color enzymatic elispot assay kit  (Cellular Technology Ltd)

Journal: Cell reports. Medicine

Article Title: Early activation of inflammatory pathways in UBA1-mutated hematopoietic stem and progenitor cells in VEXAS.

doi: 10.1016/j.xcrm.2023.101160

Figure Lengend Snippet: Figure 1. Myeloid dominance and activa- tion of the inflammatory pathways in VEXAS BMMNCs (A) Experimental workflow. BMMNC samples from patients and healthy donors were subjected to multi-color flow cytometry to profile hematopoi- etic stem and progenitor cell (HSPC) sub- populations, and to ELISpot assay to quantify BMMNCs secreting TNF-a or IFN-g. BMMNCs and FACS-sorted LineageCD34+ cells were subjected to colony forming assay and single-cell RNA sequencing (scRNA-seq) using the 10x Ge- nomics platform. scRNA-seq libraries were sequenced on the Illumina NovaSeq system before data analysis, including single-cell tran- scriptome profiling (gene expression, gene muta- tion, and cell-cell interaction) and single-cell T cell receptor/B cell receptor (scTCR/BCR) profiling. (B) A Uniform Manifold Approximation and Pro- jection (UMAP) plot of single-cell gene expression in BMMNCs of all patients and healthy donors. Cells are colored by types (HSPC, erythroblast, neutrophil, monocyte, T cell, NK cell, B cell, plasma cell, eosinophil, and dendritic cell). A bar chart shows percentages of these cell populations in individual patients and healthy donors. The co- lor legend is the same as that in the UMAP plot. A dot plot showing a myeloid (erythroblast, neutro- phil, monocyte, and dendritic cell) vs. lymphoid (T cell, B cell, NK cell, and plasma cell) ratio in pa- tients and healthy donors. Data are presented as mean values ± standard error of the mean (SEM). p values with the two-sided unpaired Mann-Whitney test are shown. (C) Heatmap showing expression of representa- tive differentially expressed genes grouped by their functional pathways in IFN-g and IFN-a signaling, TNF-a via NF-kB signaling, inflamma- tory response, E2F targets, and apoptosis, be- tween BMMNCs from VEXAS patients (n = 9) and healthy controls (n = 4). Values are presented as log2 fold-changes (log2FC). (D) Gene set enrichment analysis (GSEA) of ex- pressed genes in BMMNC subpopulations of VEXAS patients, including neutrophils, mono- cytes, erythroblasts, T cells, B cells, and NK cells. Normalized enrichment scores for the GSEA pathways are plotted, showing higher enrichment of the inflammatory pathways in neutrophils and monocytes than those in lymphoid cells. (E) Representative ELISpot wells showing TNF-a secretion by BMMNCs from two VEXAS patients and two healthy donors in a second batch of the validation cohort, in triplicate. Bottom, quantifi- cation of TNF-a-, IFN-g-, and TNF-a/IFN-g-posi- tive spots in BMMNCs plated (VEXAS patients n = 5 and healthy donors n = 2, in triplicate). Data are presented as mean values ± standard error of the mean (SEM). p values with the two-sided unpaired Mann-Whitney test are shown.

Article Snippet: ELISpot assay to check IFN-g and TNF-a secreted by human BMMNCs IFN-g and TNF-a secretion from BMMNCs of VEXAS patients and healthy donors were measured using the Human IFN-g/TNF-a Double-Color Enzymatic ELISPOT Assay kit (Cat# SKU:hIFNgTNFa-2M, ImmunoSpot) in two separate experiments in triplicate (4 patients versus 3 healthy donors for a 1st batch, and 5 patients versus 2 healthy donors for a 2nd batch), according to the manufacturer’s protocol.

