Cell Subsets In Peripheral Blood Research Articles

Regulatory T cell subsets in peripheral blood of celiac disease patients and TLR2 expression: correlation with oxidative stress

The study was carried out to study expression of Toll like receptors 2 (TLR2), natural/inducible Treg and Interferon-γ alongside oxidative stress and understand their significance in pediatric samples. Influence of oxidative stress on Celiac Disease was analysed by evaluating lipid peroxidation, reduced glutathione, Glutathione peroxidase, etc. A comparison was performed among CD patients, CD patients on gluten free diet (GFD), and healthy controls. Peripheral nTregs exhibited a similar pattern of reduced numbers in CD and GFD cases when compared to healthy controls. On the other hand, inducible Tregs were much lower in GFD patients as compared to CD patients. Expression of TLR2 on iTregs was elevated in CD and GFD, however, expression on nTregs was unaltered in all the three groups. The inflammatory cytokine IFN-γ positive Treg cells were found to be elevated in CD as compared to control group. Oxidative stress was elevated in CD as compared to healthy controls while that in GFD samples was lower in comparison to CD. The levels of LPO, activities of enzymes SOD and Catalase were higher in CD and GFD samples when compared to controls. However, enzyme Glutathione peroxidase and reduced glutathione levels declined in both CD and GFD groups as compared to controls. This report highlights the effect of elevated oxidative stress in CD on reduced traffic of iTregs toward periphery. A strong correlation was observed between the cytokine IFN-γ and TLR2 expression in movement of iTregs in CD patients.

Chemokine receptor co-expression reveals aberrantly distributed TH effector memory cells in GPA patients

BackgroundPersistent expansion of circulating CD4+ effector memory T cells (TEM) in patients with granulomatosis with polyangiitis (GPA) suggests their fundamental role in disease pathogenesis. Recent studies have shown that distinct functional CD4+ TEM cell subsets can be identified based on expression patterns of chemokine receptors. The current study aimed to determine different CD4+ TEM cell subsets based on chemokine receptor expression in peripheral blood of GPA patients. Identification of particular circulating CD4+ TEM cells subsets may reveal distinct contributions of specific CD4+ TEM subsets to the disease pathogenesis in GPA.MethodPeripheral blood of 63 GPA patients in remission and 42 age- and sex-matched healthy controls was stained immediately after blood withdrawal with fluorochrome-conjugated antibodies for cell surface markers (CD3, CD4, CD45RO) and chemokine receptors (CCR4, CCR6, CCR7, CRTh2, CXCR3) followed by flow cytometry analysis. CD4+ TEM memory cells (CD3+CD4+CD45RO+CCR7-) were gated, and the expression patterns of chemokine receptors CXCR3+CCR4-CCR6-CRTh2-, CXCR3-CCR4+CCR6-CRTh2+, CXCR3-CCR4+CCR6+CRTh2-, and CXCR3+CCR4-CCR6+CRTh2- were used to distinguish TEM1, TEM2, TEM17, and TEM17.1 cells, respectively.ResultsThe percentage of CD4+ TEM cells was significantly increased in GPA patients in remission compared to HCs. Chemokine receptor co-expression analysis within the CD4+ TEM cell population demonstrated a significant increase in the proportion of TEM17 cells with a concomitant significant decrease in the TEM1 cells in GPA patients compared to HC. The percentage of TEM17 cells correlated negatively with TEM1 cells in GPA patients. Moreover, the circulating proportion of TEM17 cells showed a positive correlation with the number of organs involved and an association with the tendency to relapse in GPA patients. Interestingly, the aberrant distribution of TEM1 and TEM17 cells is modulated in CMV- seropositive GPA patients.ConclusionsOur data demonstrates the identification of different CD4+ TEM cell subsets in peripheral blood of GPA patients based on chemokine receptor co-expression analysis. The aberrant balance between TEM1 and TEM17 cells in remission GPA patients, showed to be associated with disease pathogenesis in relation to organ involvement, and tendency to relapse.

The relationship of pharmacodynamics (PD) and pharmacokinetics (PK) to clinical outcomes in a phase I study of OX40 agonistic monoclonal antibody (mAb) PF-04518600 (PF-8600).

3027 Background: PF-8600 is a novel fully human IgG2 agonistic mAb against human OX40, a TNF receptor superfamily member expressed primarily on activated T cells. This ongoing phase 1 study (NCT02315066) is investigating the safety, efficacy, PK and PD of PF-8600 in patients (pts) with solid tumors. Methods: PF-8600 (0.01–10 mg/kg) was given IV every 14d. Expression of free/total OX40 receptor, proliferation marker ki67 and activation markers HLA-DR/CD38 were measured in T cell subsets in peripheral blood by flow cytometry in all pts. CD4, CD8, OX40 and FOXP3 were evaluated in paired tumor biopsies (bx), collected from a subset of pts (≥0.1 mg/kg) at baseline (BL) and Wk6, by immunohistochemistry. Results: As of 21Sep2016, 48 pts with melanoma (n = 14), hepatocellular carcinoma (HCC, n = 19), head and neck squamous cell (n = 6) or renal cell carcinoma (n = 9) enrolled in the dose-escalation cohorts (0.01-3 mg/kg). No immune related adverse events (AE) were reported. The most frequent treatment related AEs in > 3 pts were fatigue (27.1%) and nausea and vomiting (8.3% each); all Gr 1-2. 2 pts had a partial response: melanoma at 0.1 (Pt1) and HCC at 0.3 (Pt2) mg/kg. 25 pts had best ORR (BOR) of stable disease (SD; 3 pts ≥24 wks). A majority of pts at 0.1, 0.3, and 3 mg/kg, including Pt1 and Pt2, had increases in ki67 and HLA-DR/CD38 expression in peripheral CD4+ central memory T cells. Pt1, Pt2 and all pts at ≥0.3 mg/kg had full receptor occupancy. Paired bx were only available from pts with BOR of SD or progressive disease. In 10 pts with available paired tumor bx and defined date of radiographic progression (rPD), longer time to rPD correlated with increases in %OX40+ in bx from BL to Wk6, regardless of dose level, tumor type or prior immunotherapy (R2= 0.52, p = 0.0188); no correlation between rPD and CD4+, CD8+ or FOXP3+ expression changes was observed. Updated efficacy, safety, PK and PD data will be presented. Conclusions: PF-8600 is well tolerated with evidence of single agent efficacy. Initial observations of PD markers that change with treatment and correlate with rPD support efforts to confirm these findings as more clinical outcomes and larger sample sizes become available. Clinical trial information: NCT02315066.

Sensitive detection of rare disease-associated cell subsets via representation learning

Rare cell populations play a pivotal role in the initiation and progression of diseases such as cancer. However, the identification of such subpopulations remains a difficult task. This work describes CellCnn, a representation learning approach to detect rare cell subsets associated with disease using high-dimensional single-cell measurements. Using CellCnn, we identify paracrine signalling-, AIDS onset- and rare CMV infection-associated cell subsets in peripheral blood, and extremely rare leukaemic blast populations in minimal residual disease-like situations with frequencies as low as 0.01%.

The study of CD14+HLA-DR-/low myeloid-drived suppressor cell (MDSC) in peripheral blood of peripheral T-cell lymphoma patients and its biological function.

Peripheral T-cell lymphoma is the generic term of a group of heterogeneous disease in non-Hodgkin's lymphoma. To investigate the quantity of CD14+HLA-DR-/low MDSC and T cell subsets in peripheral blood of peripheral T cell lymphoma (PTCL) patients, and explore the biological functions of CD14+HLA-DR-/low MDSC in peripheral T-cell lymphoma. Flow cytometry was used to determine CD14+HLA-DR-/low MDSC and T cell subsets in peripheral blood of 33 peripheral T-cell lymphoma patients and 23 healthy cases; CD3 +T cells, CD14+HLA-DR-/low MDSC cells and HLA-DR+ cells were selected with magnetic activated cell sorting; Biotin-labeled micro magnetic beads (CD2 / CD3 / CD28) were used to stimulate amplification of CD3 + T cells, which were co-cultured and analyzed by flow cytometry for the effect of different concentrations of CD14+HLA-DR-/low MDSC on the proliferative activity of autologous CD3+ T cells. 1. CD14+HLA-DR-/low cells / CD14+ monocytes and CD4+ CD25+regulatory T cells Treg /CD4+Tlymphocytes were 3.28% (0.40% -17.74%) and 8.81% (0.76% -78.11 %) respectively, higher than the healthy control group with statistically significant difference (P<0.05);. 2. CD4+CD25+ Treg /CD4+Tlymphocyte and CD14+HLA-DR-/CD14+ monocytes in nucleated cells of lymphoma patients were 8.81% (0.76%-78.11%) and 3.28% (0.40% -17.74%) respectively, which was in positive correlation (correlation coefficient r = 0.510), with statistically significant difference (P = 0.002); 3. When the ratio of CD14+HLA-DR-/low and T cells was 1: 1, the proliferation inhibition of MDSC on autologous T cell is the strongest; after adding nor-NOHA, an ARG1 inhibitor, proliferation of T cells was recovered to 75.09 ± 5.18 %, IDO inhibitor 1-MT made the proliferation rate of T cells recovered to 84.05 ± 2.93%, and the proliferation rate of T cells was recovered to 97.80 ± 2.20% after combination of the IDO inhibitor and ARG1 inhibitor. CD14+HLA-DR-/lowMDSC inhibit the proliferation of T lymphocytes, which may be related ARG1 and IDO.

Caspase1 Inhibitor Ac-YVAD-CMK Prevents and Treats the Acute Graft Versus Host Disease in Mice

To explore the effect of Caspase 1 inhibitor Ac-YVAD-CMK on acute graft-versus-host disease (aGVHD) after allogeneic hematopoietic stem cell transplantation(allo-HSCT) and its mechanism. Experiments were divided randomly into 3 groups: allogeneic hematopoietic stem cell transplantation combined with splenic cell infusion group (TS group, n=12), allogeneic hematopoietic stem cell transplantation combined with splenic cell infusion plus injection of low dose Caspase 1 inhibitor group (TS+low dose of C group, n=16) and plus high dose Caspase1 inhibitor (TS+high dose of C group, n=19). The body weight of mice in each group was dynamically detected, and the clinical manifestation of GVHD and score of aGVHD were determined, and the chimerism rate of mice was detected after transplantation for 14 days. Th1, Th2 and Th17 cells in peripheral blood were examined by flow cytometry. Peripheral proinflammatory cytokines IL-1β, IFN-γ, IL-1α and IL-18 were examined by enzyme-linked immunosorbent assay(ELISA). The tissues sections of GVHD target organs (liver, lung, colon and skin) were stained with HE for histopathologic examination and histopathologic score. Ac-YVAD-CMK could alleviate murine aGVHD and pathological injury, decreare the incidence and severity of aGVHD in recipient mice. The detection of Th cell subsets in peripheral blood by flow cytometry showed that compared with TS group, the Th1 cell ratio in TS+low dose of C and TS+high dose of C groups was significantly reduced (P<0.05), while the Th2 and Th17 cell ratio was significantly enhanced (P<0.05) in TS+low dose of high dose of C groups. The detection of peripheral inflamematory cytokines by ELISA demonstrated that the inflammatory cytokines including IL-1β,IFN-γ,IL-18 and IL-1α were reduced significantly (P<0.05). Ac-YVAD-CMK can improve aGVHD by inhibiting Caspase 1 and reducing the release of some inflammatory cytokines, thereby alleviated the aGVHD pathological damage.

P2.01-044 Baseline Peripheral Blood Cell Subsets Associated with Survival Outcomes in Advanced NSCLC Treated with Nivolumab in Second-Line Setting

Nivolumab is the first checkpoint inhibitor approved for the treatment of Squamous (Sq) and non-Squamous (non-Sq) metastatic NSCLC in second-line setting, showing a survival benefit in randomized phase III trials. The aim of this study is to investigate the potential role of baseline peripheral blood cells in relation to clinical outcomes following nivolumab treatment. From November 2015 to to June 2016, we evaluated 45 patients with Sq (n = 10) and non-Sq (n = 35) NSCLC, previously treated with first-line platinum-based chemotherapy, that received nivolumab 3 mg/kg IV on day 1 of each 2 week treatment cycle. Clinical characteristics (T-Stage, lymph nodes involvement, M-Stage) were assessed. Total numbers of leukocytes, myeloid-derived suppressor cells (MDSCs), including both monocytic (Mo-MDSC) and granulocytic (Gr-MDSC) type, regulatory T cells (T-regs), and serum lactate dehydrogenase (LDH) were assessed. Endpoints were correlation with objective response (OR), progression-free survival (PFS, categorized as ≤ 3 or > 3 months) and overall survival (OS). Tumor response was assessed using RECIST criteria, version 1.1, at week 9 and every 6 weeks thereafter until disease progression. Statistical methods were based on univariate analyses (Wilcoxon rank test). The median PFS of the overall study population was 3 months. Data about Gr-MDSCs (identified by flow cytometry as Lin-CD15+CD14-CD11b+HLA-DRlow/-), Mo-MDSCs (Lin-CD14-CD11b+HLA-DRlow/-) and absolute eosinophil counts (AEC) were available in 37/45 patients (82%) of treated patients. Baseline absolute numbers of Gr-MDSCs, Mo-MDSCs and AEC were greater in patients with good prognosis (PFS > 3 months) and better outcomes. In particular among patients with shorter PFS, the median numbers of Gr-MDSCs, Mo-MDSCs and AEC were significantly lower than those detected in patients with longer PFS (4 vs 13 cell/μl, p=0.01; 4 vs 21 cell/μl, p=0.06; 55 vs 155 cell/μl; p=0.02, respectively). No correlation was observed between T-regs, LDH, absolute neutrophil, monocyte, lymphocyte counts and clinical outcomes. A baseline blood signature characterized by low levels of Gr-MDSCs, Mo-MDSCs and AEC is associated with poor outcome (median PFS ≤ of 3 months) in 67.6% of patients treated with nivolumab. In contrast, patients (32.4%) with high levels of these three biomarkers showed a median PFS significant longer than 3 months. Overall survival analysis is ongoing.

Defining Fundamental B Cell-Subset Dysfunction in Myeloma

One of the most prominent features of multiple myeloma (MM) has been immune deficiency which predisposes patients to infectious complications and suppresses development of anti-MM immune responses. We and others have previously described the T cell dysfunction in Th1, Treg and Th17 cells, plasmacytoid dendritic cells and myeloid-derived suppressor cells (MDSC). However, the most fundamental and long identified deficiency is in the humoral immune response. Suppression of uninvolved immunoglobulins (UIgs) have been well described (i.e. suppression of serum IgA and IgM in IgG myeloma); and antibody responses to vaccination have been inadequate. However, very limited information is available regarding B cell function and how UIgs are suppressed in myeloma. We have now evaluated six different B cell subsets (B1a, B1b, B2, Breg, IRA-B, and MZ) in peripheral blood (PBMC) and bone marrow (BM) to understand alterations in B cell immune function in MM. We have observed significantly lower ratio of B2 (normal B cell-subset) and B1a (natural antibody-producing cells) subsets (10±4 vs 57±17; p < 0.05) and B2 and Breg (regulatory B cell-subset) subsets (14±4 vs 45±13; p< 0.05) in PBMC from MM patients (N=19) compared with healthy donor (N=33) respectively. Similar results were observed in BM samples from MM patients (N=18) compared with healthy donors (N=12); B2/B1a subset (2.4±0.6 vs 8±1.3; p < 0.05) and B2/Breg subset (8±1.4 vs 43.7±8.4; p< 0.05) respectively. To understand whether MM cells directly or indirectly alter B cell-subsets, we incubated myeloma cells (N=4) with healthy donor PBMCs, and analyzed B cell subsets after 3 days. We observed significant elevation in B1 subset (2.5 fold of control) and reduced B2 subset (89±3% of control). When we incubated PBMCs with IL-17A over-expressing MM cells (N=3), we observed further significant reduction in B2 subset (74% of control). When normal PBMCs are cultured in IL-17A (N=4) we observed significantly increased IL-10-producing Breg subset (28% of control). Similarly, co-culture of healthy B cells with MDSC led to significant increase (3.8 times) in Breg cell- population (N=3) compared with control group. To study the impact of B cell dysfunction on T cell function in MM, we activated normal PBMC via anti-CD3 antibody, in the presence or absence of B cells, and measured intra-cellular IFN-γ levels in CD69+ cells. We observed that the absence of B cells significantly inhibited interferon-producing T cells compared to control (by 43%; p<0.05). Importantly, following removal of CD25+ cells (Tregs and activated memory T cells), with or without B cells, we did not observe any difference in the inhibition of IFN-γ, indicating that B cells influence memory T cells rather than naïve T cells for the production of IFN-γ. To evaluate impact of lenalidomide on this interaction, we stimulated purified normal donor CD45RO memory T cells with Th1 polarizing cocktail in the presence or absence of purified normal B cells or B cells from MM patient (MM-B) in presence of lenalidomide and observed thatlenalidomide significantly improved MM-B cell-mediated IFN-γ-producing Th1 responses (by 32%, p<0.05) compared to normal B cell-mediated Th1 responses. In an effort to evaluate whether any therapy may improve the B cell function, we cultured normal PBMCs in the presence of lenalidomide (N=9) and observed reduction in Breg subset by 40% of control. To evaluate the effect of therapy on B cell-subsets in MM, we analyzed B cell subsets in PBMC from newly-diagnosed and lenalidomide-treated MM patients and observed that lenalidomide-treated group showed significant (p<0.05) improvement in B cell subsets (increased B2 and lower B1 cells) even before clinical response. These results suggest that immunomodulatory agents may be able to re-program humoral immunity in these patients. In summary, we report that the myeloma cell driven skewed B cell subset distribution with consequent B cell dysfunction drives the observed abnormalities in humoral/cell mediated immunity. The current therapeutic interventions, besides providing deep clinical responses, may also improve B cell function with impact on long term outcome. DisclosuresNo relevant conflicts of interest to declare.

One-Time Administration of Recombinant Human Erythropoietin Decreases B Cell in Human Peripheral Blood

Introduction:Erythropoietin (EPO) is hematopoietic factors participating in red blood cell production by accelerating cell proliferation and inhibition of apoptosis of erythroblast. It is reported that EPO has pleiotropic effects including anti-apoptotic action for some cells, antioxidant action, vascularization action, and promoting cell division in addition to stimulation of erythropoiesis as well, whereas there are conflicting results of small cohorts as to its effect on blood immune cells. We analyzed peripheral white blood cell subsets in patients who received one bolus administration of recombinant human erythropoietin (rHuEPO) to examine the effect of EPO on human immune system.Materials and Methods:One hundred twenty eight autologous blood donors (male/female 64/64) in Gunma University Hospital were enrolled in this study after written informed consent. All the patients had no infections or inflammation. Fifty six patients were administered rHuEPO (Epoetin alpha or Epoetin beta, 24,000 IU) once after blood donation because of low hemoglobin concentration (EPO group) and 72 were not administered (non-EPO group). Peripheral blood samples were obtained at the time of the first phlebotomy and after 7 days from the same patient.We measured the number of WBC, lymphocytes, myeloid dendritic cells (mDC), plasmacytoid dendritic cells (pDC), CD4+ T cells, CD8+ T cells, Natural killer (NK) cells, B cells, monocytes, and neutrophils of peripheral blood before and after rHuEPO administration by flow cytometry. Also we compared EPO group with non-EPO group. Paired and unpaired Student’s t-test were used to compare absolute counts and percentages of each cell, P values < 0.05 were considered significant. This study was approved by the research ethics committee of our hospital.Results:In EPO group, absolute number and percentage of lymphocyte in WBC decreased significantly after rHuEPO administration from 1754.9 ± 535.4/µl to 1627.2 ± 467.7/µl, (p<0.01) and from 30.9 ± 8.00% to 28.2 ± 6.77% (p<0.01), respectively. The numbers of whole WBC, mDC, pDC, monocyte and neutrophil did not show significant changes. In regards to lymphocyte subset, absolute number of CD8+ T cell significantly decreased from 358.9 ± 257.0/µl to 311.5 ± 210.9/µl (p=0.04) and that of NK cell from 290.6 ± 157.6/µl to 257.4 ± 141.8/µl (p<0.01), but their percentages did not change. B cells significantly decreased in absolute number from 258.3 ± 154.2 /µl to 221.6 ± 129.2 /µl (p<0.01) and also in percentages from 14.8 ± 6.92% to 13.6 ± 6.07% (p<0.01). Regarding B cell subsets, absolute number of Naïve B cells significantly decreased from 171.3 ± 93.5 /µl to 153.0 ± 84.2 /µl (p<0.01); moreover other B cell subsets, such as transitional B cells, memory B cells and marginal zone B cells, also showed a trend of decrease, so that the percentage of Naïve B cell did not change in total B cells. These suggested that whole B cell decreased, not a specific subset of B cell. In non-EPO group, there was no significant change in any type of immune cells. When compared the rate of change between before and after rHuEPO treatment, B cell number significant decreased in EPO group compared to non-EPO group (0.87 vs 0.98, p<0.01). Multiple regression analysis revealed rHuEPO administration significantly influenced B cell number (p=0.015); however age, sex, disease status and the type of rHuEPO preparations (epoetin alfa or beta) did not influence.Conclusion:These findings suggested that just one administration of rHuEPO influenced human immune system, especially via reduction of B cell in peripheral blood. DisclosuresNo relevant conflicts of interest to declare.

Effect of PPARγ agonist (rosiglitazone) on the secretion of Th2 cytokine in asthma mice.

