Levels Of HLA-DR Research Articles

P6301Pro-inflammatory intermediate monocytes relate to right ventricular pressure in heart failure of adult congenital heart disease

Abstract Background Heart failure (HF) is a leading cause of mortality in adults with congenital heart disease (ACHD). ACHD is characterized by predominant right heart disease. In non-congenital heart disease inflammation and its mediators such as monocytes/macrophages play an important pathophysiological role in HF. We aimed to evaluate the role of circulating monocyte subsets in ACHD-HF. Methods This cross-sectional study includes 209 ACHD outpatients (mean age: 35.3±11.0 years; NYHA class I/II/III-IV 58.3%/19.6%/13%; male: 59.8%) and 21 healthy controls (age: 29.8±12.6 years; male: 47.6%). Patients with clinical signs of infection, inflammatory diseases or malignancies were excluded. Multivariate analysis was used to relate blood monocyte subsets to NYHA class and echocardiographically derived parameters of right and left ventricular function. Results Compared to control, ACHD had significantly higher circulating levels of pro-inflammatory HLA-DR+CD14++CD16+ intermediate monocytes (24.0±3.3 vs. 43.6±1.7 cells/μL; p<0.001). NT-proBNP was independently associated with reduced left (p<0.0001) and right (p<0.001) ventricular function, diastolic dysfunction (p=0.04) and vena cava diameter (p=0.02). Independent predictors of NYHA class were intermediate monocytes (p=0.022), plasma noradrenaline (p=0.002), albumin (p=0.001) and NT-proBNP (p<0.001). Elevated right ventricular systolic pressure (>35 mmHg) was independently associated with both, higher intermediate monocyte counts (OR 1.36; 95% CI: 1.13–1.62; p=0.001) and low oxygen saturation (OR 0.8; 95% CI: 0.7–0.92; p=0.001), even after multivariable adjustment for age, sex and NYHA class. Conclusions Right ventricular pressure and oxygen saturation are linked to elevated intermediate monocytes, suggesting an important link between inflammation and HF in ACHD. Circulating blood intermediate monocytes represent a promising biomarker in ACHD. Acknowledgement/Funding German Heart Foundation (Deutsche Herzstiftung e.V.)

Phenotypic analysis of monocytes and CD4+ T cells in hepatitis E patients with or without pregnancy

High mortality in pregnant women is a characteristic of hepatitis E virus (HEV) infection. Role of monocytes/T cells in HEV infection during pregnancy is still unclear. We compared CD14+monocytes and CD4+T cells by flow-cytometry in hepatitis-E patients including 13 pregnant (Antenatal care, ANC), 25 non-ANC patients and respective controls (12 and 20). Non-ANC-patients showed significantly higher frequency of monocytes with increased expression of CD80, CD86 and HLA-DR than control individuals (p < 0.001).Healthy pregnancy was associated with increased frequency of monocytes with higher CD80 expression and lower levels of HLA-DR (p < 0.05) compared to non-ANC controls. ANC-patients exhibited elevated levels of monocytes (p < 0.01) with higher expression of CD80 (p < 0.001) and reduced levels of HLA-DR and CD86 (p < 0.05) when compared with non-ANC patients. TLR2 and TLR4 surface expression on monocytes was higher in non-ANC-patients (p < 0.00) and lower in the ANC-patients (p < 0.01). Healthy-ANCs exhibited lower TLR4 expression on monocytes (p < 0.05). HEV infection did not change the frequency of CD4+ and CD4+CD28+T cells in patients’ group (p > 0.05). Compared to respective controls, CD137+ and CD152+CD4+T cells were higher (p < 0.05) in both patients' categories. Higher levels of CD152+CD4+T cells (p < 0.001) was noted in healthy pregnant women. Among patients' groups, the CD4+T cells and their subpopulation were not different (p > 0.05).We found higher and reduced levels of circulating inflammatory cytokines (IL12, TNFα, IL6 and IL8; miliplex-assay) in non-ANC and ANC-patients respectively. In conclusion, on contrary to the classical activation of CD14+monocytes in the non-ANC-patients, impaired response was evident in the ANC-patients while the CD4+T cell populations were similar in the patient groups.

Characterization of Magnitude and Antigen Specificity of HLA-DP, DQ, and DRB3/4/5 Restricted DENV-Specific CD4+ T Cell Responses.

