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

BACKGROUND: 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). METHODS: 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. RESULT: 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. CONCLUSION: 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.

Tumor infiltrating NK cell (TINK) subsets and functional molecules in patients with breast cancer.

INTRODUCTION: NK cells have been introduced as the main innate arm of immunity against malignancies. Recent advances introduced new subsets of, and new effector molecules on NK cells suggesting new paradigms for NK cell functions in tumor immunity. Considering these new paradigms, in the current research we investigated the frequency of tumor infiltrating NK cell (TINK) subsets and their functional molecules in breast tumor tissues by flowcytometry method. METHODS: Breast tumor tissues were obtained from 32 untreated patients with breast cancer. The tissues were then minced mechanically to acquire a single cell suspension and surface-stained with monoclonal antibodies against CD3, CD56, CD11b, CD27, NKG2A, NKG2D and CXCR3. For intracellular staining (ICS), the surface-stained cells were then fixed, permeabilized and stained with anti-Perforin and anti-Granzyme B antibodies. The samples were run and the data were acquired on a four-color flowcytometer. RESULTS: The cell suspension derived from tumor tissue encompassed 3.10 ± 0.52 % CD3-CD56+(bright/dim) total NK cells. Based on the conventional classification the percentages of cytotoxic (CD3- CD56dim) and regulatory (CD3- CD56bright) NK cells were respectively 1.74 ± 0.24 % and 1.36 ± 0.48 %. According to the new classification the percentages of cytotoxic (CD3- CD56+ CD11b+ CD27-), regulatory (CD3-CD56+ CD11b+/- CD27+) and tolerant (CD3-CD56+ CD27- CD11b-) NK cells were respectively 0.48 ± 0.07, 1.55 ± 0.34 and 1.15 ± 0.51. A significant higher frequency of total NK cells (CD3-CD56+ (bright/dim)) in the breast tumor tissues of the patients whose tumor draining lymph nodes (TDLNs) has not been yet involved by tumor cells (LN- patients) compared with the ones with lymph nodes involvement (LN+) (5.91 ± 1.79 % Vs. 2.20 ± 0.20 %, P < 0.004). Furthermore, NK cells with overexpressed activating receptor; NKGD2 (CD3- CD56+(bright/dim) NKG2D+ NK cells) was observed to be elevated in LN- patients compared with the LN+ ones (70.01 ± 7.96 Vs. 42.5 ± 4.81, P < 0.011). Correlation analysis revealed the percentages of conventional regulatory NK cells (CD3- CD56bright) in breast tumor tissue to be in positive correlation with the tumor size (R = 0.380, P < 0.04). The mean percentage of this cell subset was also observed to be higher in patients with T3 tumor size compared with smaller T1 tumor size (1.61 ± 0.20 % vs. 0.75 ± 0.15 %, P < 0.023. CONCLUSION: Our observations suggest that accumulation of NK cells as well as the expression of activating NKG2D receptor by TINKs may play roles in breast tumor regression especially in the LN- patients. As the tumor growths and the size of tumor increases the accumulation of regulatory NK cells may facilitate the tumor improvement. These observations may have implications in cancer NK cell-based immunotherapy.

Granzyme A inhibition reduces inflammation and increases survival during abdominal sepsis.

Aims: Peritonitis is one of the most common causes of sepsis, a serious syndrome characterized by a dysregulated systemic inflammatory response. Recent evidence suggests that Granzyme A (GzmA), a serine protease mainly expressed by NK and T cells, could act as a proinflammatory mediator and could play an important role in the pathogenesis of sepsis. This work aims to analyze the role and the therapeutic potential of GzmA in the pathogenesis of peritoneal sepsis. Methods: The level of extracellular GzmA as well as GzmA activity were analyzed in serum from healthy volunteers and patients with confirmed peritonitis and were correlated with the Sequential Organ Failure Assessment (SOFA) score. Peritonitis was induced in C57Bl/6 (WT) and GzmA-/- mice by cecal ligation and puncture (CLP). Mice were treated intraperitoneally with antibiotics alone or in combination serpinb6b, a specific GzmA inhibitor, for 5 days. Mouse survival was monitored during 14 days, levels of some proinflammatory cytokines were measured in serum and bacterial load and diversity was analyzed in blood and spleen at different times. Results: Clinically, elevated GzmA was observed in serum from patients with abdominal sepsis suggesting that GzmA plays an important role in this pathology. In the CLP model GzmA deficient mice, or WT mice treated with an extracellular GzmA inhibitor, showed increased survival, which correlated with a reduction in proinflammatory markers in both serum and peritoneal lavage fluid. GzmA deficiency did not influence bacterial load in blood and spleen and GzmA did not affect bacterial replication in macrophages in vitro, indicating that GzmA has no role in bacterial control. Analysis of GzmA in lymphoid cells following CLP showed that it was mainly expressed by NK cells. Mechanistically, we found that extracellular active GzmA acts as a proinflammatory mediator in macrophages by inducing the TLR4-dependent expression of IL-6 and TNFα. Conclusions: Our findings implicate GzmA as a key regulator of the inflammatory response during abdominal sepsis and provide solid evidences about its therapeutic potential for the treatment of this severe pathology.

