PI3Kγ is a molecular switch that controls immune suppression (original) (raw)

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Acknowledgements

This work was supported by NIH grants R01CA126820 (J.A.V.), T32HL098062 (M.M.K.), T32CA009523 (S.G.) and T32CA121938 (S.G.), the CAPES Foundation and Ministry of Education of Brazil (C.F.) and by Ralph and Fernanda Whitworth and the Immunotherapy Foundation (J.A.V. and E.E.C.). The authors thank J. Lee and S. Schoenberger for HPV+MEER HNSCC and SSCVII cells.

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Authors and Affiliations

1. Moores Cancer Center, University of California, San Diego, La Jolla, 92093, California, USA
   Megan M. Kaneda, Karen S. Messer, Natacha Ralainirina, Hongying Li, Christopher J. Leem, Sara Gorjestani, Gyunghwi Woo, Abraham V. Nguyen, Camila C. Figueiredo, Philippe Foubert, Michael C. Schmid, Ezra E. W. Cohen & Judith A. Varner
2. Department of Family Medicine and Public Health University of California, Department of Family Medicine and Public Health University of California, San Diego, La Jolla, 92093, California, USA
   Karen S. Messer & Hongying Li
3. Dep. Biologia Celular, UERJ, Rio de Janeiro, 20550-013, Brazil
   Camila C. Figueiredo
4. Infinity Pharmaceuticals, Cambridge, 02139, Massachusetts, USA
   Melissa Pink, David G. Winkler, Matthew Rausch, Vito J. Palombella, Jeffery Kutok & Karen McGovern
5. Department of Pediatrics, University of California, San Diego, La Jolla, 92093, California, USA
   Kelly A. Frazer
6. Institute for Genomic Medicine, University of California, San Diego, La Jolla, 92093, California, USA
   Kelly A. Frazer
7. Department of Pharmacology, University of California, San Diego, La Jolla, 92093, California, USA
   Xuefeng Wu & Michael Karin
8. Center for Computational Biology and Bioinformatics, Institute for Genomic Medicine, University of California, San Diego, La Jolla, 92093, California, USA
   Roman Sasik
9. Department of Medicine, University of California, San Diego, La Jolla, 92093, California, USA
   Ezra E. W. Cohen & Judith A. Varner
10. Department of Pathology, University of California, San Diego, La Jolla, 92093, California, USA
    Judith A. Varner

Authors

1. Megan M. Kaneda
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2. Karen S. Messer
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3. Natacha Ralainirina
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4. Hongying Li
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5. Christopher J. Leem
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6. Sara Gorjestani
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7. Gyunghwi Woo
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8. Abraham V. Nguyen
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9. Camila C. Figueiredo
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10. Philippe Foubert
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11. Michael C. Schmid
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12. Melissa Pink
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13. David G. Winkler
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14. Matthew Rausch
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15. Vito J. Palombella
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16. Jeffery Kutok
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17. Karen McGovern
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18. Kelly A. Frazer
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19. Xuefeng Wu
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20. Michael Karin
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21. Roman Sasik
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22. Ezra E. W. Cohen
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23. Judith A. Varner
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Contributions

TCGA analysis was performed by H.L. and K.S.M., RNA sequencing by K.A.F., M.M.K., S.G. and R.S., flow cytometry by M.M.K. and N.R., in vitro studies by M.M.K., N.R., S.G., G.W., C.C.F., A.V.N. and M.C.S., and animal studies by M.M.K., N.R., C.L. and P.F. M.P., V.J.P., J.K., K.M., M.R. and D.G.W. provided IPI-549 and carried out experiments for Fig.1c, Extended Data Fig. 8a–b. ML120B was contributed by X.W. and M.K. The project was directed by E.E.W.C., K.S.M. and J.A.V. The manuscript was written by J.A.V. and M.M.K.

Corresponding author

Correspondence toJudith A. Varner.

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Competing interests

M.P., V.J.P., J.K., K.M., M.R. and D.G.W. are former employees of Infinity Pharmaceuticals and J.A.V. received research support from Infinity Pharmaceuticals.

