Percentage Of Natural Killer Cells Research Articles

The alterations in peripheral lymphocyte subsets predict the efficacy and prognosis of immune checkpoint inhibitors in hepatocellular carcinoma.

Background: Immune checkpoint inhibitor (ICI) treatments are promising therapies for hepatocellular carcinoma (HCC) patients. However, not all HCC patients benefit from immunotherapy. Therefore, it is urgent to explore markers for the clinical efficacy and prognosis of immunotherapy for liver cancer. This study aimed to investigate changes in peripheral blood lymphocyte subsets after immunotherapy and to assess their predictive and prognostic value. Methods: Sixty-one patients with advanced HCC were enrolled. Peripheral blood samples were collected from HCC patients before and after ICI treatment, and lymphocytes were detected by flow cytometry. The rank sum test, chi-square test, Kaplan‒Meier curve, and Cox regression model were used to determine the relationship between the changes in the percentages of peripheral blood lymphocyte subsets and clinicopathological characteristics, clinical efficacy, progression-free survival (PFS) and overall survival (OS). Results: After ICI treatment, the percentage of CD3+CD8+ T cells increased, and the percentage of B cells decreased. The changes in memory T cells percentages varied according to different immune efficacy groups. Age, history of hepatitis B infection, first-line therapy, and distant metastasis influenced the proportion of peripheral blood lymphocyte subsets in patients with advanced HCC. Furthermore, univariate analysis demonstrated that high percentage changes in the natural killer (NK) cells percentage change predicted longer PFS and OS. Conclusions: ICI treatment alters the percentage of peripheral blood lymphocyte subsets in immunotherapy-treated HCC patients. Changes in the proportion of lymphocyte subsets are influenced by variances in immunological response and clinicopathological features. A high degree of NK cells percentage change in HCC patients treated with ICI represents an independent prognostic predictor.

Tumor immune microenvironment analysis of non-small cell lung cancer development through multiplex immunofluorescence.

Emerging evidence has underscored the crucial role of infiltrating immune cells in the tumor immune microenvironment (TIME) of non-small cell lung cancer (NSCLC) development and progression. With the implementation of screening programs, the incidence of early-stage NSCLC is rising. However, the high risk of recurrence and poor survival rates associated with this disease necessitate a deeper understanding of the TIME and its relationship with driver alterations. The aim of this study was to provide an in-depth analysis of immune changes in early-stage NSCLC, highlighting the significant transitions in immune response during disease progression. Tumor tissues were collected from 105 patients with precancerous lesions or stage I-III NSCLC. Next-generation sequencing (NGS) was used to detect cancer driver alterations. Multiplex immunofluorescence (mIF) was performed to evaluate immune cell density, percentage, and spatial proximity to cancer cells in the TIME. Next Among these patients, 64 had NGS results, including three with adenocarcinoma in situ (AIS), 10 with minimally invasive adenocarcinoma (MIA), and 51 with stage I invasive cancers. Additionally, three patients underwent neoadjuvant immuno-chemotherapy and tumor tissue specimens before and after treatment were obtained. Patients with stage I invasive cancer had significantly higher density (P=0.01) and percentage (P=0.02) of CD8+ T cells and higher percentages of M1 macrophages (P=0.04) and immature natural killer (NK) cells (P=0.041) in the tumor parenchyma compared to those with AIS/MIA. Patients with mutated epidermal growth factor receptor (EGFR) gene exhibited decreased NK cell infiltration, increased M2 macrophage infiltration, and decreased aggregation of CD4+ T cells near tumor cells compared to EGFR wild-type patients. As NSCLC progressed from stage I to III, CD8+ T cell density and proportion increased, while PD-L1+ tumor cells were in closer proximity to PD-1+CD8+ T cells, potentially inhibiting CD8+ T cell function. Furthermore, M1 macrophages decreased in density and proportion, and the number of NK cells, macrophages, and B cells around tumor cells decreased. Additionally, patients with tertiary lymphoid structures (TLSs) had significantly higher proportion of M1 macrophages and lymphocytes near tumor cells, whereas those without TLS had PD-L1+ tumor cells more densely clustered around PD-1+CD8+ T cells. Notably, neoadjuvant immuno-chemotherapy induced the development of TLS. This study offers an in-depth analysis of immune changes in NSCLC, demonstrating that the transition from AIS/MIA to invasive stage I NSCLC leads to immune activation, while the advancement from stage I to stage III cancer results in immune suppression. These findings contribute to our understanding of the molecular mechanisms underlying early-stage NSCLC progression and pave the way for the identification of potential treatment options.

The Effect of Aerobic Exercise and Low-Impact Pilates Workout on the Adaptive Immune System.

Growing evidence indicates the pronounced effects of physical activity on immune functions, which may largely depend on the type of exercise, intensity, and duration. However, limited information is available regarding the effects of low-impact exercises, especially on the level of adaptive immune system. Our study aimed to investigate and compare the changes in a broad spectrum of lymphocyte subtypes after 14 weeks of aerobic-type total-body-shaping workouts (TBSW) and Pilates workouts (PW) among healthy individuals. We determined the percentages of peripheral natural killer cells and different T and B lymphocyte subtypes with flow cytometry. At the end of the exercise program, significant changes in naïve and memory lymphocyte ratios were observed in TBSW group. Percentages of naïve cytotoxic T (Tc) cells elevated, frequencies of memory Tc and T-helper cell subsets decreased, and distribution of naïve and memory B cells rearranged. Proportions of activated T cells also showed significant changes. Nonetheless, percentages of anti-inflammatory interleukin (IL)-10-producing regulatory type 1 cells and immunosuppressive CD4+CD127lo/-CD25bright T regulative cells decreased not only after TBSW but also after PW. Although weekly performed aerobic workouts may have a more pronounced impact on the adaptive immune system than low-impact exercises, both still affect immune regulation in healthy individuals.