Granzyme B levels and granzyme B polymorphisms in peripheral blood of patients with endometriosis: a preliminary study.

The chronic course of endometriosis suggests that the immune system may play a role in its aetiology. There may be resistance to cell lysis, as well as an immune defect underlying endometriosis. Granzyme B is a serine protease that is secreted by Natural Killer (NK) cells and cytotoxic T lymphocytes during a cellular immune response and can induce apoptosis. The aim of this study was to evaluate the relationship between both Granzyme B levels and Granzyme B gene polymorphisms in endometriosis patients. Women between the ages of 20 - 45 were included in the study. The patients were divided into two groups: those diagnosed with endometriosis and those who had not been diagnosed with endometriosis. In the blood samples, Granzyme B gene polymorphisms and serum levels of Granzyme B were studied. There was no difference between the groups in terms of median Granzyme B levels and the presence of AA, AG, and GG genotypes. There was a difference in median granzyme levels for the control group; the GG genotype was found at a lower frequency. The immune defect within endometriosis-related immune cells may not be exclusively due to Granzyme B. Other mediators that are secreted from immune cells may have additive effects.IMPACT STATEMENTWhat is already known on this subject? NK cells are cytotoxic and inhibit the implantation of autologous endometrial cells that are spilled into the peritoneum by retrograde menstruation. Thus, a reduction in NK cell activity may facilitate the progression of endometriosis. The literature review reveals that there are studies suggesting that NK cell activity may be insufficient in endometriosis. Granzyme B is a serine protease that is secreted by NK cells and cytotoxic T lymphocytes during a cellular immune response.What do the results of this study add? Granzyme B is one of the cytotoxic granules in NK and cytotoxic T lymphocyte cells and its genetic polymorphisms were tested in endometriosis. We found that median Granzyme B levels were significantly different in patients with the GG genotype in the control group, compared to those with the AA and AG genotype. However, this difference was not detected between the control and endometriosis groups.What are the implications of these findings for clinical practice and/or further research? Our results contribute to uncovering the pathogenesis of endometriosis since there are no previous studies in the literature regarding this topic. Although we did not find a difference, our results will inform further studies made on this topic. Studies with different molecules and an increased number of patients are needed. The immune defect of endometriosis may not be due exclusively to Granzyme B. Other mediators that are secreted from immune cells may have mutual effects and interactions.

Hidden Granzyme B-Mediated Injury in Chronic Active Antibody-Mediated Rejection.

OBJECTIVES: Antibody-mediated injury in chronic active antibody-mediated rejection, possibly with other effector T cells, may play a role in graft injury. The role of inflammatory cells in the inflammation and fibrosis and tubular atrophy region has been recently advocated in the progression of injury. Cytotoxic T cells play a prominent role in T-cell-mediated rejection; however, the possible role of cytotoxic T cells in circulation and the intragraft compartment in chronic active antibody-mediated rejection, a common immunological cause of long-term graft failure, has not been well-studied. MATERIALS AND METHODS: We measured the frequency of circulating cytotoxic T cells with flow cytometry, serum granzyme B level by enzyme-linked immunosorbent assay and intragraft granzyme B+ cell, and mRNA by immunohistochemistry and real-time polymerase chain reaction in biopsy tissue from living donor renal allograft recipients with stable graft function and chronic active antibody-mediated rejection. RESULTS: The frequency of CD3+ and CD3+CD8+ T cells was similar in both stable graft function patients and chronic active antibody-mediated rejection patients. The frequency of CD3+CD8+granzyme B+ cytotoxic T cells was significantly lower in peripheral blood. Serum granzyme B level and intragraft number of granzyme B+ cells (counts/mm²) were also significantly higher in the chronic active antibody-mediated rejection group compared with that of patients with stable graft function. The intragraft granzyme B+ T cell count was positively correlated with serum creatinine and 24-hour urine proteinuria but negatively correlated with estimated glomerular filtration rate. CONCLUSIONS: Granzyme B mediates covert graft injury in patients with chronic active antibody-mediated rejection in addition to antibody-mediated injury.

