved discrimination of lymphocytes from monocytes, resulted in decreased NK cell percentages, likely because CD16+ and/or CD56+ monocytes were included in the CD56+CD16+ NK cell gate with conventional gating, and although less significant, resulted in an increased percentage of B cells, since relatively larger B cells were likely gated out by more restrictive light scatter gating used with the conventional gating approach. The change in NK and B cell percentages were more pronounced in samples from patients below a year of age, and in patients who were relatively lymphopenic. These data suggest that addition of CD14 to conventional lymphocyte phenotyping panels that utilize CD45 versus side scatter gating results in significant improvement in the accuracy of lymphocyte gating, and accurate quantification of NK and B cells particularly in samples from infants and lymphopenic individuals.

Overactivated MX1 Positive Natural Killer Cells Promote the Progression of Sepsis-Induced Acute Respiratory Distress Syndrome.

Natural killer (NK) cells are key regulators of immune defense in sepsis-induced acute respiratory distress syndrome (ARDS), yet the characteristics of NK cell clusters in ARDS remain poorly understood. A prospective and observational study enrolled septic patients with ARDS or not was conducted to determine the percentage of NK cells via flow cytometry. The transcriptomes of peripheral blood mononuclear cells (PBMCs) from healthy controls, patients with sepsis only, and patients with sepsis-induced ARDS were profiled. Vitro experiments were performed to confirm the mechanism mediating MX1+NK cell infiltration. A total of 115 septic patients were analyzed, among whom 63 patients developed ARDS and 52 patients did not. Decreased NK percentages were found in sepsis with ARDS patients (%, 7.46±4.40 vs 11.65±6.88, P=0.0001) compared with sepsis-only patients. A lower percentage of NK cells showed a significant increase in 28-day mortality. Single-cell sequencing analysis revealed distinct characteristics of NK cells in sepsis-induced ARDS, notably the identification of a unique cluster defined as MX1+NK cells. Flow cytometry analysis showed an elevated percentage of MX1+NK cells specifically in individuals with sepsis-induced ARDS, compared with patients with sepsis only. Pseudo-time analysis showed that MX1+NK cells were characterized by upregulation of type I interferon-induced genes and other pro-inflammatory genes. MX1+NK cells can respond to type I interferons and secrete type I interferons themselves. Ligand-receptor interaction analysis also revealed extensive interaction between MX1+NK cells and T/B cells, leading to an uncontrolled inflammatory response in ARDS. MX1+NK cells can respond to type I interferons and secrete type I interferons themselves, promoting the development of sepsis-induced ARDS. Interfering with the infiltration of MX1+NK cells could be a therapeutic approach for this disease. Due to the limited sample size, a larger sample size was needed for further exploration.

A comparative study of general and severe mycoplasma pneumoniae pneumonia in children

ObjectivesThe increasing prevalence of severe Mycoplasma pneumoniae pneumonia (SMPP) poses a significant threat to the health of children. This study aimed to characterise and assess the outcomes in children with SMPP.MethodsWe retrospectively analysed children hospitalised for M. pneumoniae pneumonia (MPP) between January and December 2022. Retrospectively, demographic, clinical, underlying diseases, laboratory and radiological findings, and treatment outcomes were collected and analysed. Disease severity was defined as severe or general according to the Guideline for diagnosis and treatment of community-acquired pneumonia in children (2019 version).ResultsOver a 12-month observation period, 417 children with MPP were enrolled, 50.6% (211/417) of whom had SMPP, with the peak incidence observed in winter. Of the 211 children with SMPP, 210 were treated and discharged with improvement, while one child with congenital heart disease died of cardioembolic stroke. A significantly higher proportion of patients with SMPP had underlying diseases, extrapulmonary complications (myocardial and digestive system involvement), and bacterial co-infection. A total of 25 (12%) children with SMPP received mechanical ventilation. The median duration of mechanical ventilation was 3 days. All children were treated with macrolide antibiotic. A significantly higher proportion of patients with SMPP received antibiotic other than macrolides, methylprednisolone sodium succinate, intravenous immunoglobulin and anticoagulation, compared with patients with general MPP (GMPP). Children with SMPP had significantly higher levels of white blood cells, neutrophil percentage, C-reactive protein, procalcitonin, interferon-γ, interleukin (IL)-2, IL-5, IL-6, IL-8, IL-10 and significantly lower percentages of lymphocytes, monocytes, and natural killer cells, compared with GMPP group.ConclusionOur findings suggest that severely ill children have more pronounced inflammatory reaction and extrapulmonary complications. For effective management of children with SMPP, hormonal, prophylactic, anticoagulant therapy, as well as the use of antibiotics other than macrolides for bacterial co-infections, could be incorporated into treatment regimens.

