Fig 1: Antibody validation. In the lung adenocarcinoma cell line A549 we detected protein bands corresponding to CXCL16 and CXCR6. Transfection with specific siRNAs caused a marked decrease of protein expression. Equal loading was ensured by staining for beta-actin
Fig 2: Immunohistochemical analyses of cancer and stromal cell CXCL16 expression and cancer cell CXCR6 in NSCLC. (a) Cancer cell CXCL16 high expression; (b) Cancer cell CXCL16 low expression; (c) stromal CXCL16 high expression; (d) stromal CXCL16 low expression; (e) Cancer cell CXCR6 high expression; (f) Cancer cell CXCR6 low expression; (g) Normal lung CXCL16 expression; (h) Normal lung CXCR6 expression
Fig 3: Intratumoral CXCR6+CD8+ T cells are pivotal for antitumor efficacy. (A) Set-up of bone marrow chimeric mice. Total bone marrow obtained from naïve CD45.1+ mice were mixed in 1:2 ratio with that from CD45.2+ WT or Cxcr6-/- mice and then were injected into lethally irradiated Rag1-/- recipient mice. Eight weeks later, MC38 cells were injected subcutaneously into chimeras. (B) Quanti?cation of CD45.1 and CD45.2 positive cells from peripheral blood (PB) and tumor gated on CD8+ cells. (C) Quanti?cation of CD45.1 and CD45.2 positive cells from tumors gated on effectors-producing CD8+ T cells. (D) Immunofluorescence staining of DAPI (blue)/CD8 (green)/CXCR6 (red) and their overlay in MSS and MSI tumor tissues. (E) Heatmap showing the relative expression of genes in intratumoral CXCR6+CD8+ T and CXCR6-CD8+ T cells. data are presented as the mean±SEM. *P<0.05, **p<0.01, and ***p<0.001. MSI, microsatellite instability; MSS, microsatellite stability.
Fig 4: High tumor expression of CXCR6 was induced by tumor tissue rather than tumor cells. (A) Timing course of proportions of CXCR6+CD8+ T cells in peripheral blood (PB) and tumor tissues from tumor burden mice. (B) Violin plot showing the expression of CXCL16 after treatment with isotype control or anti-CD40 agonist. (C) Western blot showing intratumoral CXCL16 expression. (D and E) Expression of CXCR6 on CD8+ T cells was detected after coculture with MC38 tumor cells (D) or conditional coculture with disrupted tumor tissues (E). Shredded tumor tissues were in upper wells and naïve T cells in lower wells with the presence of IL-2 recombinant cytokine and CD3/CD28 functional antibodies. (F) Timing course of expression of induced CXCR6 in vitro after conditional coculture with shredded tumor tissues. (G) Schematic diagram of inducing expression of CXCR6 in vivo. Mice were inoculated subcutaneously with B16OVA tumor cells. A week later, naïve OT-I T cells were injected into tumor load mice by tail vein and then detected OT-I+CXCR6+CD8+ T cells in PB and tumor tissue on day 2/4/7. (H) Representative density showing the proportion of CXCR6+CD8+ T cells in Pb and tumor tissue on day 4. Displayed flow graph were gated on OT-I positive cells. Data are presented as the mean±SEM. *P<0.05, **p<0.01, and ***p<0.001. Single-cell RNA sequencing data were analyzed on website http://crcleukocytecancer-pkucn/. CXCR6, C-X-C motif chemokine receptor 6; NS, no significant.
Fig 5: In vivo recall responses to anti-PD-1 in Cxcr6-/- mice are markedly compromised. (A) t-SNE plot showing distribution of T cell subsets before (left) or after (right) treatment with isotype control or anti-CD40 agonist. (B) Violin plot showing the expression of CXCR6 after treatment with isotype control or anti-CD40 agonist. (C) Schematic diagram of the anti-PD-1 treatment schedule. Mice were inoculated subcutaneously with MC38 tumor cells and were intraperitoneally treated with 200 µg of isotype control or anti-PD-1 antibodies on days 10/13/16 after tumor inoculation. (D) Tumor growth (n=6–9). (E) Proportions of intratumoral CD8+ T cells and cytokines produced was quantified from WT and Cxcr6-/- mice treated with isotype or anti-PD-1. Data are presented as the mean±SEM. *P<0.05, **p<0.01, and ***p<0.001, ****p<0.0001; single cell RNA sequencing data were analyzed on website http://crcleukocytecancer-pkucn/.
Supplier Page from Abcam for Anti-CXCR6 antibody