Granzyme B Expression in Visceral Adipose Tissue Associates With Local Inflammation and Glyco-Metabolic Alterations in Obesity.

Granzyme B (GrB) is a serine protease produced by immune and non-immune cells, able to promote multiple processes, like apoptosis, inflammation, extracellular matrix remodeling and fibrosis. GrB expression in visceral adipose tissue (VAT) was associated with tissue damage, local inflammation and insulin resistance in obesity murine model, but there is no data in humans. Aim of this study was to explore the expression of GrB in VAT from obese subjects in relation to adipose tissue injury, inflammation, metabolic alterations and GrB circulating levels. For this purpose, 85 obese individuals undergoing bariatric surgery and 35 healthy subjects (as control) were recruited at Sapienza University, Rome, Italy. Study participants underwent clinical work-up and routine biochemistry. mRNA expression of GrB in VAT and of a panel of VAT inflammatory markers was analyzed by real-time PCR. Serum GrB levels were measured by Elisa Affymetrix EBIO. We observed that 80% of obese patients expressed GrB mRNA in VAT, and GrB VAT expression was associated with the presence of local inflammation and glucose homeostasis alterations. Moreover, GrB serum levels, which were higher in obese subjects compared to non-obese healthy individuals, were associated with GrB expression in VAT and glyco-metabolic impairment. Our data show, for the first time in humans, that obese subjects with "sick" fat and altered glucose tolerance exhibit GrB expression in VAT, and suggest that GrB might contribute to obesity-related VAT inflammatory remodeling and glucose homeostasis dysregulation. Moreover, increased circulating GrB levels might represent a possible peripheral marker of VAT dysfunction in metabolic diseases.

Characterization of immunoreactivity with whole-slide imaging and digital analysis in high-grade serous ovarian cancer.

Ovarian cancer is the most lethal of gynecological cancers with 5-year survival rate of ca. 45%. The most common histologic subtype is high-grade serous carcinoma, which typically is presented with advanced stage and development of chemoresistance. Therefore, new treatment options, including immunotherapies, are needed. Understanding the features of the immune cell populations in the tumor microenvironment is essential for developing personalized treatments and finding predictive biomarkers. Digital image analysis may enhance the accuracy and reliability of immune cell infiltration assessment in the tumor microenvironment. The aim of this study was to characterize tumor microenvironment in a retrospective cohort of high-grade serous carcinoma samples with whole-slide imaging and digital image analysis. Formalin-fixed paraffin-embedded high-grade serous carcinoma tumor tissue samples (n = 67) were analyzed for six immunohistochemical stainings: CD4, CD8, FoxP3, granzyme B, CD68, and CD163. The stained sample slides were scanned into a digital format and assessed using QuPath 0.1.2 and ImageJ software. Staining patterns were associated with clinicopathological data. The higher numbers of intraepithelial CD8+, CD163+, and granzyme B+ immune cells were associated with survival benefit when analyzed individually, while high levels of both CD8+ and granzyme B+ tumor-infiltrating lymphocytes were an independent prognostic factor in the Cox multivariate regression analysis (median progression-free survival; hazard ratio = 0.287, p = 0.002). Specimens taken after administration of neoadjuvant chemotherapy presented with lower FoxP3+ tumor-infiltrating lymphocyte density (Fisher's exact test, p = 0.013). However, none of the studied immunomarkers was associated with overall survival or clinical factors. Tumors having high amount of both intraepithelial CD8+ and granzyme B+ tumor-infiltrating lymphocytes showed better progression-free survival, possibly reflecting an activated immune state in the tumor microenvironment. The combined positivity of CD8 and granzyme B warrants further investigation with respect to predicting response to immune therapy. Neoadjuvant chemotherapy may have an effect on the tumor microenvironment and therefore on the response to immuno-oncologic or chemotherapy treatments.

Peripheral Cytokine Levels Differ by HPV Status and Change Treatment-Dependently in Patients with Head and Neck Squamous Cell Carcinoma.