Background: Dengue Virus (DENV) associated disease is a major public health problem. Assessment of HLA class II restricted DENV-specific responses is relevant for immunopathology and definition of correlates of protection. While previous studies characterized responses restricted by the HLA-DRB1 locus, the responses associated with other class II loci have not been characterized to date. Accordingly, we mapped HLA-DP, DQ, and DRB3/4/5 restricted DENV-specific CD4 T cell epitopes in PBMCs derived from the DENV endemic region Sri Lanka.Methods: We studied 12 DP, DQ, and DRB3/4/5 alleles that are commonly expressed and provide worldwide coverage >82% for each of the loci analyzed and >99% when combined. CD4+ T cells purified by negative selection were stimulated with pools of HLA-predicted binders for 2 weeks with autologous APC. Epitope reactive T cells were enumerated using IFNγ ELISPOT assay. This strategy was previously applied to identify DRB1 restricted epitopes. In parallel, membrane expression levels of HLA-DR, DP, and DQ proteins was assessed using flow cytometry.Results: Epitopes were identified for all DP, DQ, and DRB3/4/5 allelic variants albeit with magnitudes significantly lower than the ones previously observed for the DRB1 locus. This was in line with lower membrane expression of HLA-DP and DQ molecules on the PBMCs tested, as compared to HLA-DR. Significant differences between loci were observed in antigen immunodominance. Capsid responses were dominant for DRB1/3/4/5 and DP alleles but negligible for the DQ alleles. NS3 responses were dominant in the case of DRB1/3/4/5 and DQ but absent in the case of DP. NS1 responses were prominent in the case of the DP alleles, but negligible in the case of DR and DQ. In terms of epitope specificity, repertoire was largely overlapping between DRB1 and DRB3/4/5, while DP and DQ loci recognized largely distinct epitope sets.Conclusion: The HLA-DP, DQ, and DRB3/4/5 loci mediate DENV-CD4 specific immune responses of lower magnitude as compared to HLA-DRB1, consistent with their lower levels of expression. The responses are associated with distinct and characteristic patterns of immunodominance, and variable epitope overlap across loci.

Deficiency in Peripheral MAIT Cells But Not in Intrahepatic MAIT Cells in Humans with CHBV Infection

Background: Chronic hepatitis B virus (CHBV) infection is a major cause of liver diseases. Mucosal-associated invariant T (MAIT) cells are important for antiviral immunity in the liver, but the distinction between intrasinusoidal MAIT cells and peripheral MAIT cells in patients with CHBV infection remains unclear. Methods: PBMC were obtained from patients with CHBV infection (n=51) and age-matched controls (n=52). Liver-associated mononuclear cells (LMCs) were collected from donors (n=32) and explanted liver (n=23) of patients to perform phenotypic, functional and TCR diversity analysis. Results: The frequency of intrasinusoidal MAIT cells in the CHBV infection group was not changed compared to that of the MAIT cells in the CHBV-negative group. However, the frequency of peripheral blood MAIT cells was severely reduced in the CHBV infection group compared to the CHBV-negative group. Moreover, IFN-γ production was intact in the intrahepatic MAIT cells of the CHBV infection patients compared to those of the control subjects post IL-12 and IL-18 stimulation, while it was reduced in the peripheral blood MAIT cells of the CHBV infection patients. More importantly, our study showed that the expression level of HLA-DR in peripheral MAIT cells was higher than that in intrasinusoidal MAIT cells in CHBV infection patients. A distinct signature of TCR usage was found in the CHBV patients. Conclusions: By analysing the phenotype, function and TCR usage of MAIT cells in the PBMC and LMC populations of patients with CHBV infection, we found distinct functions and activities in the MAIT cells of the liver compared with those of the peripheral blood. Funding Statement: YG is supported by Natural Science Foundation of Guangdong Province (Grant number: 2018A030313019) and National Natural Science Foundation of China (Grant number:31800758). Declaration of Interests: All authors disclose no conflicts of interest. Ethics Approval Statement: This study was fully approved by the First Affiliated Hospital of Sun Yat-sen University ethical board. All participants provided written informed consent before sample collection.

