- Letter
- Published:
PI3Kγ is a molecular switch that controls immune suppression
- Megan M. Kaneda1,
- Karen S. Messer1,2,
- Natacha Ralainirina1,
- Hongying Li1,2,
- Christopher J. Leem1,
- Sara Gorjestani1,
- Gyunghwi Woo1,
- Abraham V. Nguyen1,
- Camila C. Figueiredo1,3,
- Philippe Foubert1,
- Michael C. Schmid1,
- Melissa Pink4,
- David G. Winkler4,
- Matthew Rausch4,
- Vito J. Palombella4,
- Jeffery Kutok4,
- Karen McGovern4,
- Kelly A. Frazer5,6,
- Xuefeng Wu7,
- Michael Karin7,
- Roman Sasik8,
- Ezra E. W. Cohen1,9 &
- …
- Judith A. Varner1,9,10
Naturevolume 539, pages437–442 (2016)Cite this article
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ACorrigendum to this article was published on 14 December 2016
Abstract
Macrophages play critical, but opposite, roles in acute and chronic inflammation and cancer1,2,3,4,5. 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 inhibitors1,2,3,4,5,6,7. 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.
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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.
Author information
Authors and Affiliations
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
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
Dep. Biologia Celular, UERJ, Rio de Janeiro, 20550-013, Brazil
Camila C. Figueiredo
Infinity Pharmaceuticals, Cambridge, 02139, Massachusetts, USA
Melissa Pink, David G. Winkler, Matthew Rausch, Vito J. Palombella, Jeffery Kutok & Karen McGovern
Department of Pediatrics, University of California, San Diego, La Jolla, 92093, California, USA
Kelly A. Frazer
Institute for Genomic Medicine, University of California, San Diego, La Jolla, 92093, California, USA
Kelly A. Frazer
Department of Pharmacology, University of California, San Diego, La Jolla, 92093, California, USA
Xuefeng Wu & Michael Karin
Center for Computational Biology and Bioinformatics, Institute for Genomