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. 2021 May 15;203(10):1230–1244. doi: 10.1164/rccm.202006-2403OC

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Figure 7. — Lung-resident macrophages (M_LR) provide costimulatory signaling to tissue-resident memory T cells (T_RM). CD69⁺, CD4⁺, and CD8⁺ T cells were isolated from lung biopsy specimens after 6 hours of ex vivo lung perfusion using an influx cell sorter and were cultured with media alone (media), with monoclonal CD3 (CD3), with monoclonal CD3 plus M_LR at an M_LR/T_RM ratio of 1:1 (CD3 + M_LR), and with CD2/CD3/CD28 beads (beads). (A) Representative flow-cytometric plot of PD1 (programed cell death receptor 1) and HOBIT (left) and TBET (T-box transcription factor TBX21) and HOBIT (right) expression of CD8⁺ T_RM cultured in media. (B) Representative histogram (left; horizontal line denotes positive gate) and paired cumulative frequency of intracellular GZMB (granzyme B) expression between PD1^hi and HOBIT^hi or TBET^hi CD8⁺ T_RM (*P = 0.02, includes samples from 7 lungs). (C) Representative flow-cytometric plot of TNFα (tumor necrosis factor α) and IFNγ production among HOBIT or TBET^hi (left) and PD1^hi (right) CD8⁺ T_RM isolated from lung biopsy specimens at 6 hours and cocultured with CD3 + M_LR. (D) Composite data comparing the proportion of CD8⁺ T_RM producing IFNγ (left) and TNFα (right) on the basis of expression of PD1 and either HOBIT or TBET (*P = 0.02, includes data from 7 lungs). (E) Representative comparison of cytokine production and LAMP1 expression for CD8⁺ PD1^hi lung T_RM under different stimulation conditions displayed as concatenated files from a single lung (top; rectangle outlines positive cells) and cumulative data (bottom; *P = 0.02; includes data from 7 lungs). (F) Representative comparison of cytokine production and LAMP1 expression for CD4⁺ PD1^hi lung T_RM under different stimulation conditions displayed as concatenated files from a single lung (top; rectangle outlines positive cells) and cumulative data (bottom; *P = 0.02; includes data from 7 lungs). max = maximum; ns = no statistically significant difference.

Figure 7. — Lung-resident macrophages (M_LR) provide costimulatory signaling to tissue-resident memory T cells (T_RM). CD69⁺, CD4⁺, and CD8⁺ T cells were isolated from lung biopsy specimens after 6 hours of ex vivo lung perfusion using an influx cell sorter and were cultured with media alone (media), with monoclonal CD3 (CD3), with monoclonal CD3 plus M_LR at an M_LR/T_RM ratio of 1:1 (CD3 + M_LR), and with CD2/CD3/CD28 beads (beads). (A) Representative flow-cytometric plot of PD1 (programed cell death receptor 1) and HOBIT (left) and TBET (T-box transcription factor TBX21) and HOBIT (right) expression of CD8⁺ T_RM cultured in media. (B) Representative histogram (left; horizontal line denotes positive gate) and paired cumulative frequency of intracellular GZMB (granzyme B) expression between PD1^hi and HOBIT^hi or TBET^hi CD8⁺ T_RM (*P = 0.02, includes samples from 7 lungs). (C) Representative flow-cytometric plot of TNFα (tumor necrosis factor α) and IFNγ production among HOBIT or TBET^hi (left) and PD1^hi (right) CD8⁺ T_RM isolated from lung biopsy specimens at 6 hours and cocultured with CD3 + M_LR. (D) Composite data comparing the proportion of CD8⁺ T_RM producing IFNγ (left) and TNFα (right) on the basis of expression of PD1 and either HOBIT or TBET (*P = 0.02, includes data from 7 lungs). (E) Representative comparison of cytokine production and LAMP1 expression for CD8⁺ PD1^hi lung T_RM under different stimulation conditions displayed as concatenated files from a single lung (top; rectangle outlines positive cells) and cumulative data (bottom; *P = 0.02; includes data from 7 lungs). (F) Representative comparison of cytokine production and LAMP1 expression for CD4⁺ PD1^hi lung T_RM under different stimulation conditions displayed as concatenated files from a single lung (top; rectangle outlines positive cells) and cumulative data (bottom; *P = 0.02; includes data from 7 lungs). max = maximum; ns = no statistically significant difference.