PD-L1-specific helper T-cells exhibit effective antitumor responses: new strategy of cancer immunotherapy targeting PD-L1 in head and neck squamous cell carcinoma

BackgroundHead and neck squamous cell carcinoma (HNSCC) originates from squamous epithelium of the upper aerodigestive tract and is the most common malignancy in the head and neck region. Among HNSCCs, oropharynx squamous cell carcinoma (OSCC) has a unique profile and is associated with human papillomavirus infection. Recently, anti-programmed cell death-1 monoclonal antibody has yielded good clinical responses in recurrent and/or metastatic HNSCC patients. Therefore, programmed death-ligand 1 (PD-L1) may be a favorable target molecule for cancer immunotherapy. Although PD-L1-expressing malignant cells could be targeted by PD-L1-specific CD8+ cytotoxic T lymphocytes, it remains unclear whether CD4+ helper T lymphocytes (HTLs) recognize and kill tumor cells in a PD-L1-specific manner.MethodsThe expression levels of PD-L1 and HLA-DR were evaluated using immunohistochemical analyses. MHC class II-binding peptides for PD-L1 were designed based on computer algorithm analyses and added into in vitro culture of HTLs with antigen-presenting cells to evaluate their stimulatory activity.ResultsWe found that seven of 24 cases of OSCC showed positive for both PD-L1 and HLA-DR and that PD-L1241-265 peptide efficiently activates HTLs, which showed not only cytokine production but also cytotoxicity against tumor cells in a PD-L1-dependent manner. Also, an adoptive transfer of the PD-L1-specific HTLs significantly inhibited growth of PD-L1-expressing human tumor cell lines in an immunodeficient mouse model. Importantly, T cell responses specific for the PD-L1241-265 peptide were detected in the HNSCC patients.ConclusionsThe cancer immunotherapy targeting PD-L1 as a helper T-cell antigen would be a rational strategy for HNSCC patients.

Resiquimod-Mediated Activation of Plasmacytoid Dendritic Cells Is Amplified in Multiple Sclerosis.

Background: Multiple sclerosis (MS) is a chronic inflammatory autoimmune disease of the central nervous system. The cause of multiple sclerosis is unknown but there are several evidences that associate the genetic basis of the disease with environmental causes. An important association between viral infection and development of MS is clearly demonstrated. Viruses have a strong impact on innate immune cells. In particular, myeloid dendritic cells (mDCs) and plasmacytoid dendritic cells (pDCs), are able to respond to viruses and to activate the adaptive immune response. Methods: In this study we mimic viral infection using synthetic single-strand RNA, Resiquimod, and we compared the response of both DC subsets derived from healthy donors and MS patients by characterizing the expression of costimulatory molecules on the DC surface. Results: We found that pDCs from MS patients express higher levels of OX40-L, HLA-DR, and CD86 than healthy donors. Moreover, we found that blood cells from MS patients and healthy donors upon Resiquimod-stimulation are enriched in a subpopulation of pDCs, characterized by a high amount of costimulatory molecules. Conclusion: Overall, these results indicate that activation of pDCs is enhanced in MS, likely due to a latent viral infection, and that costimulatory molecules expressed on pDCs could mediate a protective response against the viral trigger of autoimmunity.

Alcohol Abstinence Does Not Fully Reverse Abnormalities of Mucosal-Associated Invariant T Cells in the Blood of Patients With Alcoholic Hepatitis.

OBJECTIVES:Alcoholic hepatitis (AH) develops in approximately 30% of chronic heavy drinkers. The immune system of patients with AH is hyperactivated, yet ineffective against infectious diseases. Mucosal-associated invariant T (MAIT) cells are innate-like lymphocytes that are highly enriched in liver, mucosa, and peripheral blood and contribute to antimicrobial immunity. We aimed to determine whether MAIT cells were dysregulated in heavy drinkers with and without AH and the effects of alcohol abstinence on MAIT cell recovery.METHODS:MR1 tetramers loaded with a potent MAIT cell ligand 5-(2-oxopropylideneamino)-6-d-ribitylaminouracil were used in multiparameter flow cytometry to analyze peripheral blood MAIT cells in 59 healthy controls (HC), 56 patients with AH, and 45 heavy drinkers without overt liver disease (HDC) at baseline and 6- and 12-month follow-ups. Multiplex immunoassays were used to quantify plasma levels of cytokines related to MAIT cell activation. Kinetic Turbidimetric Limulus Amebocyte Lysate Assay and ELISA were performed to measure circulating levels of 2 surrogate markers for bacterial translocation (lipopolysaccharide and CD14), respectively.RESULTS:At baseline, patients with AH had a significantly lower frequency of MAIT cells than HDC and HC. HDC also had less MAIT cells than HC (median 0.16% in AH, 0.56% in HDC, and 1.25% in HC). Further, the residual MAIT cells in patients with AH expressed higher levels of activation markers (CD69, CD38, and human leukocyte antigen [HLA]-DR), the effector molecule granzyme B, and the immune exhaustion molecule PD-1. Plasma levels of lipopolysaccharide and CD14 and several cytokines related to MAIT cell activation were elevated in patients with AH (interferon [IFN]-α, interleukin [IL]-7, IL-15, IL-17, IL-18, IL-23, IFN-γ, and tumor necrosis factor α). Decreased MAIT cell frequency and upregulated CD38, CD69, and HLA-DR correlated negatively and positively, respectively, with aspartate aminotransferase level. MAIT cell frequency negatively correlated with IL-18. HLA-DR and CD38 levels correlated with several cytokines. At follow-ups, abstinent patients with AH had increased MAIT cell frequency and decreased MAIT cell activation. However, MAIT cell frequency was not fully normalized in patients with AH (median 0.31%).DISCUSSION:We showed that HDC had a reduction of blood MAIT cells despite showing little evidence of immune activation, whereas patients with AH had a severe depletion of blood MAIT cells and the residual cells were highly activated. Alcohol abstinence partially reversed those abnormalities.