Additional information

Reviewer Information Nature thanks F. Balkwill, M. de Palma and the other anonymous reviewer(s) for their contribution to the peer review of this work.

Extended data figures and tables

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

a–e, Association of IL12A (P = 0.026), IL12B (P = 0.039), IFNG (P = 0.002), CD8A (P = 0.001) and IL6 (P = 0.001) with survival in 97 HPV+ HNSCC patients (log-rank test). f, Multivariate immune signature for 720 lung adenocarcinoma samples from patients from KM plotter cohorts (P = 0.001; log-rank test). g, Multivariate immune signature in 876 gastric cancer samples from patients from KM plotter cohorts (P = 0.001; log-rank test). h, Western blotting of PI3Kγ in B cells, T cells, macrophages (MΦ) and LLC, PyMT and MEER tumour cells. i, Kaplan–Meier survival plot of wild-type (WT) and _Pik3cg_−/− mice inoculated with LPS (endotoxin). P = 0.05, log-rank test. j, Pro-inflammatory cytokine mRNA expression in bone marrow from wild-type and _Pik3cg_−/− LPS-injected mice. n = 4 biological replicates; **P < 0.001; ***P < 0.0001; one-sided ANOVA with Tukey’s post-hoc test. k, Circulating inflammatory cytokine levels in _Pik3cg_−/− and wild-type mice 24 h after endotoxin administration. n = 4 biological replicates; *P < 0.01; **P < 0.001; one-sided ANOVA with Tukey’s post-hoc test. l, Tumour volume of implanted HPV− (SCCVII) carcinomas (n = 15 biological replicates) from vehicle or PI3Kγ-inhibitor-treated mice. Arrow, start of drug treatment; P = 0.001; _t_-test. 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 (200×) in wild-type and _Pik3cg_−/− mice. n = 8 biological replicates; P = 0.007; t_-test. o, Kaplan–Meier survival plot of mice bearing orthotopic PyMT tumours treated with vehicle or the PI3Kγ inhibitor IPI549 initiated as indicated by the arrow (n = 10). p, In vitro LLC tumour cell survival in the presence of gemcitabine. q, Volume of LLC tumours implanted in wild-type and Pik3cg_−/− mice treated with saline or gemcitabine. n = 10 biological replicates; **P < 0.001; ***P < 0.0001. All experiments were performed two or more times. j–l, m, q, Data are shown as mean ± s.e.m.

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

a, Gating strategy for flow cytometry analysis of myeloid cell populations in peripheral blood leukocytes. b, Representative flow cytometry analysis and quantification of myeloid cell populations in peripheral blood (PB) of naive and LLC tumour-bearing mice. n = 3 biological replicates; P < 0.008; _t_-test. c, Flow cytometry analysis of myeloid cell populations on days 0, 7, 14 and 21 after subcutaneous inoculation with Lewis lung carcinoma cells (n = 3 biological replicates). d, Quantification of populations from c. e, Flow cytometry analysis of Ly6G, CCR2, CX3CR1, CD206, CD11c, F4/80 and CD45 expression in myeloid cell populations from c (n = 3 biological replicates). f, Relative immune response transcript levels ± s.e.m. in tumour-derived myeloid cells and tumour cells (CD11b−Gr1− cells) isolated at day (d)0 (n = 3), d7 (n = 5), d14 (n = 3) or d21 (n = 4) after LLC cell inoculation (P < 0.002, d21 versus d0). n, biological replicates. g Flow cytometry analysis of CD11b+ myeloid cell populations in wild-type and _Pik3cg_−/− LLC, PyMT and MEER tumours (n = 3 biological replicates). h, Quantification of CD11b+ myeloid cell populations (P = 0.001; _t_-test) from g. i, Flow cytometry analysis of CD11b+ myeloid cell populations in vehicle and PI3Kγ-inhibitor-treated PyMT, MEER and SCCVII tumours (n = 3 biological replicates). j, Quantification of CD11b+ myeloid cell populations from i. All experiments were performed two or more times. b, d, h, j, Data are shown as mean ± s.e.m.