To explore the effect of PPARγ agonist (rosiglitazone) on the secretion of Th2 cytokines and the proportion of immune cell subsets in asthma mice. Ovalbumin (OVA)-sensitized mice were used to build asthma models. Those mice were divided into the normal control group, model group and rosiglitazone group. Differences of the changes in lung histopathology of mice in the three groups were observed through hematoxylin and eosin (HE) strain, and the numbers of the total cells, eosinophils and neutrophils in BALF of mice in the three groups were compared. ELISA and real-time PCR were employed to detect the protein levels of interleukin (IL)-5, IL-13, IL-4 and IL-10 and mRNA level, respectively. Flow cytometry number was implied to analyze the proportion of immune cell subsets in peripheral blood of mice. Compared with the mice in the control group, and mice of the model group, the infiltration of inflammatory cells in BALF increased, bronchial smooth muscle became thickened, a large amount of collagen deposited, the secretion of Th2 cytokine increased significantly, the ratio of regulatory T cells (Treg) decreased, the ratio of T17 cells rose distinctly; while in mice of the rosiglitazone group, the changes of their lung histopathology were improved obviously, the number of infiltration of inflammatory cells declined, the thickened smooth muscle relieved, the deposition of collagen decreased, the secretion of Th2 cytokine was inhibited, the ratio of Treg went up, and the increased of the ratio of T17 cells was inhibited but still not return to normal level. Rosiglitazone can regulate the proportion of Treg and Th17 cells and inhibit the secretion of Th2 cytokines, which inhibit the airway inflammatory response for asthma mice effectively.

Status of stem cells in diabetic nephropathy: predictive and preventive potentials.

BackgroundRecruitment of stem cells to sites of tissue injury constitutes an important mechanism aimed at tissue repair and regeneration. However, it is not clear how the diabetic milieu affects the viability of endogenous stem cells. Thus, we tested the hypothesis that diabetes mellitus is associated with increased apoptosis which, in turn, contributes to reduction in stem cells and the manifestation of type 2 diabetic nephropathy.MethodsSixteen-week-old male obese type 2 diabetic db/db mice, and their appropriate controls, were used for assessment of the status of endothelial progenitor cells (EPCs), mesenchymal stem cells (MSCs), and hematopoetic stem cells (HSCs) in the peripheral blood and renal tissue using specific cell markers. Further, we explored whether diabetic animals display greater apoptosis of stem cell subsets.ResultsThe peripheral blood cells of db/db mice displayed reduction in EPCs (p < 0.05) compared to those of db/m controls. Further, kidney cells prepared from experimental groups also showed reductions in EPCs, MSCs, and HSCs. We also observed increased apoptosis of stem cell subsets in cells prepared from kidneys of db/db than those of db/m mice.ConclusionsThe present study shows a similar pattern of decline in stem cell subsets in peripheral blood and kidneys of db/db mice, an effect likely related to increased apoptosis. Collectively, the results suggest that apoptosis of stem cells likely contributes to eventual manifestation of renal failure in diabetes mellitus. Monitoring of blood levels of stem cell subsets could predict failure of their reparative and protective effects and eventual manifestations of diabetic complications.

330 Characterization of multiple B cell subsets in peripheral blood of psoriasis patients identifies a correlation of regulatory B cells and disease severity

328 Assessment of psoriasis using label-free ultra-broadband optoacoustic mesoscopy J Aguirre, M Schwarz, N Garzorz-Stark, F Lauffer, D Soliman, A Buehler, K Eyerich and V Ntziachristos 1 Institute for Biological and Medical Imaging, Technical University of Munich and Helmholtz Center Munich, Munich, Germany and 2 Department of Dermatology and Allergy, Technical University of Munich, Munich, Germany Psoriasis is one of the most prevalent widespread chronic inflammatory skin disease but modern imaging methods provide only limited view on the disease specific hallmarks. In clinical practice, standard tools to assess disease severity and drug efficacy are based on visual scores that are subjective and inconsistent among different examiners. Here, we introduce paradigm shifting ultra-broadband optoacoustic mesoscopy (OPAM) for visualizing psoriatic skin beyond the reach of current dermatological imaging. Implemented in handheld mode, we show visualization of optical absorption contrast resolving for the first time vascular morphology in the dermis and quantifying inflammatory landmarks such as blood volume, epidermal thickness and characterization of capillary architecture in label free mode. Investigating the mesoscopic appearance of psoriasis in six different patients, we found that OPAM cross sectional images and 3D reconstructions examined for morphological skin alterations, capillary loop elongation, acanthosis and changes in dermal vasculature correlated with the values obtained from histopathological skin samples of the respective patients. Besides, we established a multidimensional computational analysis combining the OPAM parameters of increased dermal blood volume, capillary loop density, acanthosis and vessel structure complexity which significantly correlated with Psoriasis Area Severity Index. Thus, we confirm that obtained imaging data reflected the main clinical signs of psoriatic inflammation namely erythema, scaling and induration of plaques. The method has enormous implications in non-invasive accurate staging, treatment evaluation and assessing a larger spectrum of diseases towards precision medicine. 329 The efficacy of 1% hydrogen peroxide cream for acne vulgaris treatment K Dzilinska, K Kisiel, M Pasikowska, E Jakimiuk, R Debowska and I Eris 1 Dermatology Centre, Miedzyleski Hospital, Warsaw, Poland and 2 Dr Irena Eris Cosmetic Laboratories, Piaseczno, Poland Acne vulgaris most commonly occurs during adolescence, affecting an estimated 80e90% of teenagers. However we observe increasing number of acne vulgaris in adults as well. The purpose of this study was to evaluate the safety and anti-inflammatory, anti-bacterial and soothing effects of topical use of 1% hydrogen peroxide cream. Hydrogen peroxide is a chemical compound that finds use as a strong oxidizer, bleaching agent and disinfectant. Cosmetic industry offers a wide range of products for this condition, however hydrogen peroxide has not been very popular so far. The study was conducted on 19 volunteers with acne vulgaris between ages 16-61 years old. The product was used daily for a week. Skin condition was analyzed using VISIA system as well as by dermatologists according to the Hellgren and Vincent scale and via a questionnaire survey completed by volunteers after one week of product usage. Skin assessment revealed the reduction in the amount of pustules by 63% and reduction of the severity of seborrhea by 32%. Instrumental skin analysis revealed significant decrease of porphyrins, pores and spots. Moreover skin texture was improved by 22%. Results of the presented study demonstrated that systematic application of 1% hydrogen peroxide cream significantly reduces skin inflammation, soothes and accelerates healing process as well as demonstrates great product tolerance. 1% hydrogen peroxide can supplement pharmacological treatment of acne vulgaris with mild to moderate course of disease.

Targeted genomic analysis reveals widespread autoimmune disease association with regulatory variants in the TNF superfamily cytokine signalling network.

BackgroundTumour necrosis factor (TNF) superfamily cytokines and their receptors regulate diverse immune system functions through a common set of signalling pathways. Genetic variants in and expression of individual TNF superfamily cytokines, receptors and signalling proteins have been associated with autoimmune and inflammatory diseases, but their interconnected biology has been largely unexplored.MethodsWe took a hypothesis-driven approach using available genome-wide datasets to identify genetic variants regulating gene expression in the TNF superfamily cytokine signalling network and the association of these variants with autoimmune and autoinflammatory disease. Using paired gene expression and genetic data, we identified genetic variants associated with gene expression, expression quantitative trait loci (eQTLs), in four peripheral blood cell subsets. We then examined whether eQTLs were dependent on gene expression level or the presence of active enhancer chromatin marks. Using these eQTLs as genetic markers of the TNF superfamily signalling network, we performed targeted gene set association analysis in eight autoimmune and autoinflammatory disease genome-wide association studies.ResultsComparison of TNF superfamily network gene expression and regulatory variants across four leucocyte subsets revealed patterns that differed between cell types. eQTLs for genes in this network were not dependent on absolute gene expression levels and were not enriched for chromatin marks of active enhancers. By examining autoimmune disease risk variants among our eQTLs, we found that risk alleles can be associated with either increased or decreased expression of co-stimulatory TNF superfamily cytokines, receptors or downstream signalling molecules. Gene set disease association analysis revealed that eQTLs for genes in the TNF superfamily pathway were associated with six of the eight autoimmune and autoinflammatory diseases examined, demonstrating associations beyond single genome-wide significant hits.ConclusionsThis systematic analysis of the influence of regulatory genetic variants in the TNF superfamily network reveals widespread and diverse roles for these cytokines in susceptibility to a number of immune-mediated diseases.Electronic supplementary materialThe online version of this article (doi:10.1186/s13073-016-0329-5) contains supplementary material, which is available to authorized users.

Abstract 3242: In depth myeloid cell characterization in the murine syngeneic CT26 colon carcinoma model by 10 color flow cytometry

Abstract The efficacy of immune-modulating anti-cancer therapeutic antibodies that have been FDA-approved in recent years, such as anti-CTLA-4 and anti-PD-1, has driven growing interest in methods that provide a mechanistic understanding of drug function. Development of new mono- and combination therapies with immune-modulatory effects requires more powerful immunophenotyping techniques capable of in depth cell characterization. To this end, using the CT26 murine syngeneic colorectal cancer model we have developed a 10 color flow cytometry antibody panel that focuses on the identification of tumor-infiltrating immune cell subsets derived from myeloid lineage precursors utilizing the high-throughput-capable 4-laser, 14-color Attune NxT Flow Cytometer with autosampler. The panel includes a combination of antibodies against CD45, CD3, CD19, CD49b, CD335, CD11b, CD11c, Ly-6G, Ly-6C, F4/80, and CD115. By excluding cells of lymphoid lineage, we show that this panel facilitates analysis of myeloid derived cells including natural killer (NK) cells, macrophages, neutrophils, dendritic cells (DCs), and monocytic or granulocytic myeloid-derived suppressor cell (mMDSCs and gMDSCs) subsets in tumor and peripheral blood. In addition, this combination of antibodies allows for a more complete analysis of MDSCs which can differentially express several disease-relevant myeloid specific markers including Ly-6G, Ly-6C, F4/80, CD11c, and CD115. In the tumor, the Ly-6G-high population demonstrates differential expression of Ly-6C, 21% Ly-6C-high (granulocytes) and 77% Ly-6C-low (mMDSCs). The majority of the granulocytic population was identified as gMDSCs and the remainder as neutrophils based on CD115 expression. Macrophages constitute 27% of Ly-6G-low cells. In blood, 98% of the Ly-6G-high population was also Ly-6C-high, and this population is predominantly neutrophils. No macrophages (Ly6G-low and F4/80+) were identified in the peripheral blood. These data confirm the expected distribution of myeloid lineages in the tissue types investigated. Finally, we show that the accuracy of analysis is enhanced by the use of fluorescence minus-one (FMO) controls to identify those markers that generate dim signals, as well as a viability dye used to exclude dead cells from analysis. Identification of additional potentially responsive immune compartments will facilitate identification and development of potential combination therapies otherwise overlooked by looking primarily at T-cells. This panel allows for a significant expansion of our ability to provide a complex description of the myeloid subset. Citation Format: Matt Thayer, David Draper, Daniel Saims, Maryland Rosenfeld-Franklin, Scott C. Wise. In depth myeloid cell characterization in the murine syngeneic CT26 colon carcinoma model by 10 color flow cytometry. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3242.