Effect of Renshen Yangying Decoction Combined with Chemotherapy on Traditional Chinese Medical Symptoms and T Cell Subsets in Patients with Cervical Cancer of Ganyu Qizhi Type

Background To confirm the effects of the combination of Renshen Yangying decoction and chemotherapy on traditional Chinese medical symptoms and T cell subsets in patients with cervical cancer (CC) of Ganyu Qizhi type.Methods: Patients with CC of Ganyu Qizhi type admitted to our hospital (n=96) were selected and evenly divided into control and observation groups. The patients in control group received chemotherapy, while those in observation group were additionally treated with Renshen Yangying decoction. The clinical efficacy, traditional Chinese medical symptom (TCMS) score, serum tumor markers, changes of serum T lymphocyte subsets and adverse effects were compared.Results: The distribution of clinical efficacy grade in all patients after treatment was statistically significant, and the clinical benefit rate (CBR) of observation group was higher than that of control group. The TCMS score, the serum carcinoembryonic antigen (CEA), squamous cell carcinoma antigen (SCCA), and cancer antigen 125 (CA125) were also decreased after treatment, and those were decreased in observation group. Levels of serum CD4+ and CD4+/CD8+, and natural killer (NK) cell percentage were higher, yet serum CD8+ cell percentage was lower after treatment, and in patients of observation group, levels of serum CD4+ and CD4+/CD8+, and NK cell percentage were higher, yet serum CD8+ cell percentage was lower. The incidence of adverse effects in the observation group was lower than that in the control group.Conclusion: Renshen Yangying decoction combined with chemotherapy in the treatment of CC of Ganyu Qizhi type can alleviate the clinical symptoms, reduce serum tumor markers, and improve the cellular immunity, with significant clinical efficacy and good safety on patients.

Percent Patient NK and T Cells at Day 28 Predict Graft Outcome after Haploidentical Hematopoietic Cell Transplant for Sickle Cell Disease

The only definitive cure for sickle cell disease (SCD) is hematopoietic cell transplant (HCT). Donor options are often limited due to the requirement of having a human leukocyte antigen (HLA)-matched donor. A haploidentical HCT from a family member has the potential to increase the donor pool for HCTs in SCD patients; however, conditioning regimens remain toxic. Reduced-intensity conditioning has the potential to successfully deplete the host immune system, reducing the rate of graft failure while also avoiding toxic side effects of traditional regimens. Natural killer (NK) cells are among the first cell lineages to repopulate following HCT, so sufficient depletion is necessary to ensure that the patient's NK cells will not target the donor cells post HCT. The primary objective of this study was to determine if a high percentage of patient NK cells at day 28 post-transplant could predict successful engraftment versus graft failure in SCD patients that have received a haploidentical HCT. Whole blood was drawn from SCD patients and their donors from two clinical trials (ClinicalTrials.gov Identifier: NCT02165007, NCT03077542) at baseline (before HCT) and at days 28, 60, 100, 180, and 365 following HCT. Peripheral blood mononuclear cells (PBMCs) were isolated via gradient-dependent lymphocyte separation and frozen for future batch analysis. Once samples from all time points were received, PBMCs were thawed and stained with fluorescent antibodies against CD3, CD4, CD8, CD45, CD56, iNKT, NKG2D, and HLA and analyzed by flow cytometry. HLA antibody optimization was achieved by screening for HLA mismatches between a patient and their haploidentical donor. Mann-Wilcoxon-Whitney rank tests were used to identify significant differences in various cell populations (CD3-CD56+ NK cells, CD3+ T cells) between patients with successful engraftment and those with graft failure. Areas under the receiver operating characteristic curve (AUCs) of the cell populations were used to determine which immune cell lineage held the highest potential to predict HCT graft failure at day 28. Statistical significance was established at p<0.05. A total of 15 patients were analyzed in this study: 11 patients had successful engraftment and 4 had graft failure. There were no statistically significant differences in the pre-HCT % NK or % T cell values in those who engrafted versus those who failed to engraft. At day 28 post-HCT, significant differences were noted between the % NK cells of all lymphocytes (p=0.0151) and % T cells of all lymphocytes (p=0.0028), as well as %-patient NK cells (p=0.0029) and %-patient T cells (p=0.0055) between the engrafted population and the graft failure population [Figure 1, A,B]. NK cells of all lymphocytes had an AUC of 0.9038 (95% Confidence Interval, CI, 0.573-1.00). T cells of all lymphocytes had an AUC of 0.9385 (95% CI 0.827-1.00). The AUC for %-patient T cells was 0.9273 (95% CI 0.779-1.00). Lastly, the AUC for %-patient NK cells was 0.9773 (95% CI 0.909-1.00). This suggested that the %-patient NK cells ROC model had the highest potential to correctly classify engraftment vs graft failure. This study showed the potential for using %-patient NK cells to predict graft failure at day 28 in SCD patients after haploidentical HCT, however, the sample size was limited. To independently validate these findings, analyses of specimens from a multisite clinical trial are being performed. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Downregulation of CD16 Surface Expression on NK Cells Is a Favorable Prognostic Factor in Acute Myeloid Leukemia

