PI3Kγ is a molecular switch that controls immune suppression

Abstract

Macrophages play critical, but opposite, roles in acute and chronic inflammation and cancer. In response to pathogens or injury, inflammatory macrophages express cytokines that stimulate cytotoxic T cells, whereas macrophages in neoplastic and parasitic diseases express anti-inflammatory cytokines that induce immune suppression and may promote resistance to T cell checkpoint inhibitors. Here we show that macrophage PI 3-kinase γ controls a critical switch between immune stimulation and suppression during inflammation and cancer. PI3Kγ signalling through Akt and mTor inhibits NFκB activation while stimulating C/EBPβ activation, thereby inducing a transcriptional program that promotes immune suppression during inflammation and tumour growth. By contrast, selective inactivation of macrophage PI3Kγ stimulates and prolongs NFκB activation and inhibits C/EBPβ activation, thus promoting an immunostimulatory transcriptional program that restores CD8⁺ T cell activation and cytotoxicity. PI3Kγ synergizes with checkpoint inhibitor therapy to promote tumour regression and increased survival in mouse models of cancer. In addition, PI3Kγ-directed, anti-inflammatory gene expression can predict survival probability in cancer patients. Our work thus demonstrates that therapeutic targeting of intracellular signalling pathways that regulate the switch between macrophage polarization states can control immune suppression in cancer and other disorders.

Extended Data Figure 1. Pro-inflammatory gene expression signatures predict survival in cancer patients

(a–e) Expression levels of IL12A, IL12B, IFNG, and CD8A, and IL6 associated with survival in HPV+ HNSCC patients. (f) Multivariate immune signature for 720 lung adenocarcinoma patients from KM plotter cohorts. (g) Multivariate immune signature in 876 gastric cancer samples from KM plotter cohorts. (h) Western blotting to detect PI3Kγ (p110γ) in B cells, T cells, macrophages (MΦ) and LLC, PyMT and MEER tumor cells. (i) Kaplan Meier survival plot of WT and p110γ−/− mice inoculated with LPS (endotoxin). (j) Pro-inflammatory cytokine mRNA expression in bone marrow from WT and p110γ/ − LPS injected animals (n=4, **p<0.001, **p<0.0001). (k) Circulating inflammatory cytokine levels in p110γ−/− and WT mice 24h after endotoxin administration (n=4, *p<0.01, **p<0.001). (l) Tumor volume of HPV− (SCCVII) head and neck (n=15) carcinomas from vehicle or PI3Kγ inhibitor-treated mice. Arrow, start of drug treatment. (m) Dose response of the effect of PI3Kγ inhibitor IPI549 on in vitro MEER cell viability. (n) Spontaneous PyMT lung metastases per high power field (200X) in WT and p110γ−/− animals (n=8). (o) Kaplan Meier survival plot of mice bearing orthotopic PyMT tumors treated with vehicle or PI3Kγ inhibitor IPI549 initiated as indicated by arrow (n=10). (p) In vitro LLC tumor cell survival in the presence of gemcitabine. (q) Volume of LLC tumors implanted in WT and p110γ−/− animals treated with saline or gemcitabine (n=10, **p<0.001, **p<0.01).

Extended Data Figure 2. Effect of PI3Kγ inhibition on tumor inflammation

(a) Gating strategy for flow cytometric analysis of myeloid cell populations in peripheral blood leukocytes. (b) Representative flow cytometric analysis and quantification of myeloid cell populations in peripheral blood (PB) of naïve and LLC tumor bearing mice (n = 3). (c) Flow cytometric analysis of myeloid cell populations on days 0, 7, 14 and 21 after subcutaneous inoculation with Lewis lung carcinoma cells (n= 3). (d) Quantification of populations from c. (e) Flow cytometric analysis of Ly6G, CCR2, CX3CR1, CD206, CD11c, F4/80 and CD45 expression on myeloid cell populations from c (n=3). (f) Relative immune response transcript levels in tumor-derived myeloid cells and tumor cells (CD11b-Gr1− cells) isolated at day 0 (n=3), d7 (n=5), d14 (n=3) or d21 (n=4) after LLC cell inoculation (p < 0.002, d21 vs d0). (g) Flow cytometric analysis of CD11b+ myeloid cell populations in WT and p110γ−/− LLC, PyMT and MEER tumors (n=3). (h) Quantification of CD11b+ myeloid cell populations from (g). (i) Flow cytometric analysis of CD11b+ myeloid cell populations in vehicle and PI3Kγ inhibitor treated PyMT, MEER and SCCVII tumors (n=3). (j). Quantification of CD11b+ myeloid cell populations from (i).

Extended Data Figure 3. Effect of PI3Kγ inhibition on TAM expression profile

(a) Heatmap of differentially expressed immune response genes in TAMs isolated from LLC tumors from WT and p110γ−/− mice (n=3, *p<0.01, lfdr <0.1) obtained by RNA sequencing. (b) Relative mRNA expression of immune response factors in HPV+ HNSCC MEER tumors from p110γ−/− and WT mice (n=4), *p=0.01. (c) Relative mRNA expression of immune response factors in CD11b+ myeloid cells isolated from PyMT tumors grown in vehicle or PI3Kγ inhibitor-treated mice (n=4), *p=0.01. (d) Fold change in mRNA expression in CD11b+Gr1− (macrophage), CD11b+Gr1lo (monocytic) and CD11b+Gr1hi (granulocytic) myeloid cells isolated from LLC tumors grown in p110γ−/− mice (n=5) and normalized to WT control (n=5), p=0.001. (e) Arginase activity in tumors and TAMs isolated from LLC tumors grown in WT and p110γ−/− mice (n=4, ***p<0.0003). (f) Protein expression of cytokines in LLC tumors and TAMs from WT and p110γ−/− mice (n=4, *p<0.01, **p<0.001, ***p<0.0001).

Extended Data Figure 4. Effect of PI3Kγ deletion on in vitro macrophage mRNA expression

(a) Relative immune response mRNA expression in p110γ−/− and WT murine macrophages stimulated by IL-4 or LLC tumor cell conditioned medium (TCM) as determined by RT-PCR (n=3, *p=0.01). (b) Heat map of differentially expressed immune response transcripts in IL-4 and IFNγ/LPS polarized murine macrophages obtained by RNA sequencing (n=3, p=0.00001). (c) Heat map of select differentially expressed immune response transcripts in in vitro polarized murine macrophages (n=3, p=0.00001). (d) Heat map of immune response transcripts in mCSF, IL-4 and IFNγ/LPS stimulated p110γ−/− murine macrophages obtained by RNA sequencing and normalized to WT (n=3, p=0.00001). (e) Heat map of select differentially expressed immune response transcripts in polarized p110γ−/− murine macrophages normalized to WT (n=3, p=0.00001). (f) Heat map of differentially expressed antigen presentation and processing mRNAs in mCSF, IL-4 and IFNγ/LPS polarized p110γ−/− murine macrophages (n=3). (g) Heat map of differentially expressed chemokine and chemokine receptor mRNAs in polarized p110γ−/− murine macrophages (n=3).

Extended Data Figure 5. Effect of PI3Kγ inhibition on murine and human macrophage polarization

(a) Relative mRNA expression of immune response transcripts in IL-4 and IFNγ/LPS stimulated vehicle and PI3Kγ inhibitor (IPI-549) treated (a) murine and (b) human macrophages (n=3, p=0.001). (c) Relative mRNA expression of M2 macrophage markers (Arg1, Fizz1 and Ym1) in WT and p110γ−/− IL4-stimulated macrophages (n=3). (d) Relative expression of MHC family members in WT and p110γ−/− IL4-stimulated macrophages (n=3). (e–f) Time course of cytokine mRNA expression in IFNγ/LPS, LPS and IL-4 stimulated (e) WT vs p110γ−/− and (f) vehicle vs. PI3Kγ inhibitor (IPI-549)-treated macrophages (n=3). (g) Relative mRNA expression in mCSF-stimulated WT vs p110γ−/− and IPI-549- vs vehicle-treated macrophages (n=3). (g) Relative nuclear RelA DNA binding activity in IFNγ/LPS stimulated WT and p110γ−/− macrophages (n=3).

Extended Data Figure 6. Mechanism of PI3Kγ mediated gene expression regulation

(a) Relative levels of phospho/total p65 and phospho/total C/EBPβ in LPS and IL-4 stimulated WT and p110γ−/− macrophages. (b) Immunoblotting to detect pThr308Akt, total Akt, phospho-p65 and total p65 in LPS and IL-4 stimulated, macrophages that were treated with vehicle or the PI3Kγ inhibitor IPI-549. (c) Relative Arg1 mRNA expression in myeloid cells transfected with constitutively active, membrane-targeted PI3Kγ (p110γCAAX) and Mtor, S6ka, Cebpb or control siRNA (n=3). (d) Validation of siRNAs from c. (e) Effect of Cebpb, Mtor or S6ka siRNAs on gene expression in WT macrophages. (f–g) Effect of rapamycin (f) or S6K inhibitor (PF4708671) (g) on macrophage mRNA expression. (h) Immunofluorescence images of CD8+ T cells in 10μm tumor sections from 3c. (i) Mean tumor volumes from tumor cells mixed with WT TAMs pretreated with the mTOR inhibitor Rapamycin or the Arginase inhibitor nor- NOHA and p110γ−/− TAMs pretreated with anti-IL12 or isotype matched control antibody (cIgG), IKKβ inhibitor (MLB120) or NOS2 inhibitor (1400W dihydrochloride) (n=10).