CD21–/low B cells in human blood are memory cells

The complement receptor 2 (CR2, CD21) is part of a complex (CD21/CD19/CD81) acting as a co-receptor to the B cell receptor (BCR). Simultaneous triggering of the BCR and CD21 lowers the threshold for B cell activation. Although CD21 is important, B cells that express low amounts or lack surface CD21 (CD21(-/low) ) are increased in conditions with chronic inflammation, e.g. autoimmune diseases. However, little is known about the CD21(-/low) B cell subset in peripheral blood from healthy donors. Here, we show that CD21(-/low) cells represent approximately 5% of B cells in peripheral blood from adults but are barely detectable in cord blood, after excluding transitional B cells. The CD21(-/low) subset can be divided into CD38(-) 24(+) and CD38(-) 24(low) cells, where most of the CD38(-) 24(+) are CD27(+) immunoglobulin (Ig)M(+) IgD(+) and the CD38(-) 24(low) are switched CD27(-) . Expression levels of additional markers, e.g. CD95 and CD62L, are similar to those on classical memory B cells. In contrast to naive cells, the majority of CD21(-/low) cells lack expression of the ABCB1 transporter. Stimulation with a combination of BCR, Toll-like receptor (TLR)-7/8 andinterleukin (IL)-2 induces proliferation and differentiation of the CD21(-/low) B cells comparable to CD21(+) CD27(+) memory B cells. The response excluding BCR agonist is not on par with that of classical memory B cells, although clearly above that of naive B cells. This is ascribed to a weaker response by the CD38(-) 24(low) subset, implying that some memory Bcells require not only TLR but also BCR triggering. We conclude that the CD21(-/low) cells in healthy donors are memory B cells.

Standardizing Flow Cytometry Immunophenotyping Analysis from the Human ImmunoPhenotyping Consortium.

Standardization of immunophenotyping requires careful attention to reagents, sample handling, instrument setup, and data analysis, and is essential for successful cross-study and cross-center comparison of data. Experts developed five standardized, eight-color panels for identification of major immune cell subsets in peripheral blood. These were produced as pre-configured, lyophilized, reagents in 96-well plates. We present the results of a coordinated analysis of samples across nine laboratories using these panels with standardized operating procedures (SOPs). Manual gating was performed by each site and by a central site. Automated gating algorithms were developed and tested by the FlowCAP consortium. Centralized manual gating can reduce cross-center variability, and we sought to determine whether automated methods could streamline and standardize the analysis. Within-site variability was low in all experiments, but cross-site variability was lower when central analysis was performed in comparison with site-specific analysis. It was also lower for clearly defined cell subsets than those based on dim markers and for rare populations. Automated gating was able to match the performance of central manual analysis for all tested panels, exhibiting little to no bias and comparable variability. Standardized staining, data collection, and automated gating can increase power, reduce variability, and streamline analysis for immunophenotyping.

Reduced numbers of mucosal DRint macrophages and increased numbers of CD103+ dendritic cells during anti-TNF-α treatment in patients with Crohn’s disease

Objective Anti-TNF-α treatment constitutes a mainstay in the treatment of Crohn’s disease (CD), but its mechanisms of action are not fully understood. We aimed to investigate the effects of adalimumab, a human monoclonal TNF-α antibody, on macrophage (MQ) and dendritic cell (DC) subsets in mucosal biopsies and peripheral blood. Material and methods Intestinal biopsies and blood samples were obtained from 12 different CD patients both before and 4 weeks after the initiation of the induction of adalimumab treatment. Endoscopic disease activity was estimated by the Simple Endoscopic Score for Crohn’s Disease. Biopsies were obtained from inflamed and non-inflamed areas. The numbers of lamina propria CD14 + DRint and CD14 + DRhi MQs, CD141+, CD141− and CD103+ DCs subsets, and circulating monocytes and DCs were analyzed using flow cytometry. Results At baseline, we observed higher numbers of DRint MQs and lower numbers of CD103+ DCs in inflamed versus non-inflamed mucosa [843 vs. 391/105 lamina propria mononuclear cells (LPMCs) (p < 0.05) and 9 vs. 19 × 105 LPMCs (p = 0.01), respectively]. After four weeks of adalimumab treatment, the numbers of DRint MQs decreased [843 to 379/105 LPMCs (p = 0.03)], whereas the numbers of CD103+ DCs increased [9–20 × 105 LPMCs (p = 0.003)] compared with baseline. In peripheral blood, no alterations were observed in monocyte or DC numbers between baseline and week 4. Conclusions In CD, mucosal inflammation is associated with high numbers of DRint MQs and low numbers of CD103+ DCs. This composition of intestinal myeloid subsets is reversed by anti-TNF-α treatment. These results suggest that DRint MQs play a pivotal role in CD inflammation.

Influence of lactulose on interventional therapy for HCC patients with hepatocirrhosis and hypersplenism

To investigate the influence of lactulose on immunity of hepatocellular carcinoma (HCC) patients with hepatocirrhosis and hypersplenism after double-interventional therapies. A total of 40 HCC patients with hepatocirrhosis and hypersplenism, hospitalized during January 2013 to June 2014, were enrolled and randomized into control group and observation group. Both groups received partial splenic embolization combined with transcatheter arterial chemoembolization. Besides, observation group orally took lactulose 30mL/d. Four days before interventional therapies and at days 1, 3, 7 and 14 after therapies, fasting venous blood was collected to detect white blood cell count, red blood cell count (RBC), and platelet count (PLT). Four days before therapies and at days 7 and 14 after therapies, the levels of alanine aminotransferase, aspartate transaminase, total bilirubin, malondialdehyde, super-oxide dismutase (SOD), IFN-γ, and IL-4 as well as the distribution of T cell subsets in peripheral blood were tested. Complications were observed after interventional therapies. Before interventional therapies the levels of white blood cell count, PLT and RBC in both groups showed no difference, while after interventional therapies the levels of PLT and RBC in both groups showed an increasing tendency (P<0.05). At day 14 after interventional therapies, the level of blood cell as well as that of SOD, IFN-γ and IL-4 in serum were significantly higher than that before therapies; meanwhile, the levels of alanine aminotransferase and total bilirubin of observation group after therapies were significantly lower than before and control group (P<0.05), the levels of CD4(+)/CD8(+), SOD and IFN-γ were all higher than before and control group (P<0.05). Oral administration of lactulose could adjust the imbalance of oxidation system/antioxidant system in HCC patients with hepatocirrhosis and hypersplenism after interventional therapies, and improve the antitumor immunity and prognosis.

Abstract A195: Tremelimimab activates CD4 and CD8+ T cells in patients with hepatocellular carcinoma

Abstract Introduction: Tremelimumab is a fully human monoclonal antibody that binds to CTLA-4 expressed on the surface of activated T lymphocytes and results in inhibition of B7-CTLA-4-mediated downregulation of T-cell activation. Patients with advanced hepatocellular carcinoma, who progressed on standard of care, were treated with tremelimumab and tumor ablation (radiofrequency ablation and transcatheter arterial chemoembolization) to augment anti-tumor immunity. Treatment was safe and feasible. Encouraging clinical activity has been seen with objective confirmed partial responses in 4/12 (33%) evaluable patients and a time to progression of 7.4 months. Here we report first results from immunoanalysis evaluating tumor biopsies, immune cell subsets in peripheral blood and serum viral loads in patients with chronic HBV or HCV infection. Methods: 20 Patients with HCC were treated with tremelimumab and tumor ablation on clinical trial NCT01853618. Tumor biopsies were taken at the time of ablation and peripheral blood mononuclear cells (PBMC) were collected before and during treatment in a subset of patients. Serum samples were tested for viral load (quantitative HCV RNA). Serum HBsAg titers were measured by chemiluminescent microparticle immunoassay (CMIA) using the ARCHITECT platform (Abbott Laboratories, Chicago, IL), as per the manufacturer's instructions. Tumor biopsies with analyzed by immunohistochemistry for CD3, CD4, CD8, CD20 and Granzyme B. Eleven-color flow cytometry was performed to study PBMC using the following antibodies: CD4, CD3, CD4, CD8, CD11c, CD14, CD19, CD20, CD25, CD38, CD45RA, CD56, CD123, CD127, CCR7, CCR4, CXCR3, PD-1, 4-1BB, TIM3, CTLA4, PD-L1, ICOS, HLA-DR. Statistical analysis was done using the Wilcoxon signed rank test. Results presented are preliminary and update data will be presented at meeting. Results: Immunohistochemical analysis of tumor biopsies demonstrated an increase in the CD3+CD8+ T cell population in tumors. CD8+ T cells stained positive for Granzyme A. No changes in the number of CD68+ macrophages were seen. Multi-color flow cytotometry PBMC revealed statistically significant changes after the 1st cycle of activated CD4+ and CD8+ T cells. There was a trend towards a reduction in CD4+ Tregs. Activated CD8+ T cells remained elevated for more than three months. Eight of 9 patients with quantifiable HCV experienced a marked reduction in viral load. Four of 4 HBV patients experienced a reduction in quantitative HepBsAg. Analysis of immune cell phenotype and more in depth analysis of tumor samples is ongoing and will be reported. Conclusions: Tremelimumab in combination with subtotal TACE or RFA leads to an accumulation of intratumoral CD8+ T cells, an activation of CD4+ and CD8+ T cells in peripheral blood and reductions in HCV viral load and HBsAg. Citation Format: Firouzeh Korangy, Mei ElGindi, Drew Pratt, David Venzon, Austin Duffy, Oxana Makarova-Rusher, Sid Kerkar, David Kleiner, Bradford Wood, Tim Greten. Tremelimimab activates CD4 and CD8+ T cells in patients with hepatocellular carcinoma. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr A195.

T Cell Response in Patients with Implanted Biological and Mechanical Prosthetic Heart Valves.

The study was aimed at assessing T cell subsets of peripheral blood from recipients of long-term functioning (more than 60 months) biological and mechanical heart valve prostheses. The absolute and relative number of CD4 and CD8 T cell subsets was analyzed: naïve (N, CD45RA+CD62L+), central memory (CM, CD45RA−CD62L+), effector memory (EM, CD45RA−CD62L−), and terminally differentiated CD45RA-positive effector memory (TEMRA, CD45RA+CD62L−) in 25 persons with biological and 7 with mechanical prosthesis compared with 48 apparently healthy volunteers. The relative and absolute number of central memory and naïve CD3+CD8+ in patients with biological prosthesis was decreased (p < 0.001). Meanwhile the number of CD45RA+CD62L−CD3+CD8+ and CD3+CD4+ was increased (p < 0.001). Patients with mechanical prosthesis had increased absolute and relative number of CD45RA+CD62L−CD3+CD8+ cells (p = 0.006). Also the relative number of CD3+CD4+ cells was reduced (p = 0.04). We assume that altered composition of T cell subsets points at development of xenograft rejection reaction against both mechanical and biological heart valve prostheses.