Introduction Acute myeloid leukemia (AML) is a malignant hematological disease characterized by poor patient outcomes and limited chemotherapy options. The rapid advancement of immunotherapy has pushed natural killer (NK) cells into the spotlight. NK cells can eliminate tumor cells through CD16-mediated antibody-dependent cellular cytotoxicity (ADCC). Previous studies have found that downregulation of CD16 occurs after coculture with malignant targets, but the effects on ADCC by NK cells is less clear. Several studies have shown that blocking CD16 cleavage increases NK cell attachment to antibody-coated target cells, degranulation (Romee et al., 2013), whereas others, have reported that blocking CD16 cleavage either did not affect ADCC and limited their ability to kill in a sequential manner (Srpan et al., 2018). This uncertainty stems from a disparity between experimental methods in vitro. Therefore, understanding the clinical significance of downregulation of CD16 surface expression on NK cells is crucial. Methods Patients diagnosed with de novo AML between 2020 and 2022 were enrolled in the study. All patients were treated in accordance with NCCN Clinical Practice Guidelines in Oncology of AML (Version 3, 2020). Bone marrow samples were collected at month 0, month 1, months 1-3, months 3-6 and months 6-12 after therapy. Number of NK (CD3-CD56+) cells and their subsets (CD16+ and CD16-) were analyzed by flow cytometry. Based on the 12-month prognosis, the patients were assigned to favorable prognosis group (complete response and survival) or unfavorable prognosis group (relapse or death). Results Totally 188 AML patients (108 males, 80 females) with a mean age of 45.5±17.9 years in this study. At month 0, month 1, months 1-3, months 3-6 and months 6-12, the mean percentage of NK cells in the bone marrow were 11.95%±0.63%，12.14%±1.40%，13.02%±1.31%,13.22%±1.25,13.30%±1.50%, respectively. There was a trend towards increased, but it was not statistically significant (P=0.12, Fig.1). There were 122 patients in the favorable prognosis group and 66 patients in the unfavorable prognosis group. The mean percentage of NK cells expressing CD16 in the bone marrow between the two groups were 74.44%±2.26% vs 74.70±2.98% at month 0 (P=0.94), 53.70±6.80% vs 52.77±10.33% at month 1 (P=0.94), 50.85±5.04% vs 71.85±6.21% at months 1-3 (P<0.01), 38.78±5.74% vs 47.05±15.05% at months 3-6 (P=0.59), and 22.94±5.70% vs 78.28±9.92% at months 6-12 (P<0.01). The level of CD16 surface expression on NK cells in the favorable prognosis group showed gradually trend of decrease (P=0.02), while it increased at months 1-3 and months 6-12 in the unfavorable prognosis group (P<0.01). The two significant peaks correspond to adverse event. Conclusions CD16 surface expression on NK cells was down-regulated in AML patients, and was markedly related to a favorable prognosis. It is similar to the previous conclusion of Srpan et al. These results indicate that CD16 may be used as a potential biomarker to predict prognosis for AML patients. Future studies will explore the visibility of other tumor, and shed new light on the development of immunological therapy for tumors. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Intratumoral Treatment with 5-Androstene-3β, 17α-Diol Reduces Tumor Size and Lung Metastasis in a Triple-Negative Experimental Model of Breast Cancer.

Breast cancer treatment failure is related to low response rates, high costs, and long-term toxicities. Thus, it is necessary to find less toxic, cheaper, and more effective treatments. In situ administration ensures drug delivery to tumor cells and decreases systemic toxic effects. The androstene-3β, 17α-diol (α-AED) reduces breast tumor cell proliferation and is an ideal candidate to treat mammary tumors. This study aims to identify the in vitro and in vivo effects of α-AED on a triple-negative mammary tumor model. An in vitro biphasic steroid effect was observed in mouse and human mammary tumor cells treated with α-AED. In this sense, cells treated with higher doses (100 and 200 μM) showed an antiproliferative effect. The α-AED administrated intratumorally reduced average tumor weight and increased the percentage of natural killer cells (NK), plasmatic, and plasmablast cells in mice tumors. Of note, VEGF levels in all α-AED-treated tumors was lower than in the control and vehicle groups. The tumor in situ increased response was reflected systemically by higher anti-4T1 IgG concentration in serum from α-AED-treated mice, but no other associated systemic changes were detected. The reduction in tumor size for the local injection of α-AED is associated with the anti-proliferative effect of this steroid, and the lower local levels of VEGF may be related to the imperceptible macroscopic metastasis in α-AED-treated mice. The above suggests that α-AED may be used in clinical studies to prove its efficacy as an alternative breast tumor treatment or in conjunction with already established therapies.

Anlotinib Benefits the αPDL1 Immunotherapy by Activating ROS/JNK/AP-1 Pathway to Upregulate PDL1 Expression in Colorectal Cancer.