Cytokines and immune mediators play an important role in the communication between immune cells guiding their response to infectious diseases or cancer. In this study, a comprehensive longitudinal analysis of serum cytokines and immune mediators in head and neck squamous cell carcinoma (HNSCC) patients was performed. In a prospective, non-interventional, longitudinal study, blood samples from 22 HNSCC patients were taken at defined time points (TP) before, during, and every 3 months after completion of (chemo)radio)therapy (CRT/RT) until 12 months after treatment. Serum concentrations of 17 cytokines/immune mediators and High-Mobility-Group-Protein B1 (HMGB1) were measured by fluorescent bead array and ELISA. Concentrations of sFas were significantly elevated during and after CRT/RT, whereas perforin levels were significantly decreased after CRT/RT. Levels of MIP-1ß and Granzyme B differed significantly during CRT/RT by HPV status. Increased HMGB1 levels were observed at recurrence, accompanied by high levels of IL-4 and IL-10. The sFas increase and simultaneous perforin decrease may indicate an impaired immune cell function during adjuvant radiotherapy. Increased levels of pro-inflammatory cytokines in HPV+ compared to HPV- patients seem to reflect the elevated immunogenicity of HPV-positive tumors. High levels of HMGB1 and anti-inflammatory cytokines at recurrence may be interpreted as a sign of immune evasion.

Angiogenic and Immune-Related Biomarkers and Outcomes Following Axitinib/Pembrolizumab Treatment in Patients with Advanced Renal Cell Carcinoma.

PURPOSE: Combined axitinib/pembrolizumab is approved for advanced renal cell carcinoma (aRCC). This exploratory analysis examined associations between angiogenic and immune-related biomarkers and outcomes following axitinib/pembrolizumab treatment. PATIENTS AND METHODS: Prospectively defined retrospective correlative exploratory analyses tested biospecimens from 52 treatment-naïve patients receiving axitinib and pembrolizumab (starting doses 5 mg twice daily and 2 mg/kg respectively, every 3 weeks). Tumor tissue, serum, and whole blood samples were collected at baseline, at cycle 2 day 1 (C2D1), and end of treatment (EOT) for blood-based samples. Clinical outcomes were objective response rate (ORR) and progression-free survival (PFS). RESULTS: Higher baseline tumor levels of CD8 showed a trend toward longer PFS (HR 0.4; P = 0.091). Higher baseline serum levels of CXCL10 (P = 0.0197) and CEACAM1 (P = 0.085) showed a trend toward better ORR and longer PFS, respectively. Patients for whom IL6 was not detected at baseline had longer PFS versus patients for whom it was detected (HR 0.4; P = 0.028). At C2D1 and/or EOT, mainly immune-related biomarkers showed any association with better outcomes. The genes CA9 (P = 0.084), HIF1A (P = 0.064), and IFNG (P = 0.073) showed trending associations with ORR, and AKT3 (P = 0.0145), DDX58 (P = 0.0726), GZMA (P = 0.0666), LCN2 (NGAL; P = 0.0267), and PTPN11 (P = 0.0287) with PFS. CONCLUSIONS: With combined axitinib/pembrolizumab treatment in patients with aRCC, mostly immune-related biomarkers are associated with better treatment outcomes. This exploratory analysis has identified some candidate biomarkers to consider in future prospective testing.

Granzyme B is correlated with clinical outcome after PD-1 blockade in patients with stage IV non-small-cell lung cancer.

BACKGROUND: A minority of patients with advanced non-small-cell lung cancer (NSCLC) benefit from treatment with immune checkpoint inhibitors (ICIs). Ineffective effector function of activated T and NK cells may lead to reduced tumor cell death, even when these activated effector cells are released from their immune checkpoint brake. Hence, in this study we aimed to assess the association of baseline serum granzyme B, as well as germline variation of the GZMB gene, with clinical outcome to programmed cell death protein 1 (PD-1) blockade. METHODS: A total of 347 patients with stage IV NSCLC who started nivolumab treatment between June 2013 and June 2017 were prospectively included. Baseline serum and whole blood was available, allowing for protein quantification and targeted DNA sequencing. Clinical outcome was based on best overall response (BOR) according to Response Evaluation Criteria in Solid Tumors, V.1.1, progression-free survival (PFS), and overall survival (OS). RESULTS: Patients with low serum levels of granzyme B had worse PFS (HR: 1.96; 95% CI: 1.12 to 3.43; p=0.018) and worse OS (HR: 2.08; 95% CI: 1.12 to 3.87; p=0.021) than patients with high baseline serum levels. To validate the findings, germline variation of GZMB rs8192917 was assessed. Patients with homozygous and heterozygous variants of GZMB rs8192917 had worse BOR (OR: 1.60; 95% CI: 1.01 to 2.52; p=0.044) and worse PFS (HR: 1.38; 95% CI:1.02 to 1.87; p=0.036) than wild types. CONCLUSIONS: A low baseline serum level of granzyme B and germline variation of GZMB was associated with worse clinical outcome in NSCLC, emphasizing the relevance and additional value of monitoring germline genetic variations which mirror cytotoxic functions of T cells in ICI therapy. TRAIL REGISTRATION NUMBER: Dutch Trial Registry (NL6828).