Age-associated B cells in early drug-naive rheumatoid arthritis patients

Abstract Rheumatoid arthritis (RA) is a chronic autoimmune disorder characterised by joint inflammation. Studies in mice and patients suffering from autoimmune diseases described a novel subset of B cells named age-associated B cells (ABCs). Moreover, a subset of synovial fluid B cells with a similar phenotype and FcRL4 expression contributes to RA pathogenesis. Newly diagnosed patients were recruited from the Newcastle Early Arthritis Clinic. B cell subsets in blood were phenotyped using flow cytometry. NanoString Immunlogy Panel was used to asses gene expression from sorted ABCs, naïve, memory and CD5+ B cells from RA patients, age-matched healthy controls and disease controls. Our work showed that there are no significant differences in the frequency of ABCs between RA patients, and disease and healthy controls. Our results also show that ABCs resemble the memory B cell phenotype regarding class-switch immunoglobulins expression. Interestingly, the FcRL4+, the Ki67+ and the T-bet expressing B cells were enriched in the ABC population. ABCs expressed high levels of HLA-DR and co-stimulatory molecules, as well as the activation marker, CD69. Gene expression analysis showed a specific gene signature for the ABCs when compared to the other B cells. KEGG analysis results showed that differentially expressed genes were enriched in the antigen presentation and the cytokine-cytokine receptor interaction pathways. Moreover, 13 genes were overexpressed in ABCs from the RA patients compared to the other control groups. This data supports the idea that ABCs have a pathogenic role in RA, potentially via T cell stimulation and migration to sites of inflammation. However, further characterisation of this subset and functional studies are needed.

CD8+T-bet+ cells as a predominant biomarker for inclusion body myositis.

Myositis is a heterogeneous group of muscular auto-immune diseases with clinical and pathological criteria that allow the classification of patients into different sub-groups. Inclusion body myositis is the most frequent myositis above fifty years of age. Diagnosing inclusion body myositis requires expertise and is challenging. Little is known concerning the pathogenic mechanisms of this disease in which conventional suppressive-immune therapies are inefficacious. Our aim was to deepen our understanding of the immune mechanisms involved in inclusion body myositis and identify specific biomarkers. Using a panel of thirty-six markers and mass cytometry, we performed deep immune profiling of peripheral blood cells from inclusion body myositis patients and healthy donors, divided into two cohorts: test and validation cohorts. Potential biomarkers were compared to myositis controls (anti-Jo1-, anti-3-hydroxyl-3-methylglutaryl CoA reductase-, and anti-signal recognition particle-positive patients). Unsupervised analyses revealed substantial changes only within CD8+ cells. We observed an increase in the frequency of CD8+ cells that expressed high levels of T-bet, and containing mainly both effector and terminally differentiated memory cells. The senescent marker CD57 was overexpressed in CD8+T-bet+ cells of inclusion body myositis patients. As expected, senescent CD8+T-bet+ CD57+ cells of both patients and healthy donors were CD28nullCD27nullCD127null. Surprisingly, non-senescent CD8+T-bet+ CD57- cells in inclusion body myositis patients expressed lower levels of CD28, CD27, and CD127, and expressed higher levels of CD38 and HLA-DR compared to healthy donors. Using classification and regression trees alongside receiver operating characteristics curves, we identified and validated a frequency of CD8+T-bet+ cells >51.5% as a diagnostic biomarker specific to inclusion body myositis, compared to myositis control patients, with a sensitivity of 94.4%, a specificity of 88.5%, and an area under the curve of 0.97. Using a panel of thirty-six markers by mass cytometry, we identify an activated cell population (CD8+T-bet+ CD57- CD28lowCD27lowCD127low CD38+ HLA-DR+) which could play a role in the physiopathology of inclusion body myositis, and identify CD8+T-bet+ cells as a predominant biomarker of this disease.

