Fig. 1

PD-L1-pHLIP_wt inhibits T cell activation in pH6.1 buffer instead of pH6.8 solutions. (a–f) Mouse lymphocytes were isolated from the spleen by Ficoll gradient centrifugation and stimulated with CD3/CD28 activator microbeads in the presence of soluble PD-L1-pHLIP_wt or HER2-pHLIP_wt (Ctrl protein) (0.1 µg/mL) for 72 h in the indicated lactate-titrated pH buffer. (a) The proliferation was determined by BrdU cell proliferation assay. (b) CD4⁺T cell population was gated and its proliferation was detected by CFSE dilution. (c) The supernatants were collected and IFN-γ as well as IL-2 production was examined by ELISA. (d,e) Soluble PD-L1-pHLIP_wt at the indicated concentrations (0.01–0.1 µg/mL) was added. The proliferation and IFN-γ production were determined by BrdU cell proliferation assay or ELISA respectively. (f) CD4⁺T cells were gated and CD25 as well as CD69 expression in this population was examined by flow cytometry. (g) Splenic CD4⁺T cells from OT II-transgenic mice were fractionated by negatively magnetic isolation and co-cultured with OVA_{323 − 339}-pulsed irradiated accessory cells for 72 h in the presence of soluble PD-L1-pHLIP_wt or Ctrl protein (0.1 µg/mL) for 72 h in the indicated lactate-titrated pH buffer. The CD4⁺T cell proliferation was determined by BrdU cell proliferation assay. The experiments were repeated three or five times. The data was pooled from these experiments, and representative plots were shown. **p < 0.01; ***p < 0.001; n.s no significance.