scholarly journals Phenotypic analysis of the pediatric immune response to SARS‐CoV ‐2 by flow cytometry

2021 ◽  
Author(s):  
Freya Sibbertsen ◽  
Laura Glau ◽  
Kevin Paul ◽  
Thomas S. Mir ◽  
Søren W. Gersting ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Immune Response ◽  
Phenotypic Analysis

scholarly journals OP0083 INFECTIOUS AND AUTOINFLAMMATORY MODIC TYPE 1 CHANGES HAVE DIFFERENT PATHOMECHANISMS

2021 ◽  
Vol 80 (Suppl 1) ◽  
pp. 45.2-45
Author(s):  
I. Heggli ◽  
R. Schüpbach ◽  
N. Herger ◽  
T. A. Schweizer ◽  
A. Juengel ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Immune Response ◽  
T Cell ◽  
Cell Populations ◽  
Gene Set ◽  
Go Enrichment ◽  
Modic Type ◽  
And Control ◽  
Infectious Etiology

Background:Modic type 1 changes (MC1) are vertebral bone marrow (BM) edema that associate with non-specific low back pain (LBP). Two etiologies have been described. In the infectious etiology the anaerobic aerotolerant Cutibacterium acnes (C. acnes) invades damaged intervertebral discs (IVDs) resulting in disc infection and endplate damage, which leads to the evocation of an immune response. In the autoinflammatory etiology disc and endplate damage lead to the exposure of immune privileged disc cells and matrix to leukocytes, thereby evoking an immune response in the BM. Different etiologies require different treatment strategies. However, it is unknown if etiology-specific pathological mechanisms exist.Objectives:The aim of this study was to identify etiology-specific dysregulated pathways of MC1 and to perform in-depth analysis of immune cell populations of the autoinflammatory etiology.Methods:BM aspirates and biopsies were obtained from LBP patients with MC1 undergoing spinal fusion. Aspirates/biopsies were taken prior screw insertion through the pedicle screw trajectory. From each patient, a MC1 and an intra-patient control aspiration/biopsy from the adjacent vertebral level was taken. If C. acnes in IVDs adjacent to MC1 were detected by anaerobic bacterial culture, patients were assigned to the infectious, otherwise to the autoinflammatory etiology.Total RNA was isolated from aspirates and sequenced (Novaseq) (infectious n=3 + 3, autoinflammatory n=5 + 5). Genes were considered as differentially expressed (DEG) if p-value < 0.01 and log2fc > ± 0.5. Gene ontology (GO) enrichment was performed in R (GOseq), gene set enrichment analysis (GSEA) with GSEA software.Changes in cell populations of the autoinflammatory etiology were analyzed with single cell RNA sequencing (scRNAseq): Control and MC1 biopsies (n=1 + 1) were digested, CD45+CD66b- mononuclear cells isolated with fluorescence activated cell sorting (FACS), and 10000 cells were sequenced (10x Genomics). Seurat R toolkit was used for quality-control, clustering, and differential expression analysis.Transcriptomic changes (n=5 + 5) of CD45+CD66b+ neutrophils isolated with flow cytometry from aspirates were analyzed as for total bulk RNAseq. Neutrophil activation (n=3 + 3) was measured as CD66b+ expression with flow cytometry. CD66bhigh and CD66blow fractions in MC1 and control neutrophils were compared with paired t-test.Results:Comparing MC1 to control in total bulk RNAseq, 204 DEG in the autoinflammatory and 444 DEG in the infectious etiology were identified with only 67 shared genes (Fig. 1a). GO enrichment revealed “T-cell activation” (p = 2.50E-03) in the autoinflammatory and “complement activation, classical pathway” (p=1.1E-25) in the infectious etiology as top enriched upregulated biological processes (BP) (Fig 1b). ScRNAseq of autoinflammatory MC1 showed an overrepresentation of T-cells (p= 1.00E-34, OR=1.54) and myelocytes (neutrophil progenitor cells) (p=4.00E-05, OR=2.27) indicating an increased demand of these cells (Fig. 1c). Bulk RNAseq analysis of neutrophils from the autoinflammatory etiology revealed an activated, pro-inflammatory phenotype (Fig 1d), which was confirmed with more CD66bhigh neutrophils in MC1 (+11.13 ± 2.71%, p=0.02) (Fig. 1e).Figure 1.(a) Venn diagram of DEG from total bulk RNAseq (b) Top enriched upregulated BP of autoinflammatory (left) and infectious (right) etiology (c) Cell clustering of autoinflammatory MC1 BM (d) Enrichment of “inflammatory response” gene set in autoinflammatory MC1 neutrophils (e) Representative histogram of CD66b+ expression in MC1 and control neutrophils.Conclusion:Autoinflammatory and infectious etiologies of MC1 have different pathological mechanisms. T-cell and neutrophil activation seem to be important in the autoinflammatory etiology. This has clinical implication as it could be explored for diagnostic approaches to distinguish the two MC1 etiologies and supports developing targeted treatments for both etiologies.Disclosure of Interests:None declared

scholarly journals Platelet and Immune Characteristics of Patients with Immune Thrombocytopaenia Non Responders to Therapeutic Treatments