Prognostic Value of Macrophage Phenotypes in Resectable Non–Small Cell Lung Cancer Assessed by Multiplex Immunohistochemistry

Macrophages are important inflammatory cells that regulate innate and adaptive immunity in cancer. Tumor-associated macrophages (TAMs) are thought to differentiate into two main phenotypes: proinflammatory M1 and protumorigenic M2. Currently, the prognostic impact of TAMs and their M1 and M2 phenotypes is unclear in non–small cell cancer (NSCLC). The present study was set up to evaluate an approach for identifying common M1 and M2 macrophage markers and explore their clinical significance in NSCLC. Using multiplex chromogenic immunohistochemistry, tissue microarrays of 553 primary tumors and 143 paired metastatic lymph nodes of NSCLC specimens were stained to detect various putative macrophage phenotypes: M1 (HLA-DR/CD68), M2 (CD163/CD68), M2 (CD204/CD68), and pan-macrophage (CD68/CK). Correlation analyses were performed to examine the relationship between TAMs and adaptive/innate immune infiltrates. HLA-DR+/CD68+M1 TAM level significantly decreased from pathological stage I to III. In a compartment-specific correlation analysis, moderate to strong correlations were observed between both TAM subsets (M1 and M2) with CD3-, CD8-, CD4-, and CD45RO-positive immune cells. Survival analyses, in both stromal and intratumoral compartments, revealed that high levels of HLA-DR+/CD68+M1 (stroma, hazard ratio [HR] = 0.73, P = .03; intratumor, HR = 0.7, P = .04), CD204+M2 (stroma, HR = 0.7, P = .02; intratumor, HR = 0.6, P = .004), and CD68 (stroma, HR = 0.69, P = .02; intratumor, HR = 0.73, P = .04) infiltration were independently associated with improved NSCLC-specific survival. In lymph nodes, the intratumoral level of HLA-DR+/CD68+M1 was an independent positive prognostic indicator (Cox model, HR = 0.38, P = .001). In conclusion, high levels of M1, CD204+M2, and CD68 macrophages are independent prognosticators of prolonged survival in NSCLC.

P054 CD103+SIRPα+ DC are specifically decreased in the inflamed colon from patients with ulcerative colitis but not with Crohn’s disease

Inflammatory bowel disease (IBD), including Crohn's disease (CD) and ulcerative colitis (UC), is a chronic inflammation of the human gastrointestinal (GI) tract. Intestinal dendritic cells (DC) are essential to maintain the balance between immunity against pathogens and tolerance towards nutrients and commensals. However, there is not much information about DC composition in the human GI tract both in health and IBD. Human GI biopsies were obtained from healthy controls and IBD patients (including UC and CD; both active and quiescent). Tissue was disaggregated and lamina propria mononuclear cells (LPMC) characterised by flow cytometry. Human intestinal DC were identified within singlet viable leucocytes as CD14-CD64-HLA-DR+CD11c+. Type 1 DC were defined as CD103+SIRPα- while type 2 DC were identified as SIRPα+ and further divided into subsets based on the expression of CD103. The proportion of total DC displayed a gradient throughout the healthy human gut as it was higher in the colon (either distal or proximal) compared with the ileum. DC proportion was further decreased in the duodenum. Type 1 (minority) and type 2 (majority) conventional DC did not change their proportion throughout the healthy gut. However, CD103+SIRPα+ DC were the main subset in the duodenum as opposed to CD103-SIRPα+ DC which were predominant in the colon and the ileum. Compared with their CD103-SIRPα+ type 2 counterparts, CD103+SIRPα+ had higher levels of HLA-DR, CD40, CD86, CCR7, CD137L, ICOSL and PD-L1. CD103+SIRPα+ were also more phagocytic and had lower expression of blood-related markers like CLA and CCR2, suggesting that they are derived from CD103-SIRPα+ DC following mucosal conditioning. Indeed, CD103+SIRPα+ numbers were increased following LPMC culture, although this process was reverted in the presence of pro-inflammatory LPS. CD103+SIRPα+ DC displayed an enhanced production of IL-10, both in resting conditions and in the presence of LPS. In IBD, type 2 DC constitutively displayed lower expression of SIRPα irrespectively of IBD-type (CD or UC) or condition (active or quiescent). Nevertheless, the inflamed colon from UC patients, but not from CD, specifically displayed lower numbers of tolerogenic CD103+SIRPα+ DC. These results were in agreement with the colonic cytokine milieu, which was much more pro-inflammatory in UC patients compared with CD. Tolerogenic PD-L1 expression and IL-10 production was associated with CD103+SIRPα+ DC, confirming therefore their tolerogenic phenotype. Human colonic DC from IBD patients constitutively display lower levels of SIRPα. The specific reduction of CD103+SIRPα+ DC in the inflamed mucosa from UC, but not CD, suggests the presence of different pathogenic mechanisms occurring in IBD.