Colorectal cancer (CRC) is one of the prevalent malignant tumors. This study is aimed at evaluating the mechanism of anlotinib (anlo) on tumor microenvironment (TME) in CRC, and its effects in combination with immune checkpoint inhibitors (ICIs) therapy. Firstly, MC38 and CT26 cells were both exposed to different gradient concentrations of anlo for 72 h, to investigate the cell viability and synergetic therapy efficacy with ICIs by CCK8. The results showed that anlo could obviously inhibit cell growth and showed no synergistic efficacy therapy in combination with αPDL1 in vitro. Then, we found the upregulation of programmed cell death ligand 1(PDL1) expression both in vitro and in vivo after anlo treatment. In vivo, anlo could enhance the percentage of natural killer (NK) cells and M1 macrophage cells and decrease the percentage of M2 macrophage cells in TME. Moreover, we explored the mechanism and we proved that anlo could activate reactive oxygen species (ROS)/c-Jun N-terminal kinase (JNK)/activator protein-1 (AP-1) signaling pathway to increase the expression levels of PDL1, IFN-α/β/γ, and CXCL2 in two cell lines in vitro. We also proved that anlo had synergistic effects with ICIs in vivo. Finally, it could also increase the mRNA and protein PDL1 expression levels in human cell lines, which was consistent with mouse CRC cell lines. However, there are still a few limitations. On one hand, the ROS/JNK/AP-1 pathway needs to be proved whether it can be activated in human cell lines. On the other hand, the mechanism behind ROS promoting phosphorylation of JNK needs to be explored.

Diagnostic Value of Natural Killer Cells, CD56+ CD16+ Natural Killer Cells, NLRP3, and Lactate Dehydrogenase in Severe/Critical COVID-19: A Prospective Longitudinal Study According to the Severe/Critical COVID-19 Definitions.

Innate immunity, as the first line of defense of our immune system, plays a crucial role in defending against SARS-CoV-2 infection and also its immunopathogenesis. We aim to investigate the immune status of natural killer (NK) cells, natural killer T (NKT) cells, and NLRP3 gene expression in COVID-19 patient blood samples. The immunophenotype of NK cell subsets and NKT cells was detected by flow cytometry and the expression of NLRP3 gene assessed by reverse transcriptase real-time polymerase chain reaction in 44 COVID-19 patients and 20 healthy individuals. The percentage of most of NK cell subpopulation and NKT cells was significantly decreased in COVID-19 patients. The percentage of CD56dim CD16- NK cell subsets, and NLRP3 gene expression increased. The percentage of total NK cells, CD56+ CD16+ NK cells, and NLRP3 gene expression had acceptable sensitivity and specificity for assisting diagnosis of severe/critical COVID-19. O2 saturation% and lactate dehydrogenase levels showed valuable diagnostic value to identify critical cases. The declined NK and NKT cells in COVID-19 patients and enhanced NLRP3 gene expression were associated with disease severity. Total NK cells, CD56+ CD16+ NK cells, and NLRP3 gene expression might be used as meaningful indicators for assisting diagnosis of severe/critical COVID-19.

Adaptive NK cell expansion and activity in ovarian cancer patients with cytomegalovirus infection and inflammation (258)

Objectives: Cytomegalovirus (CMV) infection has been shown to expand a subset of natural killer (NK) cells called adaptive NK cells (CD57+NKG2C+) that are long-lived, have enhanced cytotoxicity, and are thought to contribute to cancer control. CMV reactivation is associated with increased inflammation, which can impact patient outcomes. We sought to evaluate the impact of CMV exposure and inflammation, as measured by C-reactive protein (CRP), on adaptive NK cells in patients with high-grade serous ovarian cancer (HGSOC) using serum and ascites samples collected at the time of surgery. Methods: A total of 30 serum samples, collected at the time of cytoreductive surgery in women with HGSOC, were measured for CMV serostatus using an immunoassay and measured for CRP with an immunoturbidimetric method. The samples were categorized as reactive or non-reactive for CMV and low or high for CRP (≤10, >10 mg/L). Thirty serum and 23 ascites samples were evaluated by flow cytometry to quantify adaptive NK cells. A CyTOF panel of 42 antibodies was used to identify adaptive NK cells, differences in NK cell maturation and functional mediators, activation markers, a number of other immune subsets (CD8 T cells, T regulatory cells, B cells, monocytes), and proliferation signatures. Functional assays were performed to assess natural cytotoxicity and antibody-dependent cytokine cytotoxicity (ADCC) by CD1O7a degranulation and IFNy production. Data were analyzed using GraphPad Prism 8.2.0, and p-values ≤ 0.05 were considered statistically significant. Results: Patients with HGSOC with evidence of CMV exposure had increased percentages of CD57+NKG2C+ adaptive NK cells in the blood and ascites compared to those without CMV exposure (Figure 1, p=0.03). CRP blood levels were not significantly different (p=0.30). The increased percentages of adaptive NK cells, observed in the blood and ascites samples that were CMV IgG+, were not significantly altered by the presence (p=0.90) or absence (p=0.90) of CRP. Conclusions: CMV exposure influences the presence of an adaptive NK cell population in HGSOC patients. CMV expanded adaptive NK cells represent a unique NK cell population that, with their enhanced cytotoxicity and memory-like attributes, could be manipulated for future NK cell-based therapies in ovarian cancer. Objectives: Cytomegalovirus (CMV) infection has been shown to expand a subset of natural killer (NK) cells called adaptive NK cells (CD57+NKG2C+) that are long-lived, have enhanced cytotoxicity, and are thought to contribute to cancer control. CMV reactivation is associated with increased inflammation, which can impact patient outcomes. We sought to evaluate the impact of CMV exposure and inflammation, as measured by C-reactive protein (CRP), on adaptive NK cells in patients with high-grade serous ovarian cancer (HGSOC) using serum and ascites samples collected at the time of surgery. Methods: A total of 30 serum samples, collected at the time of cytoreductive surgery in women with HGSOC, were measured for CMV serostatus using an immunoassay and measured for CRP with an immunoturbidimetric method. The samples were categorized as reactive or non-reactive for CMV and low or high for CRP (≤10, >10 mg/L). Thirty serum and 23 ascites samples were evaluated by flow cytometry to quantify adaptive NK cells. A CyTOF panel of 42 antibodies was used to identify adaptive NK cells, differences in NK cell maturation and functional mediators, activation markers, a number of other immune subsets (CD8 T cells, T regulatory cells, B cells, monocytes), and proliferation signatures. Functional assays were performed to assess natural cytotoxicity and antibody-dependent cytokine cytotoxicity (ADCC) by CD1O7a degranulation and IFNy production. Data were analyzed using GraphPad Prism 8.2.0, and p-values ≤ 0.05 were considered statistically significant. Results: Patients with HGSOC with evidence of CMV exposure had increased percentages of CD57+NKG2C+ adaptive NK cells in the blood and ascites compared to those without CMV exposure (Figure 1, p=0.03). CRP blood levels were not significantly different (p=0.30). The increased percentages of adaptive NK cells, observed in the blood and ascites samples that were CMV IgG+, were not significantly altered by the presence (p=0.90) or absence (p=0.90) of CRP. Conclusions: CMV exposure influences the presence of an adaptive NK cell population in HGSOC patients. CMV expanded adaptive NK cells represent a unique NK cell population that, with their enhanced cytotoxicity and memory-like attributes, could be manipulated for future NK cell-based therapies in ovarian cancer.