Peripheral NK cells identified as the predictor of response in extensive-stage small cell lung cancer patients treated with first-line immunotherapy plus chemotherapy.

Although immunotherapy improves outcomes in extensive-stage small-cell lung cancer (ES-SCLC), the search for biomarkers predicting treatment success is crucial. Natural killer (NK) cells are potential indicators in various cancers, however, their precise role in ES-SCLC prognosis remains unclear. In this retrospective study, 33 patients with ES-SCLC treated with first-line immuno-chemotherapy were enrolled. The peripheral NK cell percentage and its longitudinal dynamics were analyzed using flow cytometry. Progression-free survival (PFS) and overall survival (OS) were calculated as hazard ratio (HR) and compared statistically. The median PFS was better in the group with normal baseline NK cell levels than the low group (7.0 vs. 4.6months; HR = 0.17; 95% CI 0.07-0.41; P < 0.0001), but there was no association with OS (14.9 vs. 10.3months; HR = 0.55; 95% CI 0.23-1.31; P = 0.171). Furthermore, the NK cell% for 95.0% of patients increased after immunochemotherapy in the clinical response group (P = 0.0047), which led to a better median PFS (6.3 vs. 2.1months; HR = 0.23; 95% CI 0.05-0.98; P < 0.0001) and OS (14.9 vs. 5.9months; HR = 0.20; 95% CI 0.04-1.02; P < 0.0001). Similar trends were observed with NK cell% changes up to disease progression, improving PFS (6.5 vs. 4.3; HR = 0.41; 95% CI 0.12-0.92; P = 0.0049) and OS (17.4 vs. 9.7; HR = 0.42; 95% CI 0.17-1.02; P < 0.0001). In patients with ES-SCLC, the percentage and changes in peripheral NK cells can predict the response to combined immunotherapy and chemotherapy.

Reduced expressions of apoptosis-related proteins TRAIL, Bcl-2, and TNFR1 in NK cells of juvenile-onset systemic lupus erythematosus patients: relations with disease activity, nephritis, and neuropsychiatric involvement.

Lupus pathogenesis is mainly ascribed to increased production and/or impaired clearance of dead cell debris. Although self-reactive T and B lymphocytes are critically linked to lupus development, neutrophils, monocytes, and natural killer (NK) cells have also been implicated. This study assessed apoptosis-related protein expressions in NK cells of patients with juvenile-onset systemic lupus erythematosus (jSLE) and relations to disease activity parameters, nephritis, and neuropsychiatric involvement. Thirty-six patients with jSLE, 13 juvenile dermatomyositis (JDM) inflammatory controls, and nine healthy controls had Fas, FasL, TRAIL, TNFR1, Bcl-2, Bax, Bim, and caspase-3 expressions in NK cells (CD3-CD16+CD56+) simultaneously determined by flow cytometry. Disease activity parameters included Systemic Lupus Erythematosus Disease Activity Index 2000 (SLEDAI-2K) score, erythrocyte sedimentation rate, C-reactive protein level, anti-double strain DNA antibody level, complement fractions C3 and C4 levels. Patients with jSLE had a profile of significantly reduced expression of TRAIL, Bcl-2, and TNFR1 proteins in NK cells when compared to healthy controls. Similar profile was observed in patients with jSLE with active disease, positive anti-dsDNA, nephritis, and without neuropsychiatric involvement. Patients with jSLE with positive anti-dsDNA also had reduced expression of Bax in NK cells when compared healthy controls and to those with negative anti-dsDNA. Yet, patients with jSLE with negative anti-dsDNA had reduced mean fluorescence intensity (MFI) of Bim in NK cells compared to healthy controls. Patients with jSLE with nephritis also had reduced MFI of Fas in NK cells when compared to those without nephritis. In addition, in patients with jSLE, the proportion of FasL-expressing NK cells directly correlated with the SLEDAI-2K score (rs = 0.6, p = 0.002) and inversely correlated with the C3 levels (rs = -0.5, p = 0.007). Moreover, patients with jSLE had increased NK cell percentage and caspase-3 protein expression in NK cells when compared to JDM controls. This study extends to NK cells an altered profile of TRAIL, Bcl-2, TNFR1, Fas, FasL, Bax, Bim, and caspase-3 proteins in patients with jSLE, particularly in those with active disease, positive anti-dsDNA, nephritis, and without neuropsychiatric involvement. This change in apoptosis-related protein expressions may contribute to the defective functions of NK cells and, consequently, to lupus development. The full clarification of the role of NK cells in jSLE pathogenesis may pave the way for new therapies like those of NK cell-based.