Impaired immune cell cytotoxicity in severe COVID-19 is IL-6 dependent.

BACKGROUNDCoronavirus disease 19 (COVID-19) is an emerging infectious disease caused by SARS-CoV-2. Antiviral immune response is crucial to achieve pathogen clearance; however, in some patients an excessive and aberrant host immune response can lead to an acute respiratory distress syndrome. The comprehension of the mechanisms that regulate pathogen elimination, immunity, and pathology is essential to better characterize disease progression and widen the spectrum of therapeutic options.METHODSWe performed a flow cytometric characterization of immune cell subsets from 30 patients with COVID-19 and correlated these data with clinical outcomes.RESULTSPatients with COVID-19 showed decreased numbers of circulating T, B, and NK cells and exhibited a skewing of CD8+ T cells toward a terminally differentiated/senescent phenotype. In agreement, CD4+ T and CD8+ T, but also NK cells, displayed reduced antiviral cytokine production capability. Moreover, a reduced cytotoxic potential was identified in patients with COVID-19, particularly in those who required intensive care. The latter group of patients also showed increased serum IL-6 levels that inversely correlated to the frequency of granzyme A-expressing NK cells. Off-label treatment with tocilizumab restored the cytotoxic potential of NK cells.CONCLUSIONThe association between IL-6 serum levels and the impairment of cytotoxic activity suggests the possibility that targeting this cytokine may restore antiviral mechanisms.FUNDINGThis study was supported by funds from the Department of Experimental and Clinical Medicine of University of Florence (the ex-60% fund and the "Excellence Departments 2018-2022 Project") derived from Ministero dell'Istruzione, dell'Università e della Ricerca (Italy).

Comparison of caspase-8, granzyme B and cytochrome C apoptosis biomarker levels in orthopedic trauma patients.

BACKGROUND: The primary objective of this study was to investigate whether or not apoptosis is induced following bone fracture, and if so, to investigate whether the extrinsic or intrinsic pathway of cell death is stimulated. METHODS: A total of 30 patients who presented at our clinic and were diagnosed with bone fracture following trauma were included in the study group. A control group was formed of 37 age and gender-matched volunteers. On the day after the fracture, blood samples taken from the patients were examined for cytochrome C, granzyme B and caspase-8 with the ELISA method. RESULTS: A total of 67 individuals were evaluated (fracture group: 30, control group: 37) in this study. Caspase-8 was found to be statistically significantly high in the patient group (0.37±0.06 ng/mL, p=0.002). No significant difference was determined between the groups in respect to cytochrome C values (p=0.173). The granzyme B values were determined to be significantly high in the patient group (52.56±8.51 pg/mL, p=0.007). CONCLUSION: These results obtained from patients with a long bone fracture demonstrated that serum caspase-8 and granzyme B levels were higher in patients than in the control group, thereby showing activation of the extrinsic pathway. However, no significant difference was determined between the groups concerning serum cytochrome C levels. This study may guide future studies designed for better understanding of the molecular pathways that govern the events during a fracture, which will be important for the future advancement of fracture treatment.

Measuring protein biomarker concentrations using antibody tagged magnetic nanoparticles.

Under physiological conditions biomarker concentrations tend to rise and fall over time e.g. for inflammation. Ex vivo measurements provide a snapshot in time of biomarker concentrations, which is useful, but limited. Approaching real time monitoring of biomarker concentration(s) using a wearable, implantable or injectable in vivo sensor is therefore an appealing target. As an early step towards developing an in vivo biomarker sensor, antibody (AB) tagged magnetic nanoparticles (NPs) are used here to demonstrate the in vitro measurement of ~5 distinct biomarkers with high specificity and sensitivity. In previous work, aptamers were used to target a given biomarker in vitro and generate magnetic clusters that exhibit a characteristic rotational signature quite different from free NPs. Here the method is expanded to detect a much wider range of biomarkers using polyclonal ABs attached to the surface of the NPs. Commercial ABs exist for a wide range of targets allowing accurate and specific concentration measurements for most significant biomarkers. We show sufficient detection sensitivity, using an in-house spectrometer to measure the rotational signatures of the NPs, to assess physiological concentrations of hormones, cytokines and other signaling molecules. Detection limits for biomarkers drawn mainly from pain and inflammation targets were: 10 pM for mouse Granzyme B (mGZM-B), 40 pM for mouse interferon-gamma (mIFN-γ), 7 pM for mouse interleukin-6 (mIL-6), 40 pM for rat interleukin-6 (rIL-6), 40 pM for mouse vascular endothelial growth factor (mVEGF) and 250 pM for rat calcitonin gene related peptide (rCGRP). Much lower detection limits are certainly possible using improved spectrometers and nanoparticles.