Novel immunoprofiling method for diagnosing SLE and evaluating therapeutic response

ObjectiveDiagnosis of SLE is based on clinical manifestations but is heterogeneous in early onset. Hence, we aimed to evaluate the feature of the immunoprofiling in patients with SLE and apply...

Low Memory T Cells Blood Counts and High Naïve Regulatory T Cells Percentage at Relapsing Remitting Multiple Sclerosis Diagnosis.

ObjectiveThe aim of this study is to assess the peripheral immune system of newly diagnosed patients with relapsing remitting multiple sclerosis (RRMS) and compare it to healthy controls (HC).MethodsThis cross-sectional study involves 30 treatment-naïve newly diagnosed patients with RRMS and 33 sex- and age-matched HC. Peripheral blood mononuclear cells were analyzed regarding: i) thymic function surrogates [T cell receptor excision circles (TRECs) and recent thymic emigrants (RTEs)]; ii) naïve and memory CD4+ and CD8+ T cells subsets; iii) T helper (Th) phenotype and chemokine receptors expression on CD8+ T cells subsets; iv) regulatory T cell (Tregs) phenotype; and exclude expression of activating/inhibitory receptors by natural killer (NK) and NKT cells. Analyses were controlled for age, sex, and human cytomegalovirus (HCMV) IgG seroprevalence.ResultsNewly diagnosed patients with RRMS and HC have equivalent thymic function as determined by similar numbers of RTEs and levels of sjTRECs, DJβTRECs, and sj/DJβTREC ratio. In the CD8+ T cells compartment, patients with RRMS have a higher naive to memory ratio and lower memory cell counts in blood, specifically of effector memory and TemRA CD8+ T cells. Interestingly, higher numbers and percentages of central memory CD8+ T cells are associated with increasing time from the relapse. Among CD4+ T cells, lower blood counts of effector memory cells are found in patients upon controlling for sex, age, and anti-HCMV IgG seroprevalence. Higher numbers of CD4+ T cells (both naïve and memory) and of Th2 cells are associated with increasing time from the relapse; lower numbers of Th17 cells are associated with higher MS severity scores (MSSS). Patients with RRMS have a higher percentage of naïve Tregs compared with HC, and lower percentages of these cells are associated with higher MSSS. Percentages of immature CD56bright NK cells expressing the inhibitory receptor KLRG1 and of mature CD56dimCD57+ NK cells expressing NKp30 are higher in patients. No major alterations are observed on NKT cells.ConclusionCharacterization of the peripheral immune system of treatment-naïve newly diagnosed patients with RRMS unveiled immune features present at clinical onset including lower memory T cells blood counts, particularly among CD8+ T cells, higher percentage of naïve Tregs and altered percentages of NK cells subsets expressing inhibitory or activating receptors. These findings might set the basis to better understand disease pathogenesis.

Supply of Antioxidants vs. Recruit Firefighters' Cellular Immune Status: A Randomized Double-Blinded Placebo-Controlled Parallel-Group Trial.

