FIGURE 7.

Hypoxia‐mediated adhesion of NK‐to‐microvascular endothelial cell recognition involves immune checkpoint molecules. A,B, PD‐1 detection on NK cells by flow cytometry. EL4 and EL4 IL2 cells labelled with anti‐murine‐PD1 antibodies strongly express PD1. B, Higher expression is detected on activated EL41L2. Means of three biological replicates are shown, *P < .05. C, Effect of hypoxia on surface PD‐L1 expression by ECs, MBMMEC and MBrMEC. Cells were cultured in normoxia (17.8% pO₂) or hypoxia (1% pO₂) for 48 h before anti‐PD‐L1 labelling. Staining is expressed as a relative MFI of PD‐L1. Means of three biological replicates are shown, * P < .05. D‐F, Effect of hypoxia on the expression vegf and vhl transcripts measured by RT‐PCR (D, E) and VEGF protein production by ELISA (F). Means of three biological replicates are shown, * P < .05. G, H, Adhesion of activated EL4 IL2 cells as compared to EL4 cells to organospecific ECs from the brain (MBrMEC) and bone marrow (MBMMEC) under the influence of hypoxia. DiO‐labelled EL4 and EL4 IL2 cells were allowed to adhere in a 5 to ratio to ECs for 20 min in normoxia (pO₂ = 17.8%) or hypoxia (pO₂ = 1%). R = number of adhering cells per endothelial cell. *P < .05. I, The selective effect of hypoxia on organospecific ECs’ recognition of the EL4IL2 cells MBrMEC and MBMMEC, cell cultured in normoxia or hypoxia for 48 h. Normoxic, DiO‐stained EL4‐IL2 cells adhered for 20 min. R is the number of EL4‐IL2 adhered per endothelial cell. Means of three biological replicates, *P < .05. J, K, Mechanism of activated PD1+ NK cells (EL4IL2) to endothelial cells (MBMMEC). Normoxic DiO‐stained EL4IL2 adhered to MBMMEC in a 5 to 1 ratio for 20 min at 20°C (J) or 4°C (K). Blocking by anti‐PD‐L1Fc silent, chimeric, and blocking mouse IgG1 was performed by incubating the MBMMEC endothelial cell culture for 1 h with 10 µg/mL anti‐PD‐L1 IgG1 before EL4IL2 cell addition, either at room temperature (J) or 4°C (K). The results are expressed as R = the number of EL4IL2 per one MBMMEC. Data are the mean values from five mice from three separate experiments *P < .05