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

a, Heat map of differentially expressed immune response genes in TAMs isolated from LLC tumours from wild-type and _Pik3cg_−/− mice (n = 3 biological replicates; local false discovery rate < 0.1) obtained by RNA sequencing. b, Relative mRNA expression of immune response factors in HPV+ HNSCC MEER tumours from _Pik3cg_−/− and wild-type mice (n = 4 biological replicates), *P = 0.01; _t_-test. c, Relative mRNA expression of immune response factors in CD11b+ myeloid cells isolated from PyMT tumours grown in vehicle or PI3Kγ-inhibitor-treated mice (n = 4 biological replicates), *P = 0.01; _t_-test. d, Fold change in mRNA expression in CD11b+Gr1− (macrophage), CD11b+Gr1lo (monocyte) and CD11b+Gr1hi (granulocyte) myeloid cells isolated from LLC tumours grown in _Pik3cg_−/− mice (n = 5 biological replicates) and normalized to wild-type control. n = 5 biological replicates; P = 0.001; one-sided ANOVA with Tukey’s post-hoc test. e, Arginase activity in tumours and TAMs isolated from LLC tumours grown in wild-type and _Pik3cg_−/− mice. n = 4 biological replicates; ***P < 0.0003; _t_-test. f, Protein concentration of cytokines in LLC tumours and TAMs from wild-type and _Pik3cg_−/− mice. n = 4 biological replicates; *P < 0.01; **P < 0.001; ***P < 0.0001; _t_-test. All experiments were performed two or more times. b–f, Data are shown as mean ± s.e.m.

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

a, Relative immune response mRNA expression in _Pik3cg_−/− and wild-type (WT) mouse macrophages stimulated by IL4- or LLC-tumour-cell-conditioned medium as determined by RT–PCR. Data are shown as mean ± s.e.m.; n = 3 biological replicates; *P = 0.01; _t_-test. b, Heat map of differentially expressed immune response transcripts in IL4- and IFNγ/LPS-polarized mouse macrophages obtained by RNA sequencing. n = 3 biological replicates; P = 0.00001. c, Heat map of select differentially expressed immune response transcripts in in vitro polarized mouse macrophages. n = 3 biological replicates; P = 0.00001. d, Heat map of immune response transcripts in mCSF-, IL4- and IFNγ/LPS-stimulated _Pik3cg_−/− mouse macrophages obtained by RNA sequencing and normalized to wild-type macrophages. n = 3 biological replicates; P = 0.00001. e, Heat map of select differentially expressed immune response transcripts in polarized _Pik3cg_−/− mouse macrophages normalized to wild-type. n = 3 biological replicates; P = 0.00001. f, Heat map of differentially expressed antigen presentation and processing mRNAs in mCSF, IL4 and IFNγ/LPS-polarized _Pik3cg_−/− mouse macrophages. n = 3 biological replicates; P = 0.00001. g, Heat map of differentially expressed chemokine and chemokine receptor mRNAs in polarized _Pik3cg_−/− mouse macrophages. n = 3 biological replicates; P = 0.00001. All experiments were performed two or more times.

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

a, b, Relative mRNA expression of immune response transcripts in IL4 and IFNγ/LPS-stimulated vehicle and PI3Kγ inhibitor (IPI-549)-treated mouse (a) and human (b) macrophages. n = 3 biological replicates; *P = 0.01; **P = 0.001; _t_-test. c, Relative mRNA expression of M2 macrophage markers (Arg1, Retnla (also known as Fizz1) and Chil3 (also known as Ym1) in wild-type and _Pik3cg_−/− IL4-stimulated macrophages (n = 3 biological replicates; P = 0.05; _t_-test). d, Relative RNA expression of MHC family members in wild-type and _Pik3cg_−/− IL4-stimulated macrophages. n = 3 biological replicates; P = 0.01; _t_-test. e, f, mRNA expression of cytokines over time in IFNγ/LPS, LPS and IL4 stimulated wild-type or _Pik3cg_−/− (e) and vehicle- or PI3Kγ-inhibitor-(IPI-549)-treated (f) macrophages (n = 3 biological replicates). g, Relative mRNA expression in mCSF-stimulated wild-type or Pik3cg−/− and IPI-549- or vehicle-treated macrophages. n = 3 biological replicates; P = 0.01; _t_-test. h, Relative nuclear RelA DNA binding activity in IFNγ/LPS stimulated wild-type and _Pik3cg_−/− macrophages. n = 3 biological replicates; P = 0.01; _t_-test. All experiments were performed two or more times.