Background: Physical exercise can affect the immune system. We studied the effect of antioxidants on hematological and immune biomarkers after heavy training. Methods: 24 well-trained and well-fed male firefighters were randomly divided into supplemented and placebo groups, and tested for immunology-related variables using venous blood samples in the fasting state, pre- (M1) and post- (M2) five weeks of daily micronutrient supplementation (15 mg of beta-carotene, 200 mg of vitamin C, 136 mg of vitamin E, 200 μg of selenium, 15 mg of zinc, 100 mg of magnesium). Total leukocytes and a differential count for five populations were determined using standard procedures (MAXM—Beckman Coulter Diagnostics; Brea, CA, USA). Lymphocyte subsets were determined through immunophenotyping. Results: Although all values were within the normal range for healthy adults and athletes in the supplemented group (SG), mean CD3+CD8+, CD8+ and CD16+CD56+ decreased (p < 0.05; small to moderate effects), while mean CD4+, CD19+ and CD4+/CD8+ increased (p < 0.05; small effects) after five-weeks. Regarding the placebo group (PG), higher total leukocyte count (p < 0.05; trivial effect) and natural killer cells percentage (CD16+CD56+; p < 0.05; moderate effect) were observed when comparing M1 and M2. Conclusions: Antioxidants supplementation did not alter well-fed male firefighters recruit firefighters’ immune cell response during the five-week physical training program.

MO429: The Association of Circulating Monocytes Subsets, NK Cells and Lymphocyte Subpopulations With Conventional and Novel Deformation Indices of Left Ventricular Function in CKD Patients With No Established Cardiovascular Disease

Abstract BACKGROUND AND AIMS Cardiovascular disease (CVD) and chronic kidney disease (CKD) are closely interrelated. Adverse immune responses have been implicated in the pathogenesis of both CVD and CKD [1, 2]. Monocyte subsets are key in atherogenesis and the inflammatory cascade occurring in heart failure [1, 2]. Likewise, the role of lymphocyte subpopulations including natural killer (NK) cells and CD4 + CD25 + regulatory T cells (Tregs) in the modulation of inflammation and immunity and subsequent cardiovascular implications have received increasing attention [2]. The implication of immune cells subsets in the abnormal myocardial remodeling as universally occurs in CKD patients even without overt CVD, remains to be clarified [3]. The aim of our study was to investigate potential correlations between blood levels of specific immune cells subsets with conventional and novel deformation-related indices of LV function in a cohort of patients with CKD without established CVD. METHOD A total of 35 stable CKD patients (mean age 66 ±12.2 years, 57% males, 20% diabetics) without established CVD were included in the study. Exclusion criteria included the history of malignancy, autoimmune disease and active or chronic infections. The peripheral blood immune cell subsets CD14++CD16-, CD14++CD16 + and CD14 + CD16++ percentage and absolute number of cells out of the total monocytes and NK cells (CD3 + CD16 + 56+), CD3-CD19 + B lymphocytes, CD3 + CD4 + T cells, CD3 + CD8 + T cells and Tregs (CD4 + CD25 + FoxP3+) absolute values and percentage out of the total lymphocytes were measured by flow cytometry. Simultaneously, conventional [left atrial volume index (LAVI), LV mass index (LVMI), LV end-diastolic volume (LVEDV), E/E’] and novel 2D speckle tracking (2DST) echocardiographic indices of LV function [global longitudinal strain (GLS), global circumferential strain (GCS), global radial strain (GRS), TWIST and UNTWIST] were assessed. RESULTS The mean estimated glomerular filtration rate (eGFR) was 28 ±15 mL/min/1.73 m2 (CKD-EPI) and the mean 24-h proteinuria was 2457 ±2399 mg. The percentage of total blood lymphocytes, as well as both percentage and absolute counts of CD4 + T-cells, were positively correlated with the eGFR (P &amp;lt; 0.05 and &amp;lt; 0.01 respectively) whereas. In addition, both NK cells percentage (P &amp;lt; 0.05) and absolute values (P &amp;lt; 0.01) showed a negative association with proteinuria. Regarding classical indices of LV function, NK counts were negatively associated with LVEDV (P &amp;lt; 0.05). An increased percentage of intermediate CD14++CD16 + monocytes was positively associated with the GRS (P &amp;lt; 0.005). Furthermore, the percentage of T-cells and CD8 + T-cells were positively associated with LV TWIST whereas a positive association was found between the percentage of CD8 + T-cells and more negative values of LV UNTWIST (P &amp;lt; 0.05). No specific significant correlations were observed between the rest immune cells subpopulations with either the classical or the novel indices of LV dysfunction. CONCLUSION The results of our study suggest that alterations of immune cells subsets correlate with subclinical markers of LV dysfunction in CKD patients without established CVD. The significance of the implication of the cellular arm of the immune system in the pathophysiological pathways of specific CVD phenotypes in CKD and the utility of immune cells subsets as tools to identify CKD patients who are at the highest risk for developing complications shall be elucidated by future research.

MO977: Circulating CD14++CD16+MONocytes, NK Cells and LYMPHOcyte Subsets Correlate with Conventional and Novel Deformation Related Indices of Left Ventricular Function in Kidney Transplant Recipients with no Established Cardiovascular Disease