Synovial fluid monocyte/macrophage subsets and their correlation to patient-reported outcomes in osteoarthritic patients: a cohort study

BackgroundChronic, low-grade inflammation of the synovium (synovitis) is a hallmark of osteoarthritis (OA), thus understanding of OA immunobiology, mediated by immune effectors, is of importance. Specifically, monocytes/macrophages (MΦs) are known to be abundantly present in OA joints and involved in OA progression. However, different subsets of OA MΦs have not been investigated in detail, especially in terms of their relationship with patient-reported outcome measures (PROMs). We hypothesized that levels of synovial fluid (SF) MΦ subsets are indicative of joint function and quality of life in patients with OA, and can therefore serve as biomarkers and therapeutic targets for OA.MethodsIn this cohort study, synovial fluid leukocytes (SFLs, N = 86) and peripheral blood mononuclear cells (n = 53) from patients with knee OA were characterized. Soluble MΦ receptors and chemokine (sCD14, sCD163, CCL2, CX3CL1) levels were detected in SF using immunoassays. Linear models, adjusted for sex, age and body mass index, were used to determine associations between SF MΦs and soluble factors with PROMs (N = 83). Pearson correlation was calculated to determine correlation between MΦ subsets, T cells and soluble factors.ResultsSF MΦs were the most abundant SFLs. Within these, the double-positive CD14+CD16+-MΦ subset is enriched in knee OA SF compared to the circulation. Importantly, MΦ subset ratios correlated with PROMs, specially stiffness, function and quality of life. Interestingly, the SF CD14+CD16+-MΦ subset ratio correlated with SF chemokine (C-C motif) ligand 2 (CCL2) levels but not with levels of sCD163 or sCD14; we found no association between PROMs and either SF CCL2, sCD163, sCD14 or CX3CL1 (which was below detection levels). All SF MΦs displayed high levels of HLA-DR, suggesting an activated phenotype. Correlation between OA SF MΦ subsets and activated CD4+ T cell subsets suggests modulation of CD4+ T cell activation by MΦs.ConclusionSF MΦ subsets are associated with knee OA PROMs and display an activated phenotype, which may lead to modulation of CD4+ T cell activation. Knee OA SF MΦ subsets could serve as knee OA function biomarkers and as targets of novel therapeutics.

The effect of methylprednisolone prophylaxis on inflammatory monocyte subsets and suppressive regulatory T cells of patients undergoing cardiopulmonary bypass.

Cardiopulmonary bypass (CPB) during open-heart surgery triggers an inflammatory response that can cause significant morbidity and mortality. Human monocytes and regulatory T (Treg) cells are phenotypically and functionally heterogeneous and have been shown to play a significant role in the inflammatory dysfunction triggered by CPB. Glucocorticoids (GCs) have been widely administered for decades in patients undergoing CPB to reduce this inflammatory response. However, it has not been clearly established how routine prophylactic administration of glucocorticoids (GCs) affects monocyte and Treg subsets. Thirty-six patient who underwent heart surgery with CPB were randomly assigned to a methylprednisolone group (MG, N = 18; 500 mg in the CPB priming) and a non-methylprednisolone group (NMG, N = 18). The circulating monocyte and Treg subsets were analyzed by flow cytometry. The MG and NMG groups had comparable percentages of monocyte subsets and similar expression levels of HLA-DR, CD86, CD64 and toll-like receptor 4 (TLR4). Remarkably, methylprednisolone increased the percentage of CD4+CD25+ Treg cells among CD4+ T cells in patients undergoing CPB, but did not increase the proportion of suppressive Treg cells, either resting or activated, in these patients undergoing CPB. Our results showed that prophylactic administration of methylprednisolone neither decreased the percentages and counts of inflammatory monocyte subsets nor did it induce the expansion of suppressive Treg cells in patients undergoing CPB. These results clarified the effects of GCs on cell-mediated immune responses and provided additional evidence in practice. Clinicaltrials.gov : NCT01296074. Registered 14 February 2011.