Immune cells at the feto-maternal interface: Comprehensive characterization and insights into term labor

Immune cells at the feto-maternal interface play an important role in pregnancy; starting at implantation, maintenance of pregnancy, and parturition. The role of decidual immune cells in induction of labor still needs to be understood. Published reports on this topic show heterogeneity in methods of cell isolation, assay, analysis and cellular characterization making it difficult to collate available information in order to understand the contribution of immune cells at term leading to parturition. In the present study, available literature was reviewed to study the differences in immune cells between the decidua basalis and decidua parietalis, as well as between immune cells in term and preterm labor. Additionally, immune cells at the decidua parietalis were isolated from term not in labor (TNL) or term in labor (TL) samples and characterized via flow cytometry using a comprehensive, high-dimensional antibody panel. This allowed a full view of immune cell differences without combining multiple studies, which must include variation in isolation and analysis methods, for more conclusive data. The ratio of cells found in decidua parietalis in this study generally matched those reported in the literature, although we report a lower percentage of natural killer (NK) cells at term. We report that CD4 expression on CD8- NK cells decreased in term labor compared to not in labor samples, suggesting that natural killer cells may be migrating to other sites during labor. Also, we report a decrease in CD38 expression on CD8+ CD57+ T cells in labor, indicative of cytotoxic T cell senescence. Our study provides a comprehensive status of immune cells at the decidua-chorion interface at term.

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.

Analysis and validation of potential ICD-related biomarkers in development of myopia using machine learning.

We aimed to identify and verify potential biomarkers in the development of myopia associated with immunogenic cell death (ICD). We download high myopia (HM) dataset GSE136701 from Gene Expression Omnibus. Differentially expressed genes in HM were identified to overlapped with ICD-related genes. Least absolute shrinkage and selection operator were used to select the Hub genes. Furthermore, the correlation between the hub genes and immune infiltration, immune response activities, and hub genes Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways analysis was investigated using Spearman's rank correlation. Prediction of the miRNAs upstream of the Hub genes was based on the TargetScan database. We used guinea pig lens-induced myopia model's scleral tissues performed quantitative real-time polymerase chain reaction. We identified overlapped with ICD-related genes (LY96, IL1A, IL33, and AGER) and two genes (LY96 and AGER) as hub genes. Single sample gene set enrichment analysis and Spearman's rank correlation revealed that hub gene expression levels in HM were significantly correlated with the infiltration percentages of CD56dim natural killer cells, macrophages, immature B cells, and the immune response activities of APC co-stimulation and Kyoto Encyclopedia of Genes and Genomes pathways, such as terpenoid backbone biosynthesis, aminoacyl-trna biosynthesis, Huntington's disease, oxidative phosphorylation; there were a few additional signaling pathways compared to normal samples. Additionally, several miRNA were predicted as upstream regulators of LY96 and AGER. LY96 was identified as a significantly differentially expressed biomarker in myopia guinea pig's scleral tissues, as verified by qPCR. LY96 was identified and verified as a ICD-related potential myopia biomarker. Molecular mechanisms or pathways involved in myopia development by LY96 requires further research.

Association of lymphocyte subsets and cytokines with bone metabolism: a retrospective, cross-sectional study

