Fig 2: PD-L1-pHLIPva inhibits T cell activation in pH6.1 and 6.8 solutions. (a–g,j,k) mouse lymphocytes were stimulated with CD3/CD28 activator microbeads in the presence of soluble PD-L1-pHLIPwt, PD-L1-pHLIPva or Ctrl protein (0.1 µg/mL) for 72 h in the indicated lactate-titrated pH buffer. (a) The lymphocyte proliferation was determined by BrdU cell proliferation assay. (b) Soluble Her2T276-652-pHLIPva, pHLIPva or PD-L1 protein (0.1 µg/mL) as controls were added parallelly. The lymphocyte proliferation was determined by BrdU cell proliferation assay. (c) CD4+ and CD8+ T cell populations were gated respectively and their proliferation was detected by CFSE dilution. (d) The supernatants were collected and IFN-γ, IL-17 A as well as IL-2 levels were examined by ELISA. (e,f) PD-L1-pHLIPva at the indicated concentrations was added. The lymphocyte proliferation and IFN-γ production in the supernatants was determined by BrdU cell proliferation assay and ELISA respectively. (g) CD4+T and CD8+T cells were gated respectively. CD25 and CD69 expression in these two populations was examined by flow cytometry. (h) Human PBMC were stimulated with CD3/CD28 activator microbeads in the presence of soluble PD-L1-pHLIPwt, PD-L1-pHLIPva or Ctrl protein (0.1 µg/mL) for 72 h in the indicated lactate-titrated pH buffer. The lymphocyte proliferation was determined by BrdU cell proliferation assay. (i) CD4+T cells were isolated from OT II-transgenic mice and co-cultured with OVA323 − 339-pulsed accessory cells for 72 h in the presence of soluble PD-L1-pHLIPwt, PD-L1-pHLIPva or Ctrl protein (0.1 µg/mL) in the indicated pH buffer. The T cell proliferation was determined by BrdU cell proliferation assay. (j,k) Monoclonal antibodies to PD-1 or isotypes (10 µg/mL) were added in the culture. The lymphocyte proliferation was determined by BrdU cell proliferation assay. IFN-γ production in the supernatants was examined by ELISA. The experiments were repeated 3 or 5 times. The data were pooled from these experiments, and representative plots were shown. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; ns no significance.

Fig 3: PD-L1-pHLIPwt 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-pHLIPwt or HER2-pHLIPwt (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-pHLIPwt 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 OVA323 − 339-pulsed irradiated accessory cells for 72 h in the presence of soluble PD-L1-pHLIPwt 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.