Asthma and elevation of anti-citrullinated protein antibodies prior to the onset of rheumatoid arthritis.

BACKGROUND: Anti-citrullinated protein antibodies (ACPA) are central to rheumatoid arthritis (RA) pathogenesis and may develop at inflamed mucosa. We investigated whether asthma, a disease of airway mucosal inflammation, was associated with elevated ACPA before RA diagnosis. METHODS: We performed a nested case-control study among women in two prospective cohorts, the Nurses' Health Study (NHS; 1976-2014) and NHSII (1989-2015). Blood was obtained on a subset (NHS: 1989-1990; NHSII: 1996-1999). Cases met 1987 ACR or 2010 ACR/EULAR RA criteria by medical record review and were classified as seropositive (ACPA+ or rheumatoid factor positivity) or seronegative by clinical laboratory testing at diagnosis. We identified RA cases with blood drawn before the date of RA diagnosis (index date), matching each to three controls by age, cohort, year, time from blood draw to index date, and menopause. Pre-RA ACPA elevation for cases was defined as >99th percentile of the control distribution on a research assay composed of autoantibodies targeting citrullinated protein epitopes or positivity on the second-generation commercial assay for cyclic citrullinated peptide. Asthma status and covariates were obtained through biennial questionnaires before blood draw. Conditional logistic regression estimated ORs and 95%CIs for RA by pre-RA ACPA and clinical serostatus, adjusted for matching factors, smoking pack-years, passive smoking, and body mass index (BMI). RESULTS: We identified 284 incident RA cases and 849 matched controls; mean age at the index date was 61.2 years (SD 10.1). Blood was drawn 9.7 years (mean; SD 5.8) before the index date. We identified 96 (33.8%) RA cases with elevated pre-RA ACPA. At blood draw, 17.7% of pre-RA ACPA+ cases and 6.3% of matched controls (p = 0.0008) reported clinician-diagnosed asthma. After adjusting for matching factors, smoking pack-years, passive smoking, and BMI, asthma was significantly associated with pre-RA ACPA+ RA (OR 3.57, 95%CI 1.58,8.04). Asthma was not associated with overall RA (OR 1.45, 95%CI 0.91,2.31), but was significantly associated with seropositive RA (OR 1.79, 95%CI 1.01,3.18). CONCLUSIONS: Asthma was strongly associated with ACPA elevation in blood drawn prior to RA diagnosis, independent of smoking. Chronic mucosal airway inflammation may contribute to ACPA development and RA pathogenesis.

Chronic infusion of interleukin-17 promotes hypertension, activation of cytolytic natural killer cells, and vascular dysfunction in pregnant rats.

Previous studies by our lab have established that placental-ischemia stimulated T-helper 17 cells (TH 17s) cause increased cytolytic natural killer (cNK) cell proliferation and activation during pregnancy; however, the exact mechanism is unknown. The objective of this study was to investigate the role of interlukin 17 (IL-17) in inducing cNK cell activation in pregnancy. We infused 150 pg/day of recombinant IL-17 into a subset of normal pregnant (NP) Sprague Dawley rats from gestation day (GD) 12-19 (NP+IL-17). On GD 19, mean arterial pressure (MAP), fetal and placental weights, cytokines, cNK cell activation, cytotoxic enzymes, and vascular reactivity were assessed. MAP significantly increased from 99 ± 3 mmHg in NP to 120 ± 1 mmHg in NP+IL-17 (P < 0.05). Fetal weight significantly decreased from 2.52 ± 0.04 g in NP to 2.32 ± 0.03 g in NP+IL-17 as did placental weight (NP: 0.65 ± 0.03 g; NP+IL-17: 0.54 ± 0.01 g, P < 0.05). Plasma levels of TNF-α increased to 281.4 ± 55.07 pg/mL in NP+IL-17 from 145.3 ± 16.03 pg/mL in NP (P < 0.05) while placental levels of VEGF decreased from 74.2 ± 6.48 pg/mg in NP to 54.2 ± 3.19 pg/mg in NP+IL-17. Total NK cells were increased in the placenta (NP: 14.3 ± 3.49%; NP+IL-17: 29.33 ± 2.76%, P < 0.05) as were cytolytic NK cells (NP: 3.31 ± 1.25%; NP+IL-17: 13.41 ± 1.81%, P < 0.05). A similar trend was observed in circulating NK cells. Plasma granzyme K increased from 3.55 ± 2.29 pg/mL in NP to 20.9 ± 7.76 pg/mL in NP+IL-17 (P < 0.05), and plasma granzyme B increased from 10.95 ± 0.64 pg/mL in NP to 14.9 ± 0.98 pg/mL in NP+IL-17(P < 0.05). In the placenta, both granzyme A (NP: 246.1 ± 16.7 pg/mg; NP+IL-17: 324.3 ± 15.07 pg/mg, P < 0.05) and granzyme B (NP: 15.18 ± 3.79 pg/mg; NP+IL-17: 27.25 ± 2.34 pg/mg, P < 0.05) increased in response to IL-17 infusion. Finally, vascular reactivity of uterine arteries was significantly impaired in response to IL-17 infusion. The results of this study suggest that IL-17 plays a significant role in the activation of cNK cells during pregnancy.

