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. 2021 Jun 30;9(6):e001649. doi: 10.1136/jitc-2020-001649

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© Author(s) (or their employer(s)) 2021. 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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TGM4 induces CD8 T-cell activation and expansion in vitro. (A) Schematic representation of the 30-day prime/boost coculture of autologous moDCs and naïve T cells. (B) Representative images of differentiated moDCs. 4X (left) and 40X (right) magnification. (C) Differential expression of functional markers on expanded populations of CD8 T cells following coculture with autologous protein-pulsed moDCs. Heatmap showing unsupervised clusters determined with the FlowSOM algorithm as described in the Materials and methods section. (D) Expanded CD8 T-cell populations defined by FlowSOM (in C) were projected onto UMAP space as described in the Materials and methods section. Colors correspond to FlowSOM populations. (E) Fold change on activated CD69⁺CD27⁺CD28⁺ EM CD8 T cells (left) and CM T cells (right) following the last 12-hour stimulation in expanded T cells. (F) Activated CD69⁺CD27⁺CD28⁺ cells as a percentage of EM CD8 T cells (left) and CM CD8 T cells (right) following in vitro expansion (as in E). (G) PD1⁺TIM3⁺ CD69⁺CD27⁺CD28⁺ cells as a percentage of EM CD8 T cells (left) and CD8 T cells (right) following in vitro expansion (as in E). (H) Gating strategy used to manually analyze TBET⁺ in activated CD69⁺CD28⁺ EM CD8 T cells defined as CCR7⁻CD45RA⁻ following coculture with autologous protein-pulsed moDCs. (I) TBET⁺ cells as a percentage of activated CD69⁺CD28⁺ EM CD8 T cells in expanded T cells (gated as in C). (J) Schematic representation of priming of naïve T cell with autologous TGM4-pulsed moDCs stimulated with PMA/ionomycin 4 hours prior analysis by flow cytometry. (K) Gating strategy used to manually analyze cytokine production on activated TBET⁺CD69⁺CD28⁺ EM CD8 T cells defined as CCR7⁻CD45RA⁻ following coculture with autologous TGM4-pulsed moDCs stimulated with PMA/ionomycin. (I) Cytokine production as a percentage of activated TBET⁺CD69⁺CD28⁺ EM CD8 T cells in expanded T cells (gated as in K). Unpaired t-tests performed; *p≤0.05, **p≤0.01, ***p≤0.001. CM, central memory; EM, effector memory; IFN-γ, interferon gamma; IL, interleukin; moDC, monocyte-derived dendritic cell; ns, not statistically significant; PAP, prostatic acid phosphatase; PSA, prostate-specific antigen; CEFT, Cytomegalovirus, Epstein-Barr virus, Influenza virus and Clostridium tetani; TAA, tumor-associated antigen; TGM4, transglutaminase 4.