<p>Representative polychromatic dot plots demonstrating the gating strategy employed to identify the immune cell profile of TILs in NSCLC. Starting at the top left, the initial gate was to obtain live cells; next, leukocytes (CD45+) and epithelial cells (CD326+) were gated; finally, CD3+ cells and CD3- cells were separated. For CD3+ cells, a size gate was applied to identify NKT cells (CD3+ CD56+), CD3+CD4+ and CD3+CD8+ T cells. T cell subsets are displayed, including naïve (CD45RA+CD197+), central memory (CM: CD45RA-CD197+), effector memory (EM: CD45RA-CD197-), and effector memory cells re-expressing CD45RA (EMRA: CD45RA+CD197-). FOXP3-expressing CD4+ T cells were gated on CD4+ T cells: naïve Treg (Fr. I: CD45RA+FOXP3lo), effector Treg (Fr. II: CD45RA-FOXP3hi), and non-Treg (Fr. III: CD45RA-FOXP3lo). For CD3- cells, a size gate was applied to identify B (CD3-CD19+) and NK cells (CD3-CD56+). The myeloid cell lineage was also gated on CD3- cells: conventional DC (HLA-DR+CD11c+), plasmacytoid DC (HLA-DR+CD11c-CD123+), macrophage (CD68+SSChi) and monocytic MDSC (HLA-DRlo CD14+CD11b+CD33+). Ki-67-expressing cells were gated in CD8+NKT, CD4+NKT, CD4+T (NT, CM, EM, EMRA), CD8+T (NT, CM, EM, EMRA), Treg, B, NK, and mMDSC. PD-1-expressing cells were gated in CD4+T (naive, CM, EM, EMRA), CD8+T (naive, CM, EM, EMRA) and Treg cells. PD-L1-expressing cells were gated in CD8+T (NT, CM, EM, EMRA), and mMDSC. CTLA-4-expressing cells were gated in Treg cells.</p>
<p>Percentage of PD-1+ CD8 T+ and PD-L1+ CD8 T+ cells in respective immune subtypes. (a, b) Data were presented as a percentage of PD-1+ cells (a) and PD-L1+ cells (b) per indicated CD8+ T cell subset in LUAD and LUSQ. % naïve CD8; percentage of PD-1+ or PD-L1+ naïve CD8+ T cell subset per total of naïve CD8+ T cells. % CM CD8; percentage of PD-1+ or PD-L1+ CM CD8+ T cell subset per total of CM CD8+ T cells. % EM CD8; percentage of PD-1+ or PD-L1+ EM CD8+ T cell subset per total of EM CD8+ T cells. % EMRA CD8; percentage of PD-1+ or PD-L1+ EMRA CD8+ T cell subset per total of EMRA CD8+ T cells. ns; not significant. * p<0.05. **P<0.01.</p>
<p>IHC of LUAD and LUSQ tissues of a representative case from immune subtypes. The antibodies against CD20 and FOXP3 are used in LUAD (a) and LUSQ tissues (b).</p>
<p>Representative polychromatic dot plots demonstrating the gating strategy employed to identify the immune cell profile of TILs in NSCLC. Starting at the top left, the initial gate was to obtain live cells; next, leukocytes (CD45+) and epithelial cells (CD326+) were gated; finally, CD3+ cells and CD3- cells were separated. For CD3+ cells, a size gate was applied to identify NKT cells (CD3+ CD56+), CD3+CD4+ and CD3+CD8+ T cells. T cell subsets are displayed, including naïve (CD45RA+CD197+), central memory (CM: CD45RA-CD197+), effector memory (EM: CD45RA-CD197-), and effector memory cells re-expressing CD45RA (EMRA: CD45RA+CD197-). FOXP3-expressing CD4+ T cells were gated on CD4+ T cells: naïve Treg (Fr. I: CD45RA+FOXP3lo), effector Treg (Fr. II: CD45RA-FOXP3hi), and non-Treg (Fr. III: CD45RA-FOXP3lo). For CD3- cells, a size gate was applied to identify B (CD3-CD19+) and NK cells (CD3-CD56+). The myeloid cell lineage was also gated on CD3- cells: conventional DC (HLA-DR+CD11c+), plasmacytoid DC (HLA-DR+CD11c-CD123+), macrophage (CD68+SSChi) and monocytic MDSC (HLA-DRlo CD14+CD11b+CD33+). Ki-67-expressing cells were gated in CD8+NKT, CD4+NKT, CD4+T (NT, CM, EM, EMRA), CD8+T (NT, CM, EM, EMRA), Treg, B, NK, and mMDSC. PD-1-expressing cells were gated in CD4+T (naive, CM, EM, EMRA), CD8+T (naive, CM, EM, EMRA) and Treg cells. PD-L1-expressing cells were gated in CD8+T (NT, CM, EM, EMRA), and mMDSC. CTLA-4-expressing cells were gated in Treg cells.</p>
