human ifn γ elispot kit  (Danaher Inc)

Journal: medRxiv

Article Title: Distinct immune signatures discriminate SARS-CoV-2 vaccine combinations

doi: 10.1101/2022.09.05.22279572

Figure Lengend Snippet: (a)Schematic representation of the study protocol. Participants were vaccinated with one of 16 COVID-19 vaccine combination regimens and donated blood at timepoints T1 (4-12 weeks after Dose 1), T2 (two weeks after Dose 2), and T3 (four weeks after Dose 2). PBMCs collected at T1 and T3 were used for IFNγ ELISpot assays and high-dimensional spectral flow cytometry analysis. Sera and plasma collected at T1, T2, and T3 were analysed for anti-S-RBD IgG levels and NAb titres, respectively. Participants were monitored for adverse events. The data collected were analysed using dimensionality reduction, FlowSOM-based clustering algorithms, and statistical testing. (b) Cohort characteristics. Mean number of days and standard deviation after Dose 1 or Dose 2 were calculated from a subset of the total participants, as indicated below each mean.

Article Snippet: IFNγ ELISpot analysis was performed using microplates pre-coated with monoclonal IFNγ-specific antibodies (Human Interferon Gamma ELISPOT Kit, Abcam).

Techniques: Enzyme-linked Immunospot, Flow Cytometry, Clinical Proteomics, Standard Deviation

Journal: medRxiv

Article Title: Distinct immune signatures discriminate SARS-CoV-2 vaccine combinations

doi: 10.1101/2022.09.05.22279572

Figure Lengend Snippet: (a,c) Normalized IFNγ production after stimulation of PBMCs with SARS-CoV-2 spike peptide pool, measured by ELISpot assay. (a) Longitudinal IFNγ response at T1 and T3. Black lines show the median. (b) Representative images of one IFNγ ELISpot assay (n=583). (c) Normalized IFNγ responses at T3 after stimulation with SARS-CoV-2 spike peptide pool. Large black dots show the median of each group, and the vertical line spans the interquartile range. The horizontal line indicates the positive cut-off threshold. P-values indicate differences between the respective group and the overall mean of all participants. (a,c) P-values were calculated using the Mann-Whitney-Wilcoxon test and the Benjamini-Hochberg method to control for multiple hypothesis testing. Only statistically significant p-values (p < 0.05) are shown. (d) UMAP showing the FlowSOM-guided manual metaclustering of T cells (CD3+) for all vaccine groups combined. (e) Frequencies of canonical T cell subsets relative to the total number of T cells for each vaccine regimen. (f,g) UMAP of the T cell compartment showing the clusters with frequencies (f) positively or (g) negatively correlated with SARS-CoV-2 spike peptide-induced IFNγ response. (h) Differential marker expression by the specific T cell subclusters compared to the canonical T cell subsets that positively and negatively correlated with SARS-CoV-2 spike peptide-induced IFNγ responses, filtered for markers with at least 0.2 differential expression. The colour shows the Spearman’s rank correlation coefficient (rs), and the bubble size shows the p-value. Only significantly correlated clusters (p < 0.00001) are displayed.

Article Snippet: IFNγ ELISpot analysis was performed using microplates pre-coated with monoclonal IFNγ-specific antibodies (Human Interferon Gamma ELISPOT Kit, Abcam).

Techniques: Enzyme-linked Immunospot, MANN-WHITNEY, Control, Marker, Expressing, Quantitative Proteomics

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: CD40L-Stimulated B Lymphocytes Are Polarized toward APC Functions after Exposure to IL-4 and IL-21.

doi: 10.4049/jimmunol.2001173

Figure Lengend Snippet: FIGURE 6. IL-4 and Combo-activated CD40B cells are efficient APC that promote T cell activation and proliferation. (A) Schematic illustration of the general protocol for recognition assays between CD40B cells or CD40L-matured MoDC and Ag-specific CD81 T cells as detailed in the Material and Methods. (B and C) Represen- tative dot plots of the expression of (B) 4-1BB or (C) IFN-g by CD81T cells cocultured with IL- 4, IL-21, Combo-CD40B cells, or MoDC pulsed with 0.001 mM of g209-2M peptide are shown in the left panel. Cells were gated on live CD31CD4CD81T cells. A dose-response curve for all peptide concentrations evaluated is shown in the right panel. T cells only or T cells stimu- lated with PMA/ionomycin were used as controls. Results are represented as mean (SD) (n = 4). (D) IFN-g secretion was quantified in coculture supernatants by ELISA to evaluate MHC-I Ag pre- sentation by IL-4, IL-21, Combo-CD40B cells, or MoDC pulsed with indicated peptide concentra- tions. T cells only or T cells stimulated with PMA/ ionomycin were used as controls. IFN-g secretion was quantified in two technical replicates for each sample and condition. Results are represented as mean (SD) (n = 8). (E) Representative ELISPOT well images containing 5 104 T cells cocultured with autologous CEF-loaded IL-4, IL-21, Combo- CD40B cells, or MoDC. The number of IFN-g1 T cells (SFU) was quantified in two technical replicates for each sample and condition and is presented in the left panel. T cells only or T cells stimulated with PMA/ionomycin were used as controls. SFU (mean [SD]) are pooled from repeated experi- ments (n = 2) from one CEF-reactive donor and are representative of two CEF-reactive donors (Supplemental Fig. 3C). The mean spot size rep- resenting IFN-g secretion was quantified in two technical replicates for each sample and condi- tion. Results are represented as mean (SD) (n = 2 CEF-reactive donor). (F) Representative histo- grams of T cell proliferation in an MLR for the APC/T cell ratio of 1:4. T cell proliferation was assessed by evaluating CTV dilution by flow cytometry after 6 d of culture with indicated stim- ulator cells or controls. Unstimulated T cells or T cells stimulated with anti-CD3, anti-CD28, and IL-2 were used as controls. Division indexes were calculated as mentioned in the Material and Meth- ods. Results are represented as mean (SD) (n = 9). T cell proliferation was assessed in two technical replicates for each sample and condition. Statistical significance of normally distributed data (Shapir- oWilk test) compared with Combo-CD40B cells was calculated using one-way (E) or two-way (BD, F) ANOVA with post hoc Tukey HSD. *p < 0.05, **p < 0.01.

Article Snippet: ELISPOT was performed as per the Human IFN gamma ELISPOT Kit’s instructions (Abcam).

Techniques: Activation Assay, Expressing, Enzyme-linked Immunosorbent Assay, Enzyme-linked Immunospot, Flow Cytometry