Techniques: Cytometry, Enzyme-linked Immunospot, RNA Sequencing, Gene Expression, Clinical Proteomics, MANN-WHITNEY, Expressing, Functional Assay, Biomarker Discovery

Journal: Cancer Discovery

Article Title: The Polarity and Specificity of Antiviral T Lymphocyte Responses Determine Susceptibility to SARS-CoV-2 Infection in Patients with Cancer and Healthy Individuals

doi: 10.1158/2159-8290.CD-21-1441

Figure Lengend Snippet: Unexposed individuals susceptible to COVID-19 exhibited a SARS-CoV-2–specific Th2 profile during the first surge of the pandemic. A, Percentage and number of patients in each cohort—pre–COVID-19 era [yes (+)/no(−)], cancer [yes (+)/no(−)], and COVID-19 [yes (+)/no(−)]—who had a SARS-CoV-2–specific cytokine release (for the prototypic cytokines) compared with VeroE6 (control, n = 279; convalescent, n = 56; Supplementary Table S1B). Fisher exact test to compare the number of cytokine-positive patients across groups. B, Outline of the prospective collection of blood samples used to identify COVID-19–resistant (yellow) versus susceptible (red) patients with cancer ( B, top; Supplementary Table S2A and S2B). Bottom, outline of the prospective collection of blood samples used for the comparison of T-cell responses in the cohort of cancer-free individuals who lived in the same household with family members who tested positive for COVID-19 during the 2020 lockdown ( G and I ). Pie chart indicating the absolute numbers (and percentage) of patients reported as contact (resistant) or infected (susceptible) or unexposed (green) during 1-year follow-up ( D ). Number of positive cytokines released by SARS-CoV-2–specific PBLs during the cross-presentation assay ( and ) in each group (unexposed, n = 153; resistant, n = 42; susceptible, n = 19). E and F, SARS-CoV-2–specific IL2 (left) and IL5 (right) secretion contrasting resistant (yellow) versus infected (red) cancer cases. E, Each dot represents the ratio of the replicate wells in one individual, and the box plots indicate medians as well as 25th and 75th percentiles for each cancer patient subset. F, The bar plots represent the percentage of positive patients (resistant, n = 42; susceptible, n = 19). Fisher exact test to compare the number of cytokine-positive patients across groups. G and H, SARS-CoV-2–specific IL2/IL5 ratios (means ± SEM) in the different subsets of healthy individuals and patients with cancer presented in B . Refer to Supplementary Fig. S3A for the waterfall plots to visualize variations in the percentages of individuals with IL2/IL5 ratios > or < 1 according to subject category. All group comparisons were performed using the two-sided Wilcoxon–Mann–Whitney test, and P < 0.05 indicates statistically significant differences. I and J, Validation cohort investigating eight additional HCW from Hospices Civils de Lyon and 10 patients with cancer from Gustave Roussy investigated in cross-presentation assays with the dual-color IFNγ/IL5 ELISpot. I, Prototypic photograph of IFNγ and IL5 dual-color ImmunoSpot of a DC/SARS-CoV-2 or VeroE6 PBL coculture (or OKT3 as positive control) for one representative resistant (left) and susceptible (right) HCW. SFC, spot-forming colony counted per 105 PBLs. J, Percentages of SARS-CoV-2–specific Th1 or Th2 cell responses determined by dual ELISPOT assay (CoV-2/VeroE6 >1.5 increase in IFNγ+ (left) or IL5+ (middle) SFC, respectively. Calculation of the IFNγ+/IL5+ SFC ratio per individual in VeroE6 or SARS-CoV-2 condition, and percentages of patients with an increased (>2×) ratio in the SARS-CoV-2 condition, in both resistant versus susceptible groups (right). Fisher exact test to compare the number of positive patients between both groups.

Article Snippet: The enumeration of antigen-specific IFNγ- and IL5-producing T cells was performed using the ImmunoSpot human IFNγ/IL-5 double-color enzymatic ELISPOT kit (Cellular Technology Limited, CTL).

Techniques: Control, Comparison, Infection, MANN-WHITNEY, Biomarker Discovery, Enzyme-linked Immunospot, Positive Control