BackgroundPrevious research has shown that lymphocytes and cytokines can mediate bone metabolism. This study explored the clinical association and predictive ability of lymphocytes and cytokines levels for bone metabolism.MethodsA total of 162 patients were enrolled in this study. The levels of N-terminal propeptide of type I procollagen (P1NP), β-collagen degradation product (β-CTX), total T lymphocytes, immature T lymphocytes, suppressor/cytotoxic T lymphocytes, helper/inducer T lymphocytes, B lymphocytes, natural killer (NK) cells, Interferon-gamma (IFN-γ), tumour necrosis factor-alpha (TNF-α), IFN-α, interleukin-1 beta (IL-1β), IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, and IL12p70 were evaluated. The relationship between these lymphocyte subsets and cytokines with bone metabolic status was examined and their predictive ability for bone metabolic status was assessed.ResultsThe principal component analysis (PCA) and correlation analysis results varied on differences in lymphocyte subsets and cytokines in various bone metabolism states. Differential analysis revealed significant differences in the absolute counts of B lymphocytes (P < 0.05), level of IL-12p70 (P < 0.05), and IL-8 (P < 0.001) at different P1NP levels. Significant differences were observed in the absolute counts of total T lymphocytes (P < 0.05), B lymphocytes (P < 0.05), the level of IL-6 (P < 0.05), the percentage of B lymphocytes (P < 0.01), and NK cells (P < 0.05) at different β-CTX levels. Furthermore, the receiver operating characteristic (ROC) curve showed that the absolute count of B lymphocytes and levels of IL-12p70 and IL-8 could be used to evaluate bone formation states, while the absolute counts of T and B lymphocytes, level of IL-6, and percentages of NK cells and B lymphocytes could be used to evaluate bone resorption states.ConclusionThe bone metabolism status changed based on the lymphocyte subsets and cytokine levels. Differentially expressed lymphocytes and cytokines could be used to distinguish bone metabolism status.

Evaluation of klotho expression on peripheral blood lymphocytes among hemodialysis patients and its possible contribution to their immuno-compromised status

Infection is the second most common cause of mortality among end-stage kidney disease (ESKD) patients. Uremic toxins are the main cause of impaired immune response among ESKD patients. Klotho gene, the anti-aging gene, encodes the transmembrane alpha klotho (αKL) protein which acts as an obligate coreceptor for fibroblast growth factor 23 (FGF23). Klotho protein may play a role in immune cell functions, particularly in anti-inflammatory response; however, its role is still incompletely understood. In the present study, we aimed to measure αKL protein expression on peripheral blood lymphocytes (PBLs) among hemodialysis (HD) patients, and we assumed that decreased αKL expression on PBLs may contribute to the impaired immunity among HD patients. This case-control study included 20 ESKD patients on regular hemodialysis for more than 3 months. Their ages ranged from 24 to 69 years. Patients with primary immunodeficiencies, those on systemic immunosuppressive drugs, those with ongoing infections or who had recently recovered from infections, and those with malignancies on active treatment were excluded. A control group of 20 normal subjects of comparable age and gender were also included. We compared αKL protein expression on PBLs by flow cytometry between both groups. Significant reductions in percentages of αKL protein expression on B lymphocytes (CD19), T lymphocytes (CD3), and natural killer cells (CD56) were observed among HD patients compared to controls. We also noticed a significant reduction in the percentages of natural killer cells among HD patients. The present study suggests that decreased αKL expression on PBLs may contribute to the immunocompromised status among HD patients, highlighting the importance of understanding the exact function of αKL protein on immune cells. This may offer a future diagnostic and therapeutic tool to improve the immune response among HD patients.

The effects of aging, sex, and tumor burden on the peripheral blood immune cell profile and absolute counts.

Background: To better assess the peripheral immune status and aid in the early diagnosis and prognosis of tumors, we compared the proportion and absolute counting of peripheral immune cell subsets in healthy individuals and tumor patients of varying ages, taking into account the impact of sex and tumor metastasis. Methods: We used peripheral blood mononuclear cell (PBMC) samples from 520 patients with various tumor types and 109 healthy volunteers. The absolute numbers of lymphocytes and monocytes were identified by an automated blood analyzer, and multi-parameter flow cytometry was used to examine the subsets of natural killer (NK) cells (CD3-CD16+CD56+), T cells (CD3+CD4+/CD8+), and mononuclear cells (CD14+) in PBMC. Results: The percentage of T cells (CD3+) in peripheral blood mononuclear cells (PBMC) was 55.83% VS 45.54% (P<0.0001) between healthy volunteers and tumor patients, showing a significant downward trend. Meanwhile, the percentages of monocytes (CD14+) and NK cells (CD3-CD16+CD56+) showed a significant upward trend. Single factor or multifactor analysis yielded identical findings on the proportion of PBMC between healthy individuals and patients with different malignancies, considering the three confounding variables of age, sex, and tumor metastasis. Conclusion: The proportion and absolute counting of acquired immune T cells, innate immune NK cells, and monocytes in PBMCs all exhibit substantial changes between cancer patients and healthy individuals, and the differences are influenced by age, sex, and tumor progression.

