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Effector functions of MSLN-specific T cells. (A) representative donor data (left panel) and summary results (median ± IQR, n=21; right panel) showing CD8 + /IFNγ + and CD4 + /IFNγ + T cells, as assessed by ICS. (B) representative donor data (left panel) and summary results (median ± IQR; n=21; right panel) showing dual (IFNγ/TNFα)-producing CD4 + and CD8 + MSLN-specific T cells, as assessed by ICS. Box plots show the median and 25th and 75th data quartiles. (C) polyfunctionality of MSLN-specific T cells as measured by <t>FluoroSpot</t> <t>(IFNγ/Granzyme</t> <t>B/TNFα)</t> (n=6). Top panel shows the total frequency (SFC/2x105 cells) of specific T cells for each of the individual effector molecules, while the bottom panel shows the proportion of cells that are single, dual or triple analyte-producing. All data represent biological replicates (i.e. independent donors). TNFα, tumor necrosis factor alpha; ICS, intracellular cytokine staining; GrB, Granzyme B.
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Effector functions of MSLN-specific T cells. (A) representative donor data (left panel) and summary results (median ± IQR, n=21; right panel) showing CD8 + /IFNγ + and CD4 + /IFNγ + T cells, as assessed by ICS. (B) representative donor data (left panel) and summary results (median ± IQR; n=21; right panel) showing dual (IFNγ/TNFα)-producing CD4 + and CD8 + MSLN-specific T cells, as assessed by ICS. Box plots show the median and 25th and 75th data quartiles. (C) polyfunctionality of MSLN-specific T cells as measured by <t>FluoroSpot</t> <t>(IFNγ/Granzyme</t> <t>B/TNFα)</t> (n=6). Top panel shows the total frequency (SFC/2x105 cells) of specific T cells for each of the individual effector molecules, while the bottom panel shows the proportion of cells that are single, dual or triple analyte-producing. All data represent biological replicates (i.e. independent donors). TNFα, tumor necrosis factor alpha; ICS, intracellular cytokine staining; GrB, Granzyme B.
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Effector functions of MSLN-specific T cells. (A) representative donor data (left panel) and summary results (median ± IQR, n=21; right panel) showing CD8 + /IFNγ + and CD4 + /IFNγ + T cells, as assessed by ICS. (B) representative donor data (left panel) and summary results (median ± IQR; n=21; right panel) showing dual (IFNγ/TNFα)-producing CD4 + and CD8 + MSLN-specific T cells, as assessed by ICS. Box plots show the median and 25th and 75th data quartiles. (C) polyfunctionality of MSLN-specific T cells as measured by <t>FluoroSpot</t> <t>(IFNγ/Granzyme</t> <t>B/TNFα)</t> (n=6). Top panel shows the total frequency (SFC/2x105 cells) of specific T cells for each of the individual effector molecules, while the bottom panel shows the proportion of cells that are single, dual or triple analyte-producing. All data represent biological replicates (i.e. independent donors). TNFα, tumor necrosis factor alpha; ICS, intracellular cytokine staining; GrB, Granzyme B.
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Effector functions of MSLN-specific T cells. (A) representative donor data (left panel) and summary results (median ± IQR, n=21; right panel) showing CD8 + /IFNγ + and CD4 + /IFNγ + T cells, as assessed by ICS. (B) representative donor data (left panel) and summary results (median ± IQR; n=21; right panel) showing dual (IFNγ/TNFα)-producing CD4 + and CD8 + MSLN-specific T cells, as assessed by ICS. Box plots show the median and 25th and 75th data quartiles. (C) polyfunctionality of MSLN-specific T cells as measured by <t>FluoroSpot</t> <t>(IFNγ/Granzyme</t> <t>B/TNFα)</t> (n=6). Top panel shows the total frequency (SFC/2x105 cells) of specific T cells for each of the individual effector molecules, while the bottom panel shows the proportion of cells that are single, dual or triple analyte-producing. All data represent biological replicates (i.e. independent donors). TNFα, tumor necrosis factor alpha; ICS, intracellular cytokine staining; GrB, Granzyme B.
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(A) FACS analysis of antigen specificity of FluTC generated from HLA-A2 + healthy donors by repetitive expansions. All samples were gated on lymphocytes, single cells, and live cells. CD8 and Flu-Pentamer stainings were performed on days 7 and 14 of antigen-specific expansion (ASE) and day 14 of rapid expansion protocol (REP). During ASE, some CD8 + T cells were expanded in the presence of unpulsed feeder cells (w/o flu-peptide) as a negative control. (B) <t>ELISA</t> to determine cytokine secretion by FluTC. MO3.13-A2-Luc cells were pulsed with diluting concentrations of flu peptide and co-cultured with FluTC. After 20 h of co-culture, the supernatant of co-culture was analyzed by ELISA to detect IFNγ (left) and <t>Granzyme</t> <t>B</t> (right) secretion by FluTC.
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Effector functions of MSLN-specific T cells. (A) representative donor data (left panel) and summary results (median ± IQR, n=21; right panel) showing CD8 + /IFNγ + and CD4 + /IFNγ + T cells, as assessed by ICS. (B) representative donor data (left panel) and summary results (median ± IQR; n=21; right panel) showing dual (IFNγ/TNFα)-producing CD4 + and CD8 + MSLN-specific T cells, as assessed by ICS. Box plots show the median and 25th and 75th data quartiles. (C) polyfunctionality of MSLN-specific T cells as measured by FluoroSpot (IFNγ/Granzyme B/TNFα) (n=6). Top panel shows the total frequency (SFC/2x105 cells) of specific T cells for each of the individual effector molecules, while the bottom panel shows the proportion of cells that are single, dual or triple analyte-producing. All data represent biological replicates (i.e. independent donors). TNFα, tumor necrosis factor alpha; ICS, intracellular cytokine staining; GrB, Granzyme B.

