Human Lung-Resident Macrophages Colocalize with and Provide Costimulation to PD1^hi Tissue-Resident Memory T Cells

Rationale: Tissue-resident memory T cells (T_RM) play a critical role in the defense against inhaled pathogens. The isolation and study of human lung tissue-resident memory T cells and lung-resident macrophages (M_LR) are limited by experimental constraints. Objectives: To characterize the spatial and functional relationship between M_LR and human lung tissue-resident memory T cells using ex vivo lung perfusion (EVLP). Methods: T_RM and M_LR were isolated using EVLP and intraperfusate-labeled CD45 antibody. Cells isolated after 6 hours of EVLP were analyzed using spectral flow cytometry. Spatial relationships between CD3⁺ and CD68⁺ cells were explored with multiplexed immunohistochemistry. Functional relationships were determined by using coculture and T-cell-receptor complex signal transduction. Measurements and Main Results: Lungs from 8 research-consenting organ donors underwent EVLP for 6 hours. We show that human lung T_RM and M_LR colocalize within the human lung, preferentially around the airways. Furthermore, we found that human lung CD8⁺ T_RM are composed of two functionally distinct populations on the basis of PD1 (programed cell death receptor 1) and ZNF683 (HOBIT) protein expression. We show that M_LR provide costimulatory signaling to PD1^hi CD4⁺ and CD8⁺ lung T_RM,, augmenting the effector cytokine production and degranulation of T_RM. Conclusions: EVLP provides an innovative technique to study resident immune populations in humans. Human M_LR colocalize with and provide costimulation signaling to T_RM, augmenting their effector function.