Effects of Changes in the Levels of Damage-Associated Molecular Patterns Following Continuous Veno-Venous Hemofiltration Therapy on Outcomes in Acute Kidney Injury Patients With Sepsis.

Background: We investigated the association of damage-associated molecular pattern (DAMP) removal with mortality in sepsis patients undergoing continuous veno–venous hemofiltration (CVVH).Methods: Circulating levels of DAMPs [mitochondrial DNA (mtDNA); nuclear DNA (nDNA); heat shock protein 70 (HSP70); and high mobility group box 1 (HMGB1)] and cytokines were measured at baseline, 6 and 12 h after initiation of CVVH. Urinary DNA levels were analyzed at baseline and end of CVVH. The expression of human leukocyte antigen (HLA)-DR was assayed at 0, 3, and 7 days after initiation of CVVH. Moreover, the effects of HSP70 and HMGB1 clearance on survival were analyzed.Results: We evaluated 43 patients with acute kidney injury (AKI) (33 sepsis patients). Twenty-two sepsis patients (67%) and three non-sepsis patients (30%) expired (P = 0.046). Significant reductions in the levels of circulating interleukin-6 (P = 0.046) and tumor necrosis factor-α (P = 0.008) were found in the sepsis group. The levels of mtDNA were increased (ND2, P = 0.035; D-loop, P = 0.003), whereas that of HSP70 was reduced (P = 0.000) in all patients during the first 12 h. The levels of DAMPs in the plasma were markedly increased after blood passage from the inlet through the dialyzer in survivor sepsis patients. The clearance rates of HSP70 and HMGB1 were good predictors of mortality [area under the curve (AUC) = 0.937, P = 0.000; AUC = 0.90, P = 0.001, respectively]. The level of HLA-DR was increased in response to higher HSP70 clearance (P = 0.006). Survival was significantly worse in groups with higher clearance rates of HSP70 and HMGB1 than the cut-off value (log-rank test: P = 0.000 for both). Higher HSP70 clearance was a significant independent predictor of mortality (odds ratio = 1.025, 95% confidence interval [CI]: 1.012–1.039, P = 0.000). The urinary nDNA (β-globin) level before CVVH was an independent risk factor for the duration of CVVH in patients with sepsis (sRE = 0.460, 95% CI: 1.720–8.857, P = 0.005).Conclusion: CVVH removes inflammatory factors, reduces urinary DAMPs, and removes plasma DAMPs. However, survival decreases in response to higher HSP70 clearance.

Vitamin D-modulated dendritic cells delay lethal graft-versus-host disease through induction of regulatory T cells

Graft-versus-host disease (GVHD) is the most lethal complication after allogeneic bone marrow transplantation (allo-BMT). Current approaches to prevent GVHD rely on donor lymphocyte/T cell depletion or general immunosuppression, leading to opportunistic infections and cancer relapse. Tolerogenic dendritic cells can induce regulatory T cells (Tregs) with the ability to suppress inflammation and prevent transplant rejection, making them an attractive cellular therapy to control GVHD. Active vitamin D (1α,25-dihydroxyvitamin D3; 1α,25(OH)2D3) promotes the generation of tolerogenic dendritic cells (1,25D3-DCs). This study aimed to determine the ability of ex vivo generated 1,25D3-DCs to trigger the expansion of Tregs that are able to control lethal xenogeneic GVHD in humanized NOD/LtSz-PrkdcscidIL2rγtm1Wjl (NSG) mice. We demonstrate that 1,25D3-DCs express lower levels of HLA-DR and costimulatory molecules, such as CD80 and CD86, and produce higher levels of IL-10 and TNF-α and lower amounts of IL-12, compared to vehicle-treated DCs. Moreover, these cells express increased levels of various co-inhibitory molecules such as PD-L1 and ILT-3 and the glycoprotein CD52 that is known to suppress T cell activation. Consequently, 1,25D3-DCs are poor stimulators of alloantigen-primed T cells, but foster the generation of antigen-specific suppressive Tregs. When adoptively transferred in humanized NSG mice, these 1,25D3-DC-induced Tregs delayed GVHD caused by the co-transferred autologous human peripheral blood mononuclear cells (PBMCs). These results indicate that 1,25D3-DC-induced Tregs can inhibit xenogeneic GVHD and maintain their immunomodulatory function under conditions of inflammation.

OMIP-051 - 28-color flow cytometry panel to characterize B cells and myeloid cells.

OMIP-051 - 28-color flow cytometry panel to characterize B cells and myeloid cells.