Abstract BACKGROUND AND AIMS Kidney transplant recipients (KTRs) carry a significant burden of cardiovascular disease (CVD), despite improvement of patterns of left ventricular (LV) remodeling following kidney transplantation. Kidney allograft dysfunction and the immunosuppressed milieu play a major adverse role in myocardial structural changes in asymptomatic KTRs. An intricate relationship exists between immune system responses with heart failure syndromes, manifesting among others with aberrant activity and counts of proinflammatory CD16+monocytes, of natural killer (NK) cells and T regulatory cells (Tregs). The associations of immune cell subpopulations with subclinical markers of CVD remain to be determined in KTRs. The aim of our study was to investigate potential correlations between blood levels of specific immune cells subsets with conventional and novel deformation related indices of LV function in KTRs. METHOD 31 stable KTRs (mean age 58 ± 9.28 years, 67% males, 13% diabetics) without established CVD were enrolled. Control population included 17 chronic kidney disease (CKD) stage 3 patients without history of CVD. Additional exclusion criteria were history of malignancy, autoimmune disease and active or chronic infections. The peripheral blood immune cell subsets CD14++CD16-, CD14++CD16+and CD14+CD16++ absolute values and percentages out of total monocytes and NK cells (CD3+CD16+56+), CD3-CD19+B lymphocytes, CD3+CD4+T cells, CD3+CD8+T cells and Tregs (CD4+CD25+FoxP3+) absolute values and percentages out of total lymphocytes were measured by flow cytometry. Simultaneously, conventional (left atrial volume index (LAVI), LV mass index (LVMI), E/E’) and novel 2D speckle tracking (2DST) echocardiographic indices of LV function [global longitudinal strain (GLS), global circumferential strain (GCS), global radial strain (GRS), TWIST AND UNTWIST] were assessed. RESULTS KTRs had a mean eGFR of 58 ± 18 mL/min/1.73 m2 (CKD-EPI) and mean 24-h proteinuria (PER) 707 ± -1185 mg/24 h. Total lymphocytes, B-cells, T-cells and CD8+T cells counts correlated positively with eGFR (P &amp;lt; 0.05). Increased non-classical CD14+CD16++ monocytes were associated with proteinuria levels (P &amp;lt; 0.01) whereas an inverse correlation was observed between the percentage of classical CD14++CD16- monocytes with PER (P &amp;lt; 0.05). When compared with CKD patients, KTRs displayed higher CD14++CD16- and lower CD14+CD16++ monocytes counts, a lower percentage of NK cells and T cells and lower Tregs (significant differences presented in Table 1). Regarding classical indices of LV function, increased total monocytes were associated with elevated LAVI (P &amp;lt; 0.05), whereas an increased percentage of the intermediate CD14++CD16+monocytes correlated positively with an increased E/E’ value. Increased NK cells were associated with more negative GCS values (P &amp;lt; 0.05). No significant correlations were observed between the rest immune cells with the classical or novel LV dysfunction indices. CONCLUSION Alterations of immune cells subsets correlate with subclinical markers of LV dysfunction in KTRs with no established CVD. Future research is required to evaluate the role of immune subpopulations as tools to identify KTRs who are at the highest risk for complications and guide interventions.

Detection of Specific Immune Cell Subpopulation Changes Associated with Systemic Immune Inflammation-Index Level in Germ Cell Tumors.

The tumor microenvironment (TME) and the host inflammatory response are closely interconnected. The interplay between systemic inflammation and the local immune response may influence tumor development and progression in various types of cancer. The systemic immune–inflammation index (SII) represents a prognostic marker for germ cell tumors (GCTs). The aim of the present study was to detect specific immune cell subpopulation changes which were associated with the SII level in chemotherapy-naïve GCT patients. In total, 51 GCT patients, prior to cisplatin-based chemotherapy, were included in the present study. Immunophenotyping of peripheral blood leukocyte subpopulations was performed using flow cytometry. The SII level was correlated with the percentage of various leukocyte subpopulations. The obtained results demonstrated that SII levels above the cut-off value of SII ≥ 1003 were associated with higher neutrophil percentages. An inverse correlation was found between the SII and the peripheral lymphocyte percentage that logically reflects the calculations of the SII index. Furthermore, the presented data also showed that in the lymphocyte subpopulation, the association with the SII was driven by T-cell subpopulations. In innate immunity–cell subpopulations, we observed a correlation between SII level and neutrophils as well as associations with eosinophil, basophil, natural killer cell and dendritic cell percentages. We suppose that the described interactions represent a manifestation of cancer-induced immune suppression. The results of the present study contribute to the elucidation of the interrelationship between tumor cells and the innate/adaptive immune system of the host.

Functionality of natural killer cells in obese asthma phenotypes.

Obesity-associated asthma (OA) is a difficult to treat asthma phenotype due to its severity and poor response to inhaled steroids. Early-onset allergic (EoOA) and late-onset non-allergic (LoOA) OA are suggested subtypes of this phenotype. Natural Killer (NK) cells are key elements of innate immunity involved in cytotoxicity and immune regulation, with uncertain role in OA pathogenesis. Early-onset allergic and LoOA patients together with obese non-asthmatic (ONA) controls have been enrolled in the study. Peripheral blood samples have been collected for analysis. Percentages of total NK cells, CD3- CD56dim and CD3- CD56bright NK cell subsets, cytotoxic activity, intracellular interferon-γ, interleukin (IL)-10, IL-13, IL-17secretion and activatory receptors (NKG2D, NKp46i and NKp44) have been investigated by flow cytometry. The effect of IL-12 and IL-23stimulation on NK cells and intracellular cytokines in different groups have also been analysed and compared with unstimulated conditions. Results of ONA (n=5, age 42±8), EoOA (n=5, age 42±10) and LoOA (n=8, age 46±8) patients have analysed. Body Mass Index has been found to be negatively correlated with CD69(p=.022, r=-0.534). NKG2D receptor has been significantly low in CD56dim cellsof asthma population (p=.046). NKp44 receptor expression has increased after IL-12stimulation in EoOA and control group (p=.02). Intracellular IL-10 content has increased in LoOA and control subjects (p=.018, p=.03) but not in the EoOA group. Intracellular IL-17level has found be higher in allergic OA group. LoOA patients showed a decreased NK cytotoxicity compared with the early-onset asthma group (p=.05). Our study suggests an impaired NK receptor expression, activation and reduced cytotoxicity in OA patients together with variances between different subtypes of this phenotype. This data would be beneficial for tailoring a more personalized treatment strategy combatting steroid resistance and frequent exacerbations in this group of patients.

