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. 2023 Jan 24;11(1):e005518. doi: 10.1136/jitc-2022-005518

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© Author(s) (or their employer(s)) 2023. 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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Cytokine production by PBMCs on ex vivo stimulation with heterologous stimuli is increased after BCG instillations. (A) Study schedule of the Tribute study. Blood was collected and PBMCs were isolated at three time points during the BCG induction cycle: pre-BCG1, BCG2 and BCG6; and two time points during each subsequent BCG maintenance cycle: pre-BCG7, BCG9, pre-BCG10, BCG12, pre-BCG13 and BCG15. Some patients discontinued with BCG (see online supplemental table 2). Light blue arrow indicates pre-BCG1 time point which is used to calculate fold change in cytokine production. Purple arrows indicate important time points for TI, as patients did not receive BCG for weeks to months and thus represent the best ‘innate immune memory’ time points. (B) TNF production by PBMCs after 24 hour stimulation with LPS at eight time points during BCG therapy compared with pre-BCG1. (C) IL-1β production by PBMCs after 24 hour stimulation with LPS at eight time points during BCG therapy compared with pre-BCG1. (D) TNF production by PBMCs after 24 hour stimulation with Pam₃Cys (P3C) at eight time points during BCG therapy compared with pre-BCG1. (E) IL-1β production by PBMCs after 24 hour stimulation with P3C at eight time points during BCG therapy compared with pre-BCG1. Fold change values for each individual patient are displayed as gray dots. Group values for each time point are displayed as median±range in fold change compared with pre-BCG1. Two-tailed matched-pairs Wilcoxon signed-rank test was used to determine statistical significance in cytokine production between time points. Statistical significance was accepted at p<0.05 and indicated as follows: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Number of data points per time point for b, c, e: pre-BCG1: 17, BCG2: 17, BCG6: 16, pre-BCG7: 14, BCG9: 13, pre-BCG10: 8, BCG12: 7, pre-BCG13: 3, BCG15: 3. Number of data points per time point for d: pre-BCG1: 15, BCG2: 15, BCG6: 13, pre-BCG7: 12, BCG9: 11, pre-BCG10: 7, BCG12: 6, pre-BCG13: 2, BCG15: 2. LPS, lipopolysaccharide; PBMC, peripheral blood mononuclear cell.