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 1089-1089
Author(s):  
Elena Monzón Manzano ◽  
Raul Justo Sanz ◽  
Diana Hernández ◽  
Teresa Álvarez Roman ◽  
Ihosvany Fernandez-Bello ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Immune Response ◽  
Sialic Acid ◽  
Research Funding ◽  
Caspase 3 ◽  
Mononuclear Cells ◽  
Ricinus Communis ◽  
The Other ◽  
Control Group ◽  
Platelet Surface

Introduction: Mechanisms leading to diminished platelet counts in immune thrombocytopaenia (ITP) appear to be multifactorial: autoantibodies, autoreactive CD8+ cytotoxic T cells, enhanced apoptosis and loss of sialic acid which mediates platelet clearance through the Ashwell-Morell receptors present in hepatocytes. Differential involvement of each of them might condition the ability of patients with ITP to respond to treatments. We aimed to examine platelet features and the immunological state of patients with ITP who do not respond to any treatment to detect the unique characteristics of this group. Methods: This was an observational, prospective and transversal study. Patients with chronic primary ITP were included: 28 ITP patients without treatment for at least 6 months (UT-ITP); 36 responders to agonists of thrombopoietin receptors (TPO-RA); and 14 ITP patients who did not respond to first- and second-line treatments (NR-ITP). A healthy control group (n=104) was also included in the study. Active caspase-3, -7, -8 or -9 were determined by flow cytometry using CaspaTag kits (Millipore, Madrid, Spain) in PRP diluted with HEPES-buffer containing 2 mM Ca2+ and 2 mM Gly-Pro-Arg-Pro (Sigma-Aldrich, Madrid, Spain) to prevent fibrin formation . Platelet surface glycan exposure was analysed by determining the binding of lectins by flow cytometry. To do so, washed platelets were incubated with 1 μg/ml Alexa fluor 488-conjugated wheat germ agglutinin lectin (WGA, Invitrogen, Spain) or with 1 μg/ml FITC-conjugated Ricinus communis agglutinin (RCA, Vector Labs, UK). WGA binds to sialic acid and N-acetylglucosaminyl residues, and RCA is a galactose-specific legume lectin which binding serves as an indirect measurement of the loss of sialic acid. Peripheral blood mononuclear cells (PBMCs) subsets were analysed by flow cytometry using specific antibodies. Experimental data was analysed using SPSS 9.0 software (SPSS Inc., Chicago, IL). Results: Platelets from TPO-RA treated and from NR-ITP patients had increased caspase-3, -7, -8 and -9 activities (Figure 1A). Platelets from NR-ITP patients exposed less sialic acid and more N-acetylglucosaminyl residues than the other groups (Figure 1B). Binding of WGA and RCA correlated with caspase activities (Table 1). Distribution of lymphocytes, monocytes and natural killer cells is shown in Table 1. NR-ITP patients had an increased proportion of B lymphocyte (LB), maybe due to a significant rise in the fraction of naive LB cells, and a diminution in LTreg subset. Whereas classical monocytes was increased, nonclassical monocyte fraction was decreased in the UT-ITP and NR-ITP groups. NR-ITP patients also presented an increased CD16+CD56bright cells fraction and a diminished NK CD16+CD56dim subset. TPO-RA-treated patients seemed to recover an immune homeostasis similar to healthy controls (monocyte and NK cells subset distribution and LTreg count similar to control group). It is of interest to note the relationship between loss of sialic acid from platelet surface glycans and Tregs count: the most reduced surface exposure of sialic acid, the less Treg count (Figure 2). Conclusions: Platelets from NR-ITP patients had more signs of apoptosis and a different composition of surface glycans, accompanied by a diminished LTreg population, a higher LB naïve percentage, and an increased CD16+CD56bright cells fraction in circulation, indicating a severe deregulation of the immune system. Since an inverse correlation was observed between loss of sialic acid and LTreg count, a potential relationship between glycan composition on the platelet surface and immune response is suggested, positing terminal sugar moieties of the glycan chains as aetiopathogenic agents in ITP. On the other hand, TPO-RA appears to have a beneficial effect on immune response. Nevertheless, one of the limitations of our study was that patients were recruited once the response to TPO-RA was achieved; therefore, a longitudinal study would provide more information regarding TPO-RA effects. This work was supported by grants from the FIS-FONDOS FEDER (PI15/01457, NB). NVB holds a Miguel Servet tenure track grant from FIS-FONDOS FEDER (CP14/00024). Disclosures Álvarez Roman: Roche: Consultancy, Speakers Bureau; Pfizer: Consultancy, Speakers Bureau; Bayer: Consultancy, Speakers Bureau; Novartis: Consultancy, Speakers Bureau; Amgen: Consultancy, Speakers Bureau; Takeda: Research Funding; NovoNordisk: Consultancy, Speakers Bureau; CSL Behring: Consultancy, Speakers Bureau; Sobi: Consultancy, Speakers Bureau. Fernandez-Bello:Novartis, Pfizer, ROCHE, Stago: Speakers Bureau. Martín:SOBI: Research Funding; Novartis, Pfizer, ROCHE, Novo Nordisk: Speakers Bureau. Rivas Pollmar:Novartis, Pfizer, ROCHE, Novo Nordisk: Speakers Bureau; SOBI: Research Funding. Canales:Novartis: Honoraria; Takeda: Speakers Bureau; iQone: Honoraria; Sandoz: Honoraria; Celgene: Honoraria; SOBI: Research Funding; Karyopharm: Honoraria; F. Hoffmann-La Roche Ltd: Honoraria, Speakers Bureau; Gilead: Honoraria; Janssen: Honoraria, Speakers Bureau. Jimenez-Yuste:Bayer, CSL Behring, Grifols, Novo Nordisk, Octapharma, Pfizer, Roche, Sobi, Shire: Consultancy, Honoraria, Other: reimbursement for attending symposia/congresses , Research Funding, Speakers Bureau. Butta:Novartis: Consultancy; Roche, Pfizer: Speakers Bureau.

scholarly journals 614 Investigating immune mediated mechanisms of PARPi resistance in BRCA1-associated triple negative breast cancer (TNBC)

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A644-A644
Author(s):  
Anita Mehta ◽  
Madeline Townsend ◽  
Madisson Oliwa ◽  
Patrice Lee ◽  
Nicholas Saccomano ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Flow Cytometry ◽  
Immune Response ◽  
T Cell ◽  
Triple Negative Breast Cancer ◽  
Triple Negative ◽  
Parp Inhibitor ◽  
Triple Combination ◽  
Tumor Clearance ◽  
Sting Pathway

BackgroundPoly(ADP-ribose) polymerase inhibitors (PARPi) have improved the outcomes of BRCA-associated breast cancer; however, treatment responses are often not durable. Our preclinical studies demonstrated that PARPi activates the cGAS/STING pathway and recruitment of anti-tumor CD8+ T-cells that are required for tumor clearance [1]. These studies contributed to development of clinical trials testing PARPi plus immune checkpoint blockade (ICB). Unfortunately, early phase trials of PARPi + ICB have not yet suggested efficacy will be superior to PARPi monotherapy. Lack of demonstrated clinical synergy between PARPi + ICB underscores the need to study the tumor microenvironment (TME) during PARPi therapy to identify optimal strategies to enhance T-cell activation. We recently showed that PARPi induces CSF-1R+ suppressive tumor associated macrophages (TAMs) that restrict antitumor immune responses, contributing to PARPi resistance [2]. Removing TAMs with anti-CSF-1R therapy in combination with PARPi significantly enhanced overall survival (OS) compared to PARPi monotherapy in preclinical models [2]. Here, we investigate how modulating TAMs can enhance PARPi + ICB.MethodsMice bearing BRCA1-deficient TNBC (K14-Cre;Brca1f/f;p53f/f) tumors were treated for 98 days with PARPi (Talazoparib) ± small molecule inhibitor of CSF-1R (ARRAY-382; CSF-1Ri) ± anti-PD-1 and then followed for survival. Flow cytometry was employed to elucidate changes in the TME after treatment.ResultsPARPi conferred a significant survival advantage over vehicle treated mice (median OS 33 v. 14 days; p=0.0034) and 2/8 PARPi-treated mice experienced complete tumor clearance at day 98. PARPi + CSF-1Ri treated mice (median OS 140 days) remarkably cleared 7/10 tumors by day 98. The addition of anti-PD-1 to PARPi did not enhance OS compared to PARPi monotherapy. The triple combination of anti-PD-1 + PARPi + CSF-1Ri has not yet significantly enhanced the median OS compared to PARPi + CSF-1Ri (ongoing; 168 v. 140 days); nor did it increase clearance of tumor by day 98 (7/10). However, the triple combination led to superior long term tumor clearance. At day 161 the triple combination exhibited 5/10 tumor free mice compared to 2/10 treated with PARPi + CSF-1Ri. To elucidate how CSR-1Ri enhanced PARPi + ICB responses, flow cytometry was performed and revealed increased expression of the co-stimulatory molecule CD80, reduced tissue resident macrophages (CX3CR1+) and lower CSF-1R expression compared to PARPi + ICB.ConclusionsThese data suggest that targeting immunosuppressive macrophages may induce a favorable anti-tumor immune response and enhance responses to PARPi plus ICB. We are currently evaluating the adaptive immune response in this context.ReferencesPantelidou, C., et al., PARP inhibitor efficacy depends on CD8+ T cell recruitment via intratumoral STING pathway activation in BRCA-deficient models of triple-negative breast cancer. Cancer Discovery, 2019: p. CD-18-1218.Mehta, A.K., et al., Targeting immunosuppressive macrophages overcomes PARP inhibitor resistance in BRCA1-associated triple-negative breast cancer. Nat Cancer, 2021. 2(1): p. 66–82.