Natural Killer Cells Infiltration in the Joints Exacerbates Collagen-Induced Arthritis.

IntroductionThe role of natural killer (NK) cells in rheumatoid arthritis remains controversial. We aimed to assess the role of NK cells in the pathogenesis of rheumatoid arthritis.Materials and MethodsThe percentage of NK cells in the peripheral blood, spleen, lymph nodes and inflamed paws from collagen-induced arthritis mice were examined through the disease progression. Correlation between the proportion of NK cells and subsets with arthritis score, histopathological changes, and bone destruction were evaluated. Adoptive cell transfer was performed to determine the effect of NKp46+NK cells on arthritis development, and the role of receptor NKp46 was explored with NKp46 knockout mice.ResultsThe percentage of NK cells in peripheral blood decreased at the late stage of the disease and negatively correlated with arthritis score. NK cells increased in the inflamed paws during arthritis development and were positively associated with arthritis score, histopathological change, and bone destruction. Adoptive transfer of NKp46+NK cells before disease onset resulted in increased NK cells infiltration in the joints, higher incidence of arthritis, more severe clinical symptoms, and more pronounced joint inflammation and bone damage. NKp46 deficiency had no significant influence on the incidence and severity of arthritis in collagen-induced arthritis mice.ConclusionsNK cell infiltration in the joints positively correlates with arthritis progression, inflammation, and bone destruction. The pathogenic role of NK cells in rheumatoid arthritis may be independent of the receptor NKp46.

Reduced NK Cell Cytotoxicity by Papillomatosis-Derived TGF-β Contributing to Low-Risk HPV Persistence in JORRP Patients

The role of natural killer (NK) cells in juvenile-onset recurrent respiratory papillomatosis (JORRP) patients remains elusive. In this study, we find increased NK cell percentage, particularly CD11b-CD27- (DN) subsets in peripheral blood of JORRP patients and associated with disease activity. RNA sequencing shows a downregulated “natural killer cell-mediated cytotoxicity” feature in JORRP tumors. We also find impaired cytotoxic capacity and lower expression of NK cell-activating receptors including NKp30 and NKp46. Higher transforming growth factor-beta 1 (TGF-β1) is found both in plasma and tumor tissues of JORRP, and anti-TGF-β1 antibody could restore NK cell cytolytic activity and upregulate NKp30 and NKG2D expression. Also, we find a significantly higher Chemokine receptor type 6 (CXCR6) on NK cells in tumors compared with that in peripheral blood. Finally, RT-PCR analysis show that both HPV6-E6-E7 and HPV11-E6-E7 overexpression leads to higher TGFB1 expression compared with control SNU-1076 cell line, and higher CXCR6 expression is detected on NK coculture with HPV11-E6-E7-overexpressing cells. In conclusion, we demonstrate that TGF-β1 by papillomatosis leads to decreased NK cell cytotoxicity through downregulating NK cell-activating receptors in JORRP patients.

Low Eosinophil Phenotype Predicts Noninvasive Mechanical Ventilation Use in Patients with Hospitalized Exacerbations of COPD.

RationaleEosinophilic inflammation is related to the progression and outcomes of acute exacerbation of chronic obstructive pulmonary disease (AECOPD). Till now, few studies have focused on low EOS in AECOPD.ObjectiveTo reveal the clinical characteristics, therapeutic responses and prognosis of patients hospitalized of AECOPD with low EOS.MethodsThe electronic database of Zhongshan Hospital, Fudan University was used. Cohort 1 included 608 patients with hospitalized AECOPD. Study population 2 consisted of 166 patients with AECOPD admission at least twice. Impact of low EOS on NIMV treatment, length of hospital stays and 12-month AECOPD-related readmission were analyzed with multivariable logistic regression model. Thirty-five hospitalized AECOPD patients were prospectively recruited as cohort 3 to explore the association between EOS and other immune cells using Spearman correlation coefficient for ranked data.ResultsEOS level was suppressed on admission in AECOPD patients, and significantly improved after hospitalized treatment (P < 0.05). For inflammatory markers, leucocytes, neutrophils and lactate dehydrogenase levels were higher, while lymphocytes, monocytes and interleukin-6 levels were lower in the low-EOS group than those in the non-low EOS group (P < 0.05). Low EOS (EOS < 50 cells/μL) was an independent risk factor of NIMV use (OR = 1.86, 95% CI = 1.26 ~ 2.73). The EOS percentage was positively correlated with the T cell percentage (r = 0.46, P < 0.05) and negatively correlated with the natural killer cell percentage (r = −0.39, P < 0.05). The patients with low EOS had lower level of CD4+ T cell (P < 0.05) than that of patients with non-low EOS.ConclusionLow EOS might be a stable phenotype in patients with hospitalized AECOPD and could be used to inform NIMV management, hyperinflammatory state and impaired immunity situation.