Figure 2. Lung cDC2 are the most potent inducers of Tfh-like cell polarization.

DC subsets were purified from non-obstructed peritumoral lung tissue and co-cultured with allogeneic naive blood CD4⁺ T-cells. (A) Percentages of ICOS⁺PD-1⁺ T-cells in the different DC/T-cell co-cultures were determined at d7 of the co-culture via flow cytometry. Summary data graph with each symbol representing an individual donor (n=10). (B) Flow cytometry histogram of OX40 staining on ICOS⁺PD-1⁺ (purple), ICOS⁻PD-1⁺ (orange), ICOS⁺PD-1⁻ (blue) and ICOS⁻ PD-1⁻ (black) T-cell subsets in cDC2/T-cell co-cultures. Representative data from 3 donors is shown. (C) Intracellular IL-21 (n=10) and CXCL13 (n=6) staining of ICOS⁺PD-1⁺ (purple), ICOS⁻PD-1⁺ (orange), ICOS⁺PD-1⁻ (blue) and ICOS⁻PD-1⁻ (black) T-cell subsets in cDC2/T-cell co-cultures after restimulation with PMA and ionomycin in the presence of Golgi-plug and Golgi-stop. Summary data graph in which each symbol represents an individual donor. (D) Percentages of ICOS⁺PD-1⁺IL-21⁺ T-cells in cDC2/T-cell and cDC1/T-cell co-cultures were determined. Summary data graph in which each symbol represents an individual donor (n=10). (E) Proportions of ICOS⁺CXCR5⁺ T-cells in the different DC/T-cell co-cultures were determined at day 4. Summary data graph in which each symbol represents an individual donor (n=6). (F) Percentages of PD-1^hiBCL6^hi cells in ICOS⁺CXCR5⁺, ICOS⁺CXCR5⁻ and ICOS⁻ CXCR5⁻ T-cell subsets in the cDC2/T-cell co-cultures were determined via flow cytometry. Summary data graph in which each symbol represents an individual donor (n=6). *p<0.05, **p<0.01, ***p<0.001, Tukey’s multiple comparison test (A, C, E and F) and paired student’s t-test (D).