Using of TLR2 and TLR4 as Biomarker of Sepsis Severity Detection

Our study has been aimed to find the relation between the expression of toll-like receptors 2, 4, level of TNF-α, IL-10 and soluble HLA-DR with the severity bacterial septic syndrome in Iraqi patients. The quantitative real-time PCR technique has been used for measure TLR2 and TLR4 gene expression in whole blood, and ELISA technique has been used for detection of cytokines TNF-α, IL-10 and soluble HLA-DR from 75 septic syndrome cases (nineteen of patients showed symptoms of systemic inflammatory response syndrome (SIRS); twenty-eight patients have sepsis, seventeen patients suffered from severe sepsis and eleven patients have septic shock) and 55 healthy controls (HC). TLR2 and TLR4 mRNA expression were higher significantly in the all patients (P&lt; 0.05), TNF-α, IL-10 and sHLA-DR serum levels were significantly elevated in the serum of patients with septic syndrome compared with controls (P

Levels of IL-17F and IL-33 correlate with HLA-DR activation in clinical-grade human bone marrow–derived multipotent mesenchymal stromal cell expansion cultures

Background aimsMultipotent mesenchymal stromal cell (MSC)-based medicines are extensively investigated for use in regenerative medicine and immunotherapy applications. The International Society for Cell and Gene Therapy (ISCT) proposed a panel of cell surface molecules for MSC identification that includes human leukocyte antigen (HLA)-DR as a negative marker. However, its expression is largely unpredictable despite production under tightly controlled conditions and compliance with current Good Manufacturing Practices. Herein, we report the frequency of HLA-DR expression in 81 batches of clinical grade bone marrow (BM)-derived MSCs and investigated its impact on cell attributes and culture environment. MethodsThe levels of 15 cytokines (interleukin [IL]-1β, IL-4, IL-6, IL-10, IL-17A, IL-17F, IL-21, IL-22, IL-23, IL-25, IL-31, IL-33, interferon-γ, soluble CD40 ligand and tumor necrosis factor-α) were determined in sera supplements and supernatants of BM-MSC cultures. Identity, multipotentiality and immunopotency assays were performed on high (>20% of cells) and low (≤20% of cells) HLA-DR+ cultures. ResultsA correlation was found between HLA-DR expression and levels of IL-17F and IL-33. Expression of HLA-DR did neither affect MSC identity, in vitro tri-lineage differentiation potential (into osteogenic, chondrogenic and adipogenic lineages), nor their ability to inhibit the proliferation of stimulated lymphocytes. DiscussionOut of 81 batches of BM-MSCs for autologous use analyzed, only three batches would have passed the ISCT criteria (<2%), whereas 60.5% of batches were compliant with low HLA-DR values (≤20%). Although a cause–effect relationship cannot be drawn, we have provided a better understanding of signaling events and cellular responses in expansion culture conditions relating with HLA-DR expression.

HLA-DR in Cytotoxic T Lymphocytes Predicts Breast Cancer Patients' Response to Neoadjuvant Chemotherapy.

Prediction of breast cancer response to Neoadjuvant Chemotherapy (NACT) is an urgent need to promptly direct non-responder patients to alternative therapies. Infiltrating T lymphocytes, namely cytotoxic T lymphocytes (CTLs) have been appointed as predictors of response. However, cancer cells have the ability to dampen CTLs' activity and thus, the prognostic value of the CTLs, per se, is debatable. Here, we disclose that more than the occurrence of CTLs, it is their activation state, revealed by HLA-DR expression, that can accurately predict response to NACT. Flow cytometry analysis of breast cancer biopsies showed that the frequency of CTLs and other lymphocytes were similar regardless disease stage and between NACT responders and non-responders. However, only breast cancer patients without axillary lymph node metastasis and NACT responders have HLA-DRhi CTLs. Interestingly, HLA-DR levels in tumor CTLs is correlated with HLA-DR levels in systemic CTLs. These HLA-DR+ CTLs produce IFN-γ and Granzyme B, enlightening their effector and probable anti-tumor activity profile. Moreover, the level of HLA-DR in CTLs is negatively correlated with the level of HLA-DR in T regulatory lymphocytes and with immunosuppressive and pro-tumor molecules in the tumor microenvironment. Hence, HLA-DR levels in CTLs is a highly sensitive and specific potential predictive factor of NACT-response, which can be assessed in blood to guide therapeutic decisions.

Maintained thymic output of conventional and regulatory T cells during human pregnancy

Maintained thymic output of conventional and regulatory T cells during human pregnancy