Increased circulating granzyme B in type 2 diabetes patients with low-grade systemic inflammation.

In metabolic diseases, like type 2 diabetes (T2D), adipose tissue (AT) is infiltrated by macrophages and other leukocytes - which secrete many bioactive peptides leading to local and systemic low-grade chronic inflammation - and undergoes remodeling and aberrant fibrosis. Granzyme B (GrB) is a serine protease produced by some leukocytes, including cytotoxic lymphocytes and macrophages. It exerts both intracellular apoptotic function and extracellular functions, leading to tissue injury, inflammation and repair. Elevated circulating GrB levels have been found in aging- and inflammation-associated diseases and a role for GrB in the pathogenesis of several chronic inflammatory diseases has been reported. Aims of this study were to investigate circulating GrB levels in T2D patients in relation to their systemic inflammatory profile and to unravel its correlates. For this cross-sectional study, we recruited 51 consecutive T2D patients referring to our diabetes outpatient clinics (Sapienza University, Rome, Italy) for metabolic evaluations, and 29 sex, age and body mass index comparable non-diabetic subjects as control group. Study participants underwent clinical work-up; fasting blood sampling was performed for routine biochemistry and for inflammatory profile (CRP, IL-2, IL-4, IL-6, IL-8, IL-10, TNF-α, IFN-γ, GM-CSF, adiponectin, WISP1); serum GrB was measured by Human Granzyme-B Platinum Elisa kit (Affymetrix EBIO). We found that T2D patients had serum levels of GrB significantly higher than the control group (10.17 ±â€¯12.6 vs 7.2 ±â€¯14.1 pg/ml, p = 0.03). Moreover, in T2D patients increased GrB correlated with unfavorable inflammatory profile, as described by elevated levels of validated adipokines such as IL-6 (p = 0.04), TNF-α (p = 0.019) and WISP1 (p = 0.005). Furthermore, multivariate linear regression analysis showed that increased GrB was associated with T2D diagnosis independently from possible confounders. In conclusion, our results show that increased levels of circulating GrB are associated with T2D diagnosis and correlates with markers of AT-linked systemic inflammation, suggesting a potential role for GrB in the inflammatory and reactive processes occurring in metabolic diseases.

Association of Perforin and Granzyme-B Levels with Hyperandrogenism in Polycystic Ovary Syndrome: A Case-Control Study.

BACKGROUND AND OBJECTIVE: Polycystic ovary syndrome (PCOS) is shown to be associated with hyperandrogenemia and has some features such as cytotoxic T-cells activation and release of perforin and granzyme-B. Present work was aimed to investigate the relation of perforin and granzyme-B to androgenic state in PCOS. MATERIALS AND METHODS: Forty three PCOS and 40 control women were recruited. After recording demographic data, sex hormone status and cardiovascular disease (CVD) risk factors were evaluated. Perforin and granzyme-B levels were measured using sandwich ELISA kits. RESULTS: Sex hormone binding globulin (SHBG) levels were lower in patients. Luteinizing hormone (LH), free testosterone (FT), dehydroepiandrosterone sulfate (DHEA-S), free androgen index (FAI), perforin and granzyme-B values were higher in PCOS group. Perforin and granzyme-B were positively correlated with FT and FAI and with each other in PCOS group. In patients, granzyme-B and perforin were related with FT and FAI, respectively. CONCLUSION: The results of present study together with evidences about the release of pro-inflammatory cytokines in insulin resistance, CVD and PCOS suggest that perforin/granzyme-B may be involved in interactions of sex hormones system in PCOS patients.

PEGylated IL-10 (Pegilodecakin) Induces Systemic Immune Activation, CD8+ T Cell Invigoration and Polyclonal T Cell Expansion in Cancer Patients.