Extended Data Figure 7. No direct effect of PI3Kγ inhibition on T cells

(a) Volumes of LLC tumors treated with vehicle + control liposomes, PI3Kγ inhibitor (IPI-549) + control liposomes, clodronate liposomes + vehicle and PI3Kγ inhibitor + clodronate liposomes (n=10). (b) Quantification of F4/80+ macrophages in tumors from a (n=3). (c) Quantification of F4/80+ macrophages in livers from a (n=3). (d) Quantification of T cells in tumors from a (n=3, *p<0.05). (e) Volumes of CT26 tumors treated with vehicle + cIgG, PI3Kγ inhibitor (IPI-549) + cIgG, anti-CD115 + vehicle and PI3Kγ inhibitor + anti-CD115 (n=15). (f) Quantification of CD11b+ myeloid cells in tumors from e (n=5). (g) Images and quantification of CD8+ T cells in WT and p110g−/− LLC tumors by IHC (n=5). (h) Flow cytometric analysis and quantification of T cell populations in tumors from WT and p110γ^−/− or IPI-549 treated animals (n=3). (i) Quantification of T cells in spleens of naïve and LLC tumor-bearing WT and p110γ−/− mice (n=3). (j) Volumes of LLC lung tumors from WT, p110γ^−/−, CD8^−/− and CD8^−/−; p110γ^−/− animals (n=12). (k) LLC tumor volume from WT and p110γ−/− animals treated with anti-CD8 antibodies or control (n=10) and percent CD8+ T cells in these tumors (n=3). (l) In vitro proliferation of T cells isolated from naïve and LLC tumor-bearing WT and p110γ−/− mice (n=3). (m) IFNγ and Granzyme B protein expression in T cells from l (n=3).

Extended Data Figure 8. PI3Kγ inhibition relieves T cell exhaustion

(a) Effect of PI3Kγ and PI3Kδ inhibitors on IFNγ expression by activated human T cells (n=3). (b) Mean weights of tumors derived from a mixture of LLC cells and WT or p110γ−/− tumor derived T cells or WT T cells pre-incubated with 10 or 100 nM PI3Kγ (IPI-549) and PI3Kδ (Cal101) inhibitors prior to implantation (n=16). (c–d) In vitro LLC tumor cell cytotoxicity induced by T cells isolated from LLC tumors from (c) WT and p110γ−/− or (d) control- and PI3Kγ inhibitor-treated mice (n=3, *p<0.001). (e) Images of TUNEL and H&E stained tumors as described in Fig. 5c. (f) Quantification of TUNEL+ cells in tumor sections from e. (g) Mean tumor volumes in WT mice derived from LLC tumor cells mixed 1:1 with CD90.2+, CD4+ and CD8+ T cells or no T cells from WT or p110γ^−/− tumor-bearing animals (n=8). (h) IL10 and TGFβ protein expression in lysates from tumor and CD90.2+, CD8+ and CD4+ T cells isolated from LLC tumors grown in WT and p110γ^−/− animals (n=3). (i) Interferon gamma and Granzyme B protein expression in PI3Kγ inhibitor or control-treated LLC tumors (n=3). (j) Ifnγ and Tgfb1 mRNA expression in T cells isolated from LLC tumors grown in WT and p110γ−/− or control- and PI3Kγ inhibitor-treated mice (n=3). (k) Relative mRNA expression of Cd4, Cd8, Grzb and Ifng in control and PI3Kγ inhibitor treated PyMT tumors (n=3). (l) Relative mRNA expression of Cd4, Cd8, Grzb, and Ifng in WT and p110γ−/− and PI3Kγ inhibitor treated HPV+ MEER tumors (n=3).

Extended Data Figure 9. PI3Kγ role in the macrophage-mediated tumor immune response

(a–b) Flow cytometric analysis of PD-L1 and PD-L2 expression on (a) tumor cells and TAMs from WT and p110γ−/− LLC tumors and (b) WT and p110γ−/− in vitro cultured IFNγ/LPS- and IL4-stimulated macrophages (n=3). (c) HPV+ HNSCC tumor growth in female WT or p110γ−/− mice that were treated with anti-PD-1 or isotype matched antibody (cIgG), as indicated by arrows, and percent change in tumor volumes between days 11 and 23. (d) HPV+ HNSCC tumor growth in female WT mice that were treated with PI3Kγ inhibitor (2.5 mg/kg TG100–115 b.i.d). in combination with anti-PD-1 or isotype matched antibody (cIgG), as indicated by arrows, and percent change in tumor volumes 11 and 29. (e) HPV− HNSCC tumor growth in mice that were treated with PI3Kγ inhibitor (2.5 mg/kg TG100–115 b.i.d) in combination with anti-PD-1 cIgG, as indicated by arrows, and percent change in tumor volumes between days 19–26. (f) Tumor rechallenge in HPV+ mice that had cleared previously HPV+ tumors (n=7–12) vs WT mice (n=5). (g) Quantification of percent CD3, CD4+ and CD8+ T cells and MHCII+ macrophages from Figure 5l. (*p<0.05, **p<0.005, ****p<0.00005).