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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 IL4-stimulated wild-type and _Pik3cg_−/− macrophages. b, Immunoblotting to detect pThr308Akt, total Akt, phospho-p65 and total p65 in LPS- and IL4- 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 (_Pik3cg_CAAX) and Mtor, S6ka, Cebpb or control siRNA. n = 3 biological replicates; P = 0.001, _t_-test. d, Validation of mRNA expression in macrophages expressing siRNAs from c (n = 3 biological replicates). e, Effect on cytokine mRNA expression in wild-type macrophages transfected with Cebpb, Mtor or S6ka siRNA. n = 3 biological replicates; *P = 0.01; **P = 0.001; _t_-test. f, g, Cytokine mRNA expression in macrophages treated with rapamycin (f) or the S6K inhibitor PF4708671 (g) (n = 3 biological replicates, P = 0.001, t-test). h, Immunofluorescence images of CD8+ T cells in 10 μm tumour sections from animals in Fig. 3b, c. i, Tumour volumes from tumour cells mixed with wild-type TAMs pretreated with the mTOR inhibitor rapamycin or the arginase inhibitor nor-NOHA and _Pik3cg_−/− TAMs pretreated with anti-IL12 or isotype-matched control antibody (cIgG), the IκKβ inhibitor MLB120 or the NOS2 inhibitor 1400W dihydrochloride (n = 10 biological replicates; P ≤0.04, one-sided ANOVA with Tukey’s post-hoc test). All experiments were performed two or more times. c–g, i, Data are shown as mean ± s.e.m.

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

a, Volumes of LLC tumours treated with vehicle + control liposomes, PI3Kγ inhibitor (IPI-549) + control liposomes, clodronate liposomes + vehicle and PI3Kγ inhibitor + clodronate liposomes. n = 10 biological replicates; P = 0.005; one-sided ANOVA with Tukey’s post-hoc test. b, Quantification of F4/80+ macrophages in tumours from a. n = 3 biological replicates; P = 0.02; one-sided ANOVA with Tukey’s post-hoc test. c, Quantification of F4/80+ macrophages in livers from a. n = 3 biological replicates; P < 0.005; one-sided ANOVA with Tukey’s post-hoc test. d, Quantification of T cells in tumours from a. n = 3 biological replicates; *P < 0.05, one-sided ANOVA with Tukey’s post-hoc test. e, Volumes of CT26 tumours treated with vehicle + cIgG, PI3Kγ inhibitor (IPI-549) + cIgG, anti-CD115 + vehicle and PI3Kγ inhibitor + anti-CD115. n = 15 biological replicates; P = 0.05; one-sided ANOVA with Tukey’s post-hoc test. f, Quantification of CD11b+ myeloid cells in tumours from e. n = 5 biological replicates; P < 0.001; one-sided ANOVA with Tukey’s post-hoc test. g, Images and quantification of CD8+ T cells in wild-type and _Pik3cg_−/− LLC tumours by immunohistochemistry. n = 5 biological replicates; P = 0.001; one-sided ANOVA with Tukey’s post-hoc test. h, Flow cytometry analysis and quantification of T cell populations in tumours from wild-type and _Pik3cg_−/− or IPI-549-treated mice. (n = 3 biological replicates; P < 0.05; _t_-test. i, Quantification of T cells in spleens of naive and LLC tumour-bearing wild-type and _Pik3cg_−/− mice. n = 3 biological replicates; P = 0.001; _t_-test. j, Volumes of LLC lung tumours from wild-type, _Pik3cg_−/−, _CD8_−/− and _CD8_−/−, _Pik3cg_−/− mice. n = 12 biological replicates; P < 0.001; one-sided ANOVA with Tukey’s post-hoc test. k, LLC tumour volume from wild-type and _Pik3cg_−/− mice treated with anti-CD8 antibodies or control (n = 10 biological replicates; P = 0.004; one-sided ANOVA with Tukey’s post-hoc test) and per cent CD8+ T cells out of CD3+ T cells in these tumours (n = 3 biological replicates; P = 0.01; _t_-test). l, In vitro proliferation of T cells (mean ± s.e.m. absorbance at 560 nm) isolated from naive and LLC tumour-bearing wild-type and _Pik3cg_−/− mice (n = 3 biological replicates). m, IFNγ and granzyme B protein expression in T cells from l (n = 3 biological replicates). All data are shown as mean ± s.e.m. and all experiments were performed two or more times.

