Journal: Frontiers in Immunology

Article Title: Moving for optimal immunity: the effect of acute high-intensity interval training on phenotype, virus specificity and chemokine receptor expression in human CD8+ T cells

doi: 10.3389/fimmu.2025.1739657

Figure Lengend Snippet: Acute HIIT selectively increases circulating virus-reactive CD8 + T cells, regardless of virus type. (A) Heatmap showing the estimated frequencies of peptide-specific CD8 + T cells per peptide. Analysis by DNA barcode-labeled peptide-MHC multimers, grouped after the respective virus. Darker colors indicate higher frequencies of peptide-specific CD8 + T cells. (B) Number of virus peptide-specific CD8 + T cells reactive against any of the 250 virus peptides. The estimated frequencies of MHC-matched, single peptide-specific CD8 + T cells were summed and multiplied by the absolute count of CD8 + T cells measured using the TBNK kit. (C) Estimated VART frequencies, summarized per virus. Plotted is the mean across all participants with a response at any timepoint on a log2 scale. (D) Numbers of EBV-specific CD8 + T cells against any EBV peptide, calculated according to (B, E) IFN-γ response to EBV peptide (GLCTLVAML). Plot shows the number of IFN-γ secreting cells per 5 x 105 PBMCs. ELISPOTS were run in technical triplicates. Spot counts are given as a difference between averages of the wells stimulated with the peptide and control wells. Samples were selected based on availability (n=5). (F) Numbers of specific CD8 + T cells against any SARS-CoV-2 peptide, calculated according to (B, D, F) Individual values overlaid with violin plots are plotted. P-values were calculated with Wilcoxon test and are Holm adjusted. (G) Proportions of CD8 + multimer + memory subsets based on flow cytometric CCR7 and CD45RA staining. (n=23) (H) Age-adjusted correlation heatmap of exercise-induced VART mobilization (defined as log2 (VART ex02/bsl)) vs. demographic, anthropometric and fitness participant characteristics. No correlations reached statistical significance. HLA genotyping revealed 22 participants matching the HLA restrictions of the peptide pool, thus n=22 participants are analyzed for panels (A-F, H) Significance levels indicated by asterisks on the graphs: ns p > 0.05, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001. EBV Ebstein Barr virus, HLA human leukocyte antigen, MHC major histocompatibility complex.

Article Snippet: 3–5 × 10 5 PBMCs were plated in IFN-γ Ab-coated ELISPOT plates and stimulated in triplicates with 5 μM of GLCTLVAML (EBV, Schafer-N) peptide for 20–24 h. After washing, biotinylated IFN-γ Ab, streptavidin-AP, and BCIP/NBT substrate (all Mabtech) were applied sequentially.

Techniques: Virus, Labeling, Control, Staining, Immunopeptidomics

Journal: Journal of Neuroinflammation

Article Title: Sez6L2 autoimmunity induces cerebellar ataxia in mice

doi: 10.1186/s12974-025-03637-7

Figure Lengend Snippet: Sez6L2 immunized mice generate Sez6L2-specific T cells. A and B Splenocyte and lymph node mixed cell cultures from Sez6L2 or sham immunized mice were left unstimulated or were stimulated with H-Sez6L2 or M-Sez6L2 protein as indicated for 24 h in an IFNγ ELISPOT assay. N = 8–10 mice (4–5 male and 4–5 female); one mouse = one culture. Cells from mice immunized with M-Sez6L2 were only stimulated with M-Sez6L2 (not H-Sez6L2) as only the reaction to M-Sez6L2 is relevant to the autoimmunity model for that group. A Representative images of ELISPOT wells. B Quantification of the number of spots per 200,000 splenocytes/lymph node cells plated. C - E Splenocyte/lymph node mixed cell cultures from Sez6L2 or sham immunized mice were stimulated with M-Sez6L2 protein for 24–72 h followed by cell surface and intracellular cytokine staining and analysis by flow cytometry. C Graphs show the percent of CD4 + cells positive for IFNγ or TNFα after stimulation in culture for 24 h. N = 8–10 (4–5 male and 4–5 female). D Graphs show the percent of CD4 + cells positive for IFNγ, TNFα, IL-4, or IL-17 A after stimulation in culture for 72 h. N = 4–5 males only. E Graphs show the percent of CD8 + cells positive for IFNγ, TNFα after stimulation in culture for 24 h. N = 8–10 (4–5 male and 4–5 female). Statistics for all graphs = Welch-ANOVAs with Dunnett’s T3 MCTs

Article Snippet: Mixed splenocyte and lymph node cells were plated on IFNγ ELISPOT pre-coated plates (Cellular Technology Limited, mIFNgp-2 M) in serum-free media at a cell density of 200,000 cells per well with or without 8 μg/mL mouse or human recombinant Sez6L2 protein.

Techniques: Enzyme-linked Immunospot, Staining, Flow Cytometry

Journal: Journal of Neuroinflammation

Article Title: Sez6L2 autoimmunity induces cerebellar ataxia in mice

doi: 10.1186/s12974-025-03637-7

Figure Lengend Snippet: Immunodominant T cell epitopes and antibody peptide epitopes from Sez6L2-immunized mice. A Splenocytes from H-Sez6L2 or sham-immunized mice were stimulated with 15-mer/10aa overlapping peptide library containing peptides from the extracellular domain of mouse and human Sez6L2 and assayed in an IFNγ ELISPOT assay. The 15-mer immunodominant peptide sequences are shown with the MHC-II core binding region predicted by IEDB Resource highlighted in red. For the peptide ELISPOT, H-Sez6L2-immunized N=5 (3 male, 2 female). B ELISA absorbance from serum of 6-week post-immunization mice tested for binding to the Sez6L2 15-mer peptide library. 12 H-Sez6L2-immunized mice were tested. For both A and B) Circle data points represent results from peptides that are identical in human and mouse Sez6L2, diamond data points are from peptides unique to humans, and triangle data points are from peptides unique to mouse. The colors represent individual mice. A schematic of Sez6L2’s domain structure is aligned to the A and B graphs. The red lines highlight the location of immunodominant T cell epitopes and the green lines highlight the antibody epitopes. C Summary table from 15-mer experiments showing peptide sequences that had positive hits in the ELISPOT and/or ELISA experiments. MHC-II core binding region predicted by IEDB Resource are highlighted in red. Overlapping peptides sharing 10 amino acids in common are underlined.

Article Snippet: Mixed splenocyte and lymph node cells were plated on IFNγ ELISPOT pre-coated plates (Cellular Technology Limited, mIFNgp-2 M) in serum-free media at a cell density of 200,000 cells per well with or without 8 μg/mL mouse or human recombinant Sez6L2 protein.

Techniques: Enzyme-linked Immunospot, Binding Assay, Enzyme-linked Immunosorbent Assay