Journal: Frontiers in Immunology

Article Title: Evaluating mesothelin as an immunotherapeutic target for endogenous T cells

doi: 10.3389/fimmu.2026.1853409

Figure Lengend Snippet: Effector functions of MSLN-specific T cells. (A) representative donor data (left panel) and summary results (median ± IQR, n=21; right panel) showing CD8 + /IFNγ + and CD4 + /IFNγ + T cells, as assessed by ICS. (B) representative donor data (left panel) and summary results (median ± IQR; n=21; right panel) showing dual (IFNγ/TNFα)-producing CD4 + and CD8 + MSLN-specific T cells, as assessed by ICS. Box plots show the median and 25th and 75th data quartiles. (C) polyfunctionality of MSLN-specific T cells as measured by FluoroSpot (IFNγ/Granzyme B/TNFα) (n=6). Top panel shows the total frequency (SFC/2x105 cells) of specific T cells for each of the individual effector molecules, while the bottom panel shows the proportion of cells that are single, dual or triple analyte-producing. All data represent biological replicates (i.e. independent donors). TNFα, tumor necrosis factor alpha; ICS, intracellular cytokine staining; GrB, Granzyme B.

Article Snippet: For the quantitation of polyfunctional cells simultaneously secreting IFNγ, GrB and/or TNFα a commercial FluoroSpot assay was used (Human IFNγ/Granzyme B/TNFα FluoroSpot Plus, Mabtech, Inc.).

Techniques: Staining

(A) FACS analysis of antigen specificity of FluTC generated from HLA-A2 + healthy donors by repetitive expansions. All samples were gated on lymphocytes, single cells, and live cells. CD8 and Flu-Pentamer stainings were performed on days 7 and 14 of antigen-specific expansion (ASE) and day 14 of rapid expansion protocol (REP). During ASE, some CD8 + T cells were expanded in the presence of unpulsed feeder cells (w/o flu-peptide) as a negative control. (B) ELISA to determine cytokine secretion by FluTC. MO3.13-A2-Luc cells were pulsed with diluting concentrations of flu peptide and co-cultured with FluTC. After 20 h of co-culture, the supernatant of co-culture was analyzed by ELISA to detect IFNγ (left) and Granzyme B (right) secretion by FluTC.

Journal: bioRxiv

Article Title: Multifaceted immune resistance landscapes in human oligodendrocytes protect against cytotoxic T cells and are dysregulated in MS brain cell subsets

doi: 10.64898/2026.04.21.719872

Figure Lengend Snippet: (A) FACS analysis of antigen specificity of FluTC generated from HLA-A2 + healthy donors by repetitive expansions. All samples were gated on lymphocytes, single cells, and live cells. CD8 and Flu-Pentamer stainings were performed on days 7 and 14 of antigen-specific expansion (ASE) and day 14 of rapid expansion protocol (REP). During ASE, some CD8 + T cells were expanded in the presence of unpulsed feeder cells (w/o flu-peptide) as a negative control. (B) ELISA to determine cytokine secretion by FluTC. MO3.13-A2-Luc cells were pulsed with diluting concentrations of flu peptide and co-cultured with FluTC. After 20 h of co-culture, the supernatant of co-culture was analyzed by ELISA to detect IFNγ (left) and Granzyme B (right) secretion by FluTC.

Article Snippet: Supernatants of FluTC-MO3.13-A2-Luc co-cultures and anti-CD3/CD28 activated FluTC were further analyzed for the detection of IFNγ (Human IFN-γ ELISA Set, BD OptEIA, #555142), TNFα (Human TNF ELISA Set, BD OptEIA, #555212), Granzyme B (Human Granzyme B ELISA development kit, Mabtech, #3485-1H-20) and TRAIL (human TRAIL/TNFSF10 DuoSet ELISA, R&D Systems, #DY375-05) ( , ).

Techniques: Generated, Negative Control, Enzyme-linked Immunosorbent Assay, Cell Culture, Co-Culture Assay