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Extended Data Figure 8 PI3Kγ inhibition relieves T cell exhaustion.

a, Expression of IFNγ in activated human T cells treated with PI3Kγ and PI3Kδ inhibitors. Data are shown as mean ± s.d.; n = 2 biological replicates. b, Tumour weights derived from a mixture of LLC cells and wild-type or _Pik3cg_−/− tumour-derived T cells or wild-type T cells pre-incubated with 10 or 100 nM PI3Kγ (IPI-549) and PI3Kδ (Cal101) inhibitors before implantation. n = 16 biological replicates; P = 0.005 (_Pik3cg_−/−); P = 0.05 (PI3Kγi); one-sided ANOVA with Tukey’s post-hoc test. c, d, LLC tumour cell cytotoxicity induced by T cells isolated from LLC tumours from wild-type and _Pik3cg_−/− (c) or control- and PI3Kγ-inhibitor-treated (d) mice. n = 3 biological replicates; *P < 0.001; _t_-test. e, Images of TUNEL and haematoxylin and eosin stained tumours implanted with WT, _Pik3cg_−/− or no T cells from tumours shown in Fig. 3h. f, Quantification of TUNEL+ cells in tumour sections from e. n = 10 biological replicates; P = 0.01; _t_-test. g, Tumour volumes in wild-type mice of tumours derived from LLC tumour cells mixed 1:1 with CD90.2+, CD4+ and CD8+ T cells or no T cells from wild-type or _Pik3cg_−/− tumour-bearing mice. n = 8 biological replicates; P = 0.001; one way ANOVA with Tukey’s post-hoc test. h, mRNA expression of IL10 (P = 0.008; _t_-test) and TGFβ (P = 0.03, _t_-test) protein expression in lysates from tumour and CD90.2+, CD8+ and CD4+ T cells isolated from LLC tumours grown in wild-type and _Pik3cg_−/− mice (n = 3 biological replicates). i, IFNγ (P = 0.13, _t_-test) and granzyme B (P = 0.004, _t_-test) protein expression in PI3Kγ-inhibitor- or control-treated LLC tumours (n = 3 biological replicates). j, Ifng and Tgfb1 mRNA expression in T cells isolated from LLC tumours grown in wild-type and _Pik3cg_−/− or control- and PI3Kγ-inhibitor-treated mice. n = 3 biological replicates; P = 0.05, _t_-test). k, Relative mRNA expression of Cd4, Cd8, Gzmb and Ifng in control- and PI3Kγ-inhibitor-treated PyMT tumours. n = 3 biological replicates; P = 0.05, _t_-test. l, Relative mRNA expression of Cd4, Cd8, Gzmb and Ifng in wild-type and _Pik3cg_−/− and PI3Kγ-inhibitor-treated HPV+ MEER tumours (n = 3 biological replicates, _t_-test). All experiments were performed two or more times. b–d, f, g–j, l, Data are shown as mean ± s.e.m.

