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. 2020 Jun 25;8(1):e000928. doi: 10.1136/jitc-2020-000928

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© Author(s) (or their employer(s)) 2020. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.

This is an open access article distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited, appropriate credit is given, any changes made indicated, and the use is non-commercial. See http://creativecommons.org/licenses/by-nc/4.0/.

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Flow cytometry analysis of 5T4-specific immune responses induced by vaccination. Freshly isolated peripheral blood mononuclear cells (PBMCs) were cultured for 2 weeks in the presence of low-dose hIL-2 and the total 5T4 peptide pool to expand relatively infrequent 5T4-specific T cells. Flow cytometric analysis was performed on cultured cells at different time points, and the percentage of polyfunctional CD4+ and CD8+ secreting IFN-γ+ and TNF-α+ was calculated by subtracting background responses (Δ value) and compared with corresponding pre-vaccination values. (A) Representative gating strategy. From left to right, top to bottom: time–lymphocytes–single cells–live cells–CD3+ T cells–CD4+ and CD8+ T cells. (B) Representative dot plot with percentages showing CD4+ (top) and CD8+ (bottom) T cells secreting IFN-γ+ and TNF-α+. (C) Percentages of evaluated patients showing a positive response in cultured PBMCs (gray bar) and relative percentages of positivity for CD4+, CD8+, and both CD4+ and CD8+ among responders (black bars). (D) Baseline Δ value percentages of IFN-γ+ and TNF-α+CD4+ and CD8+ T cells are compared with corresponding peak Δ value percentages obtained after vaccination in each individual patient analyzed. BL: baseline. (E) Fold increase of peak Δ value percentages over baseline of polyfunctional CD4+ and CD8+ T cells induced by vaccination are shown. Lines represent median.