scholarly journals 883 An anti-carcinoma monoclonal antibody (mAb) NEO-201 can also target human acute myeloid leukemia (AML) cell lines in vitro

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A925-A925
Author(s):  
Alessandra Romano ◽  
Nunziatina Parrinello ◽  
Sara Marino ◽  
Enrico La Spina ◽  
Massimo Fantini ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Functional Analysis ◽  
Epithelial Cells ◽  
Cell Line ◽  
Cell Lines ◽  
Lymphoblastic Leukemia ◽  
Phase 1 ◽  
Adcc Activity ◽  
Phenotypic Analysis

BackgroundNEO-201 is an IgG1 mAb targeting variants of CEACAM5/6 and has demonstrated tumor sensitivity and specificity in epithelial cells. Functional analysis has revealed that NEO-201 can engage innate immune effector mechanisms including ADCC and CDC to directly kill tumor cells expressing its target. A recent Phase 1 clinical trial at the NCI has determined both safety and recommended Phase 2 dosing. We have also seen the expression of the NEO-201 target on hematologic cells, specifically Tregs and neutrophils. Due to epitope being expressed both on malignant epithelial cells as well as several hematologic cells, we designed this study to explore the reactivity of NEO-201 against hematological neoplastic cells in vitro.MethodsPhenotypic analysis was conducted by flow cytometry. Cell lines used were six AML (HL60, U937, MOLM13, AML2, IMS-M2 and OCL-AML3), two multiple myelomas (MM) (OPM2, MM1.S), two acute lymphoblastic leukemia (ALL) (SUP-B15, RPMI8402) and four mantle cell lymphoma (MCL) (Jeko-1, Z138, JVM2 and JVM13). Markers used for flow cytometry analysis were CD15, CD45, CD38, CD138, CD14, CD19 and NEO-201. Functional analysis was performed by evaluating the ability of NEO-201 to mediate ADCC activity against AML cell lines using human NK cells as effector cells.Results5 of 6 AML cell lines tested bind to NEO-201 and the% of positive cells were 47%, 99.5%,100%,100% and 97.8% for HL60, U937, MOLM13, AML3 and IMS-M2, respectively. The% of positive cells in the two MM cell line were 99% and 18% for OPM2 and MM1.S, respectively. NEO-201 binding was not detected in the two ALL and the four MCL cell lines tested. Functional analysis has demonstrated that NEO-201 can mediate ADCC activity against the AML cell line (HL60) tested.ConclusionsThis study demonstrates that NEO-201 mAb’s target is expressed in most of the AML cell lines tested in vitro. In addition, we have shown it can mediate ADCC activity against HL60 cells (AML). Together, these findings provide a rationale for further investigation of the role of NEO-201 in AML as well as MM, further exploring patient PBMCs and bone marrow samples.

scholarly journals Immune Checkpoint Receptors Tim-3 and PD-1 Regulate Monocyte and T Lymphocyte Function in Septic Patients

2018 ◽  
Vol 2018 ◽  
pp. 1-8 ◽  
Author(s):  
Qi Xia ◽  
Li Wei ◽  
Yuntao Zhang ◽  
Jifang Sheng ◽  
Wei Wu ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Immune Response ◽  
T Lymphocytes ◽  
Signaling Pathway ◽  
T Lymphocyte ◽  
Receptor Blockade ◽  
Lymphocyte Function ◽  
Significant Elevation ◽  
Programmed Cell Death 1 ◽  
Checkpoint Receptors

We aim to investigate the effects of Tim-3 and programmed cell death-1 (PD-1) on the monocytes and T lymphocytes in septic patients. Expression of Tim-3 and PD-1 on the CD3, CD4, and CD8 lymphocytes and monocytes was determined using flow cytometry. CBA technique was utilized to determine the expression of cytokines in the lymphocyte supernatant in addition to the IL-10 and TNF-αpositivity in monocytes in the presence of Tim-3 and/or PD-1 receptor blockade. Compared with the normal control, significant elevation was observed in the expression of PD-1 on CD3 (P=0.004), CD4, and CD8 monocytes. Blockade of the Tim-3 signaling pathway contributed to the significant elevation of IL-10 and TNF-αin the supernatant of T lymphocytes in the septic patients, while the PD-1 signaling pathway blockade only triggered the obvious elevation of TNF-αin the T lymphocytes. Blockade of Tim-3 and PD-1 induced the positivity of IL-10- and TNF-α-expressing cells in the peripheral monocytes. Significant changes were noticed in the Tim-3 and PD-1 in the T lymphocytes and monocytes. Blockade of Tim-3 and PD-1 contributed to the function of lymphocytes and monocytes. In the septic process, Tim-3 and PD-1 played crucial roles in the immune response of T lymphocytes and monocytes.

Elevated CD54 Expression Renders CD4+ T Cells Susceptible to Natural Killer Cell-Mediated Killing

2019 ◽  
Vol 220 (12) ◽  
pp. 1892-1903 ◽  
Author(s):  
Xi Chen ◽  
Huihui Chen ◽  
Zining Zhang ◽  
Yajing Fu ◽  
Xiaoxu Han ◽  
...  
Keyword(s):  
Immune Response ◽  
T Cells ◽  
Nk Cells ◽  
Natural Killer ◽  
Cd4 T Cells ◽  
Nk Cell ◽  
Killer Cell ◽  
Adaptive Immune Response ◽  
Phenotypic Analysis

Abstract Background Natural killer (NK) cells are an important type of effector cell in the innate immune response, and also have a role in regulation of the adaptive immune response. Several studies have indicated that NK cells may influence CD4+ T cells during HIV infection. Methods In total, 51 HIV-infected individuals and 15 healthy controls participated in this study. We performed the flow cytometry assays and real-time PCR for the phenotypic analysis and the functional assays of NK cell-mediated deletion of CD4+ T cells, phosphorylation of nuclear factor-κB (NF-κB/p65) and the intervention of metformin. Results Here we detected high CD54 expression on CD4+ T cells in HIV-infected individuals, and demonstrate that upregulated CD54 is associated with disease progression in individuals infected with HIV. We also show that CD54 expression leads to the deletion of CD4+ T cells by NK cells in vitro, and that this is modulated by NF-κB/p65 signaling. Further, we demonstrate that metformin can suppress CD54 expression on CD4+ T cells by inhibiting NF-κB/p65 phosphorylation. Conclusions Our data suggest that further studies to evaluate the potential role of metformin as adjunctive therapy to reconstitute immune function in HIV-infected individuals are warranted.

scholarly journals Antibiotic Therapy Does Not Alter the Humoral Response to Vaccination for Porcine Circovirus 2 in Weaned Pigs

2019 ◽  
Vol 6 (2) ◽  
pp. 51
Author(s):  
Jonathan E. Fogle ◽  
Jenna A. Scott ◽  
Glen W. Almond
Keyword(s):  
Flow Cytometry ◽  
Immune Response ◽  
Antibiotic Therapy ◽  
Humoral Immunity ◽  
Immune Cell ◽  
Humoral Response ◽  
Porcine Circovirus ◽  
Cell Populations ◽  
Antibiotic Administration ◽  
Weaned Pigs

Recent reports suggest that antibiotic therapy may either reduce or enhance the immune response to various porcine vaccines. Based upon these findings, we asked if antibiotic therapy alters immune cell populations, as measured by flow cytometry and/or vaccine-specific humoral immunity, as measured by sample to positive (S/P) antibody ratios. Here, we investigated the immuno-modulatory effects of enrofloxacin, ceftiofur, and tulathromycin on the immune response to a Mycoplasma hyopneumoniae (M. hyopneumoniae) and porcine circovirus type 2 (PCV-2) combination vaccine in weaned pigs. Maternal antibody likely interfered with the induction of immunity to M. hyopneumoniae. Antibiotic administration did not affect immune cell populations, as assessed by flow cytometry and did not affect the induction of humoral immunity to PCV-2.

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