Splenic B Cell Lymphoma Innate Immune Cells: Detection of Red Versus White Pulp Lymphoma Differences in NK Cell Populations By High-Parameter Flow Cytometry

Introduction Splenic B-cell lymphomas, (SLs, total incidence ~1:100,000/year) comprise four distinct indolent B-cell non-Hodgkin lymphomas. Three predominantly involve the red pulp of the spleen (classical hairy cell leukemia (HCL), hairy cell leukemia variant (HCLv) and splenic diffuse red pulp small B-cell lymphoma (SDRPL)) and the fourth predominantly involves the white pulp (splenic marginal zone lymphoma (SMZL)). HCL is usually diagnosed by detection of an activation mutation in BRAF and responds well to treatment. We focused our study on the remaining three diseases, which have overlapping clinical presentations with only subtle differences in their morphology and phenotypes, but have considerable differences in prognosis, response to treatment and outcome. The white pulp lymphoma SMZL generally has good treatment responses and longer survival, whereas the red pulp SLs, HCLv and SDRPL are less responsive to treatments with shorter survival. Cells of the tumor microenvironment (TME) may have distinct characteristics between these three diseases. We hypothesized that TME innate immune cells from red pulp SLs (HCLv, and SDRPL) would be similar to each other and distinct from the white pulp SL, SMZL.In this study, we report that natural killer (NK) cells, which are important innate immune cells critical for immune surveillance, vary in proportions among red and white pulp SLs. Methods We studied splenectomy specimens from patients with SMZL (n=8), HCLv (n=8), SDRPL (n=7), and controls (healthy, post-trauma, n=8). Cryo-preserved bulk splenic suspension cell isolates from fresh (&amp;lt;4 hour) surgical specimens and intact formalin-fixed paraffin embedded (FFPE) tissue sections were used. Human specimen collection and usage was conducted with written informed consent after approval of the Institutional Research Subjects Review Board. FFPE tissue sections were studied by immunohistochemical (IHC) staining and splenic suspension cells were analyzed by high-parameter fluorescence flow cytometry using a Cytek Aurora spectral flow cytometer with a 25-color myeloid and a 28-color NK/B-cell panel. Results IHC characterization showed an extensive network of innate immune cells as reflected by CD163 + macrophages. Using high-parameter flow cytometry, we focused on innate immune cells in the non-B- non-T-cell (CD3 -CD19 -) population. The average proportions of this population were: NK cells 25-36%, monocyte/macrophages 20-34%, dendritic cells 5-11%, and granulocytes 2%. Among the patient groups, the median percentage of NK cells was greater for controls (34%) and SMZL (35%) as compared to red pulp lymphomas (HCLv (28%), SDRPL (24%)). Next, we examined NK subtypes: 1) cytotoxic NK cells (CD56 +CD16 +), 2) cytokine-secreting weakly cytotoxic NK cells (CD56 +CD16 -), and 3) undefined NK cells (CD56 -CD16 +) as a median percentage of total NK cells. The major subtype for control and SMZL was CD56 +CD16 - with values of 58 and 44%, respectively. This subtype comprises only 38%-40% of red pulp SLs. Proportionally the CD56 +CD16 + subtype was the largest for red pulp SLs at 52-57% as compared to 30% for control and 38% for SMZL. The remaining CD56 -CD16 + NK cell subtype was generally higher among red pulp SLs (HCLv (9%), SDRPL (12%)) as compared to control (6%) and SMZL (4%) patients. The increase in cytotoxic CD56 +CD16 + subtype among red pulp SLs was confirmed by measuring the percentage of granzymeB +perforin + cells among mature cytotoxic NK cells (CD56 +CD16 +/ CD57 +NKG2a -). SLs had higher proportions of this NK phenotype (78-86% compared to control 69%) and red pulp SLs had the most granzymeB +perforin + proportions with 82 and 86% for HCLv and SDRPL, respectively. Discussion In summary, spleens from patients with SMZL had similar percentages of total NK cells to controls, with lower percentages in red pulp SLs. All SLs had a higher percentage of cytotoxic NK cells than control, with the red pulp SLs having the most. These data suggest that monoclonal antibody treatments targeting SL antigen, such as anti-CD20, may be effective by activating cytotoxic NK cell to target lymphoma cells by antibody-dependent cellular cytotoxicity. Thus, uncovering the characteristics of the TME may aid the development of non-invasive diagnostic procedures and/or precision therapies.