PI3Kγ Activates Integrin α₄ and Promotes Immune Suppressive Myeloid Cell Polarization during Tumor Progression

Blockade of the PI3Kγ–integrin α₄ pathway promotes infiltration and maturation of CD11c⁺ DC in the tumor microenvironment. A, Representative images of CD11c⁺ DCs (green, arrowhead) and CD11b⁺ myeloid cells (red, arrow) in cryosections from LLC and Panc02 tumors and LLC tumor draining lymph nodes from WT and α4Y991A mice. Scale bar, 100 μm. B, Quantitative analysis of CD11b⁺ and CD11c⁺ cells in LLC and Panc02 tumors in WT and α4Y991A mice from A, expressed as pixels per field (n = 5). **, P < 0.01 vs. WT. C, Quantitative analysis of CD11b⁺ and CD11c⁺ cells in LLC tumor-draining lymph nodes in WT and α4Y991A mice from A, expressed as pixels per field (n = 5). **, P < 0.01 vs. WT. Scale bar, 100 μm. D, Representative images of CD11c⁺ DCs and CD11b⁺ myeloid cells in LLC tumors implanted in integrin α4Y991A mice transplanted with WT or α4Y991A BM and in WT mice transplanted with α4Y991A or WT BM. Scale bar, 100 μm. E and F, Quantitative analysis of (E) CD11b⁺ and (F) CD11c⁺ pixels in tumors from D (n = 5). G, Quantitative analysis of CD11c⁺MHCII⁺ mature DCs in Panc02 tumors assessed by flow cytometry in WT, α4Y991A, and PI3Kγ^–/− mice (n = 5). **, P < 0.01 vs. WT. H, Representative FACs plots of MHCII expression in CD11c⁺ cells from G. I, Quantification of CD11c⁺MHCII⁺ mature DC infiltration in LLC tumors assessed by flow cytometry in WT, α4Y991A, and PI3Kγ^–/− mice and mice treated with anti-α₄ or PI3Kγ inhibitor TG100-115 (n = 5); **, P < 0.01. J and K, Quantification of the ratio between mature DC (CD11c⁺/MHCII⁺) and immature DC (CD11c⁺/MHCII⁻) in Panc02 (J) and LLC (K) tumors from (G and I; n = 5) *, P < 0.05; **, P < 0.01. L, Mean fluorescence intensity of CD11c, CD80, and CD86 in CD11b⁺ cells from d14 LLC tumors from WT, PI3Kγ, and α4Y991A animals (n = 4). *, P < 0.05 for differences between WT and mutants. Error bars, SEM.

Blockade of the PI3Kγ–integrin α₄ pathway repolarizes tumor-associated myeloid cells. A and B, mRNA expression of immune related cytokines in (A) LLC and (B) Panc02 tumors from WT, α4Y991A, and PI3Kγ^–/− mice (n = 4) *P < 0.05. C–D, mRNA expression of immune related cytokines in LLC tumors from mice that were treated with (C) anti-α₄ versus isotype matched control antibody or (D) PI3Kγ inhibitor TG100-115 versus control (n = 4); **, P < 0.01. E, mRNA expression of immune-related cytokines in LLC tumor–associated myeloid cells from WT, α4Y991A, and PI3Kγ^–/− mice (n = 4); **, P < 0.05. F, mRNA expression of immune-related cytokines in in vitro IL4-stimulated WT and α4Y991A macrophages (n = 4); *, P < 0.05. Error bars, SEM.

Integrin α₄ activation controls IL10 expression. A, Il10 mRNA levels ± SEM in CD11b⁻ and CD11b⁺ cell populations from LLC tumors (n = 3). B, IL10 protein concentration (pg/mg total protein) ± SEM in LLC tumors from WT and α4Y991A animals (n = 3); *, P < 0.05. C,Il10 mRNA levels ± SEM in tumors from WT and α4Y991A bone marrow–transplanted animals (n = 3); *, P < 0.05. D, Mean LLC tumor volumes ± SEM from mice that were treated with two doses of anti-IL10 or cIgG (n = 6); *, P < 0.05.E, Percent CD11c⁺MHC⁺ DCs in tumors from D (n = 6); *, P < 0.05. F, Quantification of the ratio of mature DC (CD11c⁺/MHCII⁺) and immature DC (CD11c⁺/MHCII⁻) in mice from D (n = 6); **, P < 0.01. G, Il12b, (H) Ifng, and (I) Cd8a mRNA expression in tumors from D (n = 3); *, P < 0.05; **, P < 0.01. J, Representative images of CD8⁺ T cells in LLC tumors from D. Scale bar, 100 μm. K, Mean CD8⁺ T cells in tumors ± SEM (pixels/field; n = 3); **, P < 0.01. Error bars, SEM.

Blockade of the PI3Kγ–integrin α₄ pathway promotes CD8⁺ T-cell infiltration and T-cell cytotoxicity. A, Representative images of CD4⁺ and CD8⁺ T-cell infiltration in LLC tumors in cryosections from WT, α4Y991A, and PI3Kγ^–/−. Scale bar, 100 μm. B, Quantitative analysis CD4⁺ and CD8⁺ T cells in tumors from WT, α4Y991A, and PI3Kγ^–/− mice (n = 5); **, P < 0.01 vs. WT. C, Flow cytometry analysis of CD4⁺ and CD8⁺ TILs from LLC tumor–bearing mice from WT, α4Y991A, and PI3Kγ^–/− animals). D, The ratio of CD8⁺ to CD4⁺ T cells in tumors from WT, α4Y991A, and PI3Kγ^–/− animals (n = 3); *, P < 0.05 vs. WT. E, Relative expression of Cd8a mRNA in TILs from LLC tumors from WT, α4Y991A, and PI3Kγ^–/− animals (n = 4); *, P < 0.05. F, mRNA expression of Ifng, Il10, and Tgfb mRNA in TILs from LLC tumors from WT, α4Y991A, and PI3Kγ^–/− animals (n = 4). G, TILs from LLC tumors from WT, α4Y991A, and PI3Kγ^–/− animals were isolated and mixed with LLC tumor cells. T cell-cytotoxic activity against parental LLC tumor cells was assessed in an ex vivo CTL assay (n = 6); *, P < 0.05 vs. WT. Error bars, SEM.