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

a, b, Flow cytometry analysis of PD-L1 and PD-L2 expression in tumour cells and TAMs from wild-type and _Pik3cg_−/− LLC tumours (a) and wild-type and _Pik3cg_−/− in vitro cultured IFNγ/LPS− and IL4-stimulated macrophages (b) (n = 3 biological replicates). c, HPV+ HNSCC tumour volume in female wild-type or _Pik3cg_−/− mice that were treated with anti-PD-1 or isotype-matched antibody (cIgG), as indicated by arrows and per cent change in tumour volumes between days 11 and 23. n = 10 biological replicates; *P = 0.01; **P = 0.001; ***P = 0.0001; ****P = 0.00001; one-sided ANOVA with Tukey’s post-hoc test). d, HPV+ HNSCC tumour volume in female wild-type mice that were treated with PI3Kγ inhibitor (2.5 mg kg−1 TG100-115 twice per day) in combination with anti-PD-1 or isotype-matched antibody (cIgG), as indicated by arrows, and per cent change in tumour volumes between days 11 and 29. n = 10 biological replicates; *P = 0.01; **P = 0.001; ***P = 0.0001; ****P = 0.00001, one-sided ANOVA with Tukey’s post-hoc test). e, HPV− HNSCC tumour volume in mice that were treated with PI3Kγ inhibitor (2.5 mg kg−1 TG100-115 twice per day) in combination with anti-PD-1 or cIgG, as indicated by arrows, per cent change in tumour volumes between days 19 and 26 and survival of treated mice. n = 10 biological replicates; *P = 0.01; **P = 0.001; ***P = 0.0001; ****P = 0.00001; one-sided ANOVA with Tukey’s post-hoc test). f, Tumour volume in HPV+ mice that had previously cleared HPV+ tumours and that were re-challenged with new HPV+ tumours (n = 7–12 biological replicates) compared to wild-type mice newly implanted with HPV+ tumours (n = 5 biological replicates). ***P = 0.0001; ****P = 0.00001; one-sided ANOVA with Tukey’s post-hoc test). g, Per cent CD3+, CD4+ and CD8+ T cells and MHCII+ macrophages from Fig. 4i. n = 3 biological replicates; *P = 0.05; **P = 0.005; ***P = 0.0005; ****P = 0.00005; one-sided ANOVA with Tukey’s post-hoc test. All experiments were performed two or more times. c–g, Data are shown as mean ± s.e.m.

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Extended Data Figure 10 PI3Kγ promotes immune suppression.

a, Comparison of median gene expression between HPV+ (n = 97) and HPV− (n = 423) cohorts indicating HPV− samples had significantly (P < 0.05, log-rank test) lower expression of adaptive immune genes and higher expression of immune suppressive and/or pro-metastasis genes. Blue, HPV− samples; red, HPV+ samples. b, Model depicting the effect of PI3Kγ inhibition on tumour immune suppression. PI3Kγ inhibition converts tumour-associated macrophages into pro-inflammatory macrophages that promote a CD8+ T cell response that suppresses tumour growth. c, Model depicting the PI3Kγ signalling pathway in macrophages. PI3Kγ activation attenuates NFκB activation and promotes mTOR-dependent C/EBPβ activation, leading to expression of immune suppressive factors and tumour growth. By contrast, PI3Kγ inhibition inhibits C/EBPβ and stimulates NFκB, leading to altered expression of pro-inflammatory immune response cytokines.

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This file contains source data for all gels used in Figure 2 and Extended Data Figures 1 and 6. (PDF 1417 kb)

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Kaneda, M., Messer, K., Ralainirina, N. et al. PI3Kγ is a molecular switch that controls immune suppression.Nature 539, 437–442 (2016). https://doi.org/10.1038/nature19834

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• Received: 14 March 2016
• Accepted: 12 September 2016
• Published: 19 September 2016
• Issue Date: 17 November 2016
• DOI: https://doi.org/10.1038/nature19834