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. 2021 Aug 20;9(8):e002279. doi: 10.1136/jitc-2020-002279

Figure 5.

Figure 5

Inhibition of PI3K signaling by ZSTK474 in CD8+ T cells enhances memory T cell differentiation. (A–F) Naive CD8+ T cells (CD45RA+CCR7+) (A–E) and effector CD8+ T cells (CD45RA-CCR7-) (F) prepared from PBMCs of healthy individuals were stimulated with anti-CD3/anti-CD28 mAb for 8 days with or without the indicated doses of ZSTK474 or Idelalisib. Representative flow cytometry staining (left) and summary (right) of the frequencies of MPECs (KLRG1-CD127+) in CD8+ T cells (A), the frequencies of activated-caspase-3+CD8+ T cells in CD8+ T cells (B), the mean fluorescence intensity (MFI) of Ki67 (C), summary of the frequencies of MPECs (KLRG1-CD127+) in CD8+ T cells (D), absolute counts of MPEC per well in 96-well plates (E) and the frequencies of MPECs (KLRG1-CD127+) in CD8+ T cells (F). The numbers in the panels indicate the frequencies in CD8+ T cells (A, B) and the MFI of Ki67 in the indicated cells with the same color (C). (G, H) CD8+ T cells in PBMCs from healthy individuals were stimulated by X-irradiated APCs pulsed with Melan-A or CMV peptide with or without the indicated doses of ZSTK474 for 8 days. Representative flow cytometry staining (left) and summaries (right) of the frequencies of antigen-specific CD8+ T cells (G) and MPECs in antigen-specific CD8+ T cells (H). The numbers in the panels indicate the frequencies in CD8+ T cells (G) and the frequencies in antigen-specific CD8+ T cells (H). (I, J) CD8+ T cell responses against cancer antigens treated with the indicated doses of ZSTK474. CD8+ T cells in PBMCs from gastric cancer patients were cultured with or without ZSTK474 in the presence of X-irradiated CD4−CD8− PBMCs as APCs pulsed with WT-1, NY-ESO-1, MAGE-A3 or MAGE-A4 peptide. Flow cytometry staining of antigen-specific CD8+ T cells (I) and MPECs in antigen-specific CD8+ T cells (J). The experiments (A–H) were performed independently at least two to three with similar results. Data are means±SE. Statistical analyses were performed by Dunnett’s test (D–H). *P<0.05; **p<0.01; ***p<0.001. APCs, antigen-presenting cells; CMV, cytomegalovirus; mAb, monoclonal antibody; MPECs, memory precursor effector cells; nd, no data; ns, not significant; PBMCs, peripheral blood mononuclear cells.