Tumor-reactive T cell exhaustion prevents the success of immune therapies. Pegilodecakin activates intratumoral CD8+ T cells in mice and induces objective tumor responses in patients. Here we report that pegilodecakin induces hallmarks of CD8+ T cell immunity in cancer patients, including elevation of interferon-γ and GranzymeB, expansion and activation of intratumoral CD8+ T cells, and proliferation and expansion of LAG-3+ PD-1+ CD8+ T cells. On pegilodecakin, newly expanded T cell clones, undetectable at baseline, become 1%-10% of the total T cell repertoire in the blood. Elevation of interleukin-18, expansion of LAG-3+ PD-1+ T cells and novel T cell clones each correlated with objective tumor responses. Combined pegilodecakin with anti-PD-1 increased the expansion of LAG-3+ PD-1+ CD8+ T cells.

Autoimmune disease associated IFIH1 single nucleotide polymorphism related with IL-18 serum levels in Chinese systemic lupus erythematosus patients.

Systemic lupus erythematosus (SLE) has heterogeneous clinical manifestations. IFIH1 (interferon induced with helicase C domain 1) as one of antiviral helicase genes mediating type I interferon production, plays an essential role in the pathogenesis of SLE. The gene variants in IFIH1 could abnormally activate antiviral defenses and increased type I interferon signaling. The present study aimed to validate associations between single nucleotide polymorphisms (SNP) in IFIH1 and the pathogenesis of SLE. In total, rs1990760, rs3747517 and rs10930046 in IFIH1 are genotyped in 400 SLE patients and 659 health controls in Chinese cohort by an improved multiplex ligation detection reaction (iMLDR) technique. Significant associations were observed between alleles of IFIH1 (rs1990760 C > T, P = 0.005, OR = 1.36, 95%CI = 1.10-1.69; rs3747517 T > C, P = 0.004, OR = 1.31, 95%CI = 1.09-1.58, respectively) and SLE susceptibility. IFIH1 rs1990760 TT genotype carriers had lower serum levels of IL-18 (P < 0.001) and granzyme B (P < 0.001) than CC and CT genotype carriers. IFIH1 rs1990760 CT genotype carriers had higher anti-dsDNA-positive than CC and TT genotype carriers. In conclusion, IFIH1 polymorphisms (rs1990760 and rs3747517) were associated with SLE susceptibility and rs1990760 risk T allele related with IL-18 and granzyme B serum levels in SLE patients.

Placental ischemia-stimulated T-helper 17 cells induce preeclampsia-associated cytolytic natural killer cells during pregnancy.

Previous studies have demonstrated that T-helper 17 (TH17) cells and cytolytic natural killer (cNK) cells are increased in women with preeclampsia. In this study we investigated the role of placental ischemia-stimulated TH17 cells in induction of cNK cells in pregnancy. We further assessed the role of TH17 cell-mediated oxidative stress in facilitation of cNK cell activation in pregnancy by treating rats with the SOD mimetic tempol. CD4+/CD25- cells were isolated from reduced uterine perfusion pressure (RUPP) rats and differentiated into TH17 cells in vitro. On day 12 of gestation ( GD12), 1 × 106 placental ischemia-stimulated TH17 cells were injected into normal pregnant (NP) rats (NP + RUPP TH17 rats), and a subset of rats were treated with tempol (30 mg·kg-1·day-1) from GD12 to GD19 (NP + RUPP TH17 + tempol rats). On GD19, cNK cells, mean arterial pressure, fetal weight, and cNK cell-associated cytokines and proteins were measured. Placental cNK cells were 2.9 ± 1, 14.9 ± 4, and 2.8 ± 1.0% gated in NP, NP + RUPP TH17, and NP + RUPP TH17 + tempol rats, respectively. Mean arterial pressure increased from 96 ± 5 mmHg in NP rats to 118 ± 2 mmHg in NP + RUPP TH17 rats and was 102 ± 3 mmHg in NP + RUPP TH17 + tempol rats. Fetal weight was 2.37 ± 0.04, 1.95 ± 0.14, and 2.3 ± 0.05 g in NP, NP + RUPP TH17, and NP + RUPP TH17 + tempol rats, respectively. Placental IFNγ increased from 1.1 ± 0.6 pg/mg in NP rats to 3.9 ± 0.6 pg/mg in NP + RUPP TH17 rats. Placental perforin increased from 0.18 ± 0.18 pg/mg in NP rats to 2.4 ± 0.6 pg/mg in NP + RUPP TH17 rats. Placental levels of granzymes A and B followed a similar pattern. Treatment with tempol did not lower placental cNK cytokines or proteins. The results of the present study identify TH17 cells as a mediator of aberrant NK cell activation that is associated with preeclampsia.