Extended Data Figure 10. PI3Kγ promotes immune suppression

(a) Comparison of gene expression between HPV+ and HPV− cohorts indicating HPV− samples had significantly (p<0.05) lower expression of adaptive immune genes and higher expression of immune suppressive/pro-metastasis genes (Blue: HPV− samples, Red: HPV+ samples.) (b) Model depicting the effect of PI3Kγ inhibition on tumor immune suppression. PI3Kγ inhibition converts tumor-associated macrophages into pro-inflammatory macrophages that promote a CD8+ T cell response that suppresses tumor growth. (c) Model depicting the PI3Kγ signaling pathway in macrophages. PI3Kγ activation restrains NFκB activation and promotes mTOR-dependent C/EBPβ activation, leading to expression of immune suppressive factors and tumor growth. In contrast, PI3Kγ inhibition inhibits C/EBPβ and stimulates NFκB, leading to altered expression of pro-inflammatory immune response cytokines.

Figure 1. PI3Kγ promotes immune suppression

(a) Multivariate immune response mRNA signature in HPV+ HNSCC patients (n=97). (b) Immune response mRNA expression in p110γ−/− or WT peritoneal macrophages (n=3). (c) Mean +/− sem tumor growth in WT, p110γ−/− and PI3Kγ-inhibitor-treated mice (n=15). Arrow, daily treatment initiation. (d–f) Mean +/− sem (d) spontaneous breast carcinoma growth and metastasis (bar, 200μm) in WT (n=21) and p110γ−/− (n=8) animals; (e) MHCII expression on WT vs p110γ−/− TAMs (n=3) and (f) fold change mRNA expression in tumors and tumor-derived CD11b+ cells from p110γ−/− and PI3Kγ inhibitor-treated mice (n=5). (g) Heatmap of immune response mRNA expression in tumors from WT and p110γ−/− mice (n=3). n=biological replicates.

Figure 2. PI3Kγ regulates NFκB and C/EBPβ during macrophage polarization

(a) Heatmap of mRNA expression in p110γ−/− vs WT macrophages (n=3). (b–c) Mean +/− sem (b) mRNA (n=3) and (c) protein (n=4) expression. (d–e) Mean +/− sem (d) p65 RelA and (e) C/EBPβ DNA-binding activity in WT and p110γ−/− macrophages (n=4). (f–g) Immunoblotting of pRelA/RelA, pC/EBPβ/C/EBPβ, and pAkt/Akt in (f) LPS and (g) IL-4 stimulated WT and p110γ−/− macrophages. (h) Immunoblotting of IRAK1, pIKKβ/IKKβ, IκBα, pTBK1/TBK1, p110γ and actin in WT and p110γ−/− macrophages. (i) Mean +/− sem mRNA expression in IKKβ inhibitor-treated macrophages (n=3). n=biological replicates.

Figure 3. Macrophage PI3Kγ suppresses T cell activation

(a) Adoptive transfer method. (b) Weights of tumors implanted with p110γ−/−, WT or no TAMs (n=8). (c) CD8+ T cells from b (n=16). (d, e) Weights of tumors implanted with (d) in vitro cultured macrophages (n=8) or (e) macrophage-conditioned medium (CM) (n=8). (f) Percent T cells in WT and p110γ−/− tumors (n=3). (g) Tumor volumes in p110γ and/or CD8−/− mice (n=6). (h–i) Weights of tumors implanted with (h) naïve or tumor-derived T cells (n=8) or (i) inhibitor-treated T cells (n=16). (j) IFNγ and Granzyme B expression in tumors and T cells from WT and p110γ−/− animals (n=3). All graphs show mean +/− sem of biological replicates.

Figure 4. PI3Kγ inhibition synergizes with anti-PD-1

(a, d) Mean +/− sem tumor volumes in anti-PD-1 (black arrows) treated (a) WT or p110γ−/− mice with HNSCC HPV+ tumors (n=13) and (d) PI3Kγ inhibitor-treated mice with HPV− HNSCC tumors (n=13). (b, e) Percent survival of mice in a, d. (c, f) Change in tumor volumes in a, d. (g–h) Heatmap (g) and graph (h) of mRNA expression from d (n=3). (i) Immune cell profiles from d. (j) Heatmap of PI3Kγ-regulated mRNA expression in HPV+HNSCC patients (n=45). (k–l) Multivariate PI3Kγ-regulated immune signature in (k) HPV+HNSCC patients (n=97) and (l) lung adenocarcinoma patients (n=507). n=biological replicates.