Journal: Frontiers in Immunology

Article Title: Divergent SARS-CoV-2-specific T cell responses in intensive care unit workers following mRNA COVID-19 vaccination

doi: 10.3389/fimmu.2022.942192

Figure Lengend Snippet: Pre-existing SARS-CoV-2-specific T cells in healthy ICU workers. (A) Study design. Blood samples were taken from intensive care unit workers with no known antecedents of COVID-19 before vaccination (n=41, Pre-v), and 1 month (n=41, Post-v1) and 10 months (n=16, Post-v10) after receiving two doses of the mRNA vaccine. Pre-pandemic healthy donors (n=20) and COVID-19 convalescent patients (n=15) with mild and severe disease were used as control groups. Antibody levels and SARS-CoV-2-reactive T cells were assayed at the indicated times. Frequencies of IFN-γ-producing T cells assayed by ELISpot assay against spike (S, orange circles), membrane (M, green circles) and nucleocapsid (N, yellow circles) proteins in samples from ICU workers (B) taken before vaccination, pre-pandemic healthy donors (C) and convalescent patients recovered from COVID-19 (D) . Data from ELISpot assays are depicted as the number of spot-forming cells (SFCs) per 1 x 10 6 PBMCs. The red dashed line shows the established cut-off (≥33.3 SFCs/10 6 cells) after subtraction of negative control values. (E) Flow cytometry dot plots from one ICU workers showing the representative gating strategy to identify antigen specific CD4 + (CD3 - CD4 + OX40 + CD137 + ) and CD8 + (CD3 - CD8 + CD69 + CD137 + ) T cells by AIM assay. A patient with severe COVID-19 was reference as positive control. DMSO and polyclonal induction with anti-CD3/CD28 antibodies were used as negative and positive controls of stimulation for each sample. Numbers in dot plots represent the percentage of AIM + cells in each indicated square. Number of CD4+ (F) , CD8 + (G) T cells or both (H) detected by AIM assay against spike (S, orange squares, n=7), membrane (M, green squares, n=9) and nucleocapsid (N, yellow squares, n=5) proteins in samples from ICU workers that were positive by ELISpot assay. Data from AIM assay are represented as stimulation index (SI, squares) and frequency (triangles). Red dashed line represents the limit of positivity for SI > 1.1. All data are represented as mean ± SEM.

Article Snippet: The cellular immune response against SARS-CoV-2 was evaluated by the ELISpot assay using the anti-IFN-γ ELISpot kit (AID ® GmbH, Strasberg, Germany) to measure counts of IFN-γ- producing T cells that had previously been stimulated with SARS-CoV-2 peptide pools.

Techniques: Control, Enzyme-linked Immunospot, Membrane, Negative Control, Flow Cytometry, Positive Control

Journal: Frontiers in Immunology

Article Title: Divergent SARS-CoV-2-specific T cell responses in intensive care unit workers following mRNA COVID-19 vaccination

doi: 10.3389/fimmu.2022.942192

Figure Lengend Snippet: Interindividual variation of the cellular immune response to SARS-CoV-2 after COVID-19 mRNA vaccination. (A) Paired graph showing the number of specific T cells reactive against the S protein was evaluated by ELISpot assay in PBMCs from ICU workers (n=41) obtained before vaccination (Pre-v) and 1 month (Post-v1) after full vaccination. Statistical comparisons were performed by Wilcoxon test. (B) Frequency of spike-specific T cells detected by ELISpot assay in samples with a positive response (“Responders”) o those with a null response (“Non-responders”). The latter were divided in “Null responders” when the number of specific T cells was below the cut-off (<33.3 SFCs/10 6 PBMCs) and “Equal to Pre-v” when the number of specific T cells is not enhanced after vaccination. (C) Representative images of IFN-γ-inducing spots in “Responders” and “Non-responders” groups. (D, E) Percentage of spike AIM + CD4 + and CD8 + T cells in seven individuals from “Responders” group and six samples that lack of cellular response to SARS-CoV-2 (“Null responders” group). Statistical comparisons were performed by two tail Mann Whitney test. (F, G) Correlation between frequencies of spike AIM + CD4 + and CD8 + T cells and the number of spike-reactive T cells evaluated by ELISpot assay in the individuals corresponding to “Responders” (n=7) and “Null responders” (n=6) groups. Statistical was performed by Spearman correlation coefficient.

Article Snippet: The cellular immune response against SARS-CoV-2 was evaluated by the ELISpot assay using the anti-IFN-γ ELISpot kit (AID ® GmbH, Strasberg, Germany) to measure counts of IFN-γ- producing T cells that had previously been stimulated with SARS-CoV-2 peptide pools.

Techniques: Enzyme-linked Immunospot, MANN-WHITNEY

Journal: Applied Microbiology and Biotechnology

Article Title: T cell immunity rather than antibody mediates cross-protection against Zika virus infection conferred by a live attenuated Japanese encephalitis SA14-14-2 vaccine

doi: 10.1007/s00253-020-10710-z

Figure Lengend Snippet: Cross-reactive cytokine responses in mouse splenocytes. a Schedule. Three weeks after the final immunization, splenocytes from adult C57BL/6 mice were collected. b The cytokines IL-2, IL-4, and IFN-γ secreted by splenocytes were determined using ELISPOT ( n = 7). The numbers of cytokine-positive cells are reported as the mean SFU/3 × 10 5 splenocytes + SD. Asterisk indicates P < 0.05; double asterisk indicates P < 0.01; triple asterisk indicates P < 0.001

Article Snippet: Three weeks after the final immunization, the cytokines IL-2, IL-4, and IFN-γ secreted by the splenocytes of C57BL/6 mice were determined using enzyme-linked immunospot (ELISPOT) kits (BD, USA) according to the manufacturer’s instructions and the previous protocol (Wang et al. , ).

Techniques: Enzyme-linked Immunospot

Journal: Experimental and Therapeutic Medicine

Article Title: LAPTM4B-35 protein is a weak tumor-associated antigen candidate

doi: 10.3892/etm.2013.1427

Figure Lengend Snippet: Enzyme-linked immunosorbent spot (ELISPOT) assay. Cytotoxic T-cell responses, assayed using an interferon-γ (IFN-γ) spot assay, against N91 peptides or HepG2 tumor cells. A fixed number of various target and effector cells (5×10 4 cells per well, effector to HepG2 target ratio of 40:1) were cultured in replicate wells overnight. PBMC, peripheral blood mononuclear cell.

Article Snippet: The CTL functions were analyzed using interferon-γ (IFN-γ) enzyme-linked immunosorbent spot (ELISPOT) assay kits (BD-Pharmingen, San Jose, CA, USA), in accordance with the manufacturer’s instructions.

Techniques: ELISpot Assay, Enzyme-linked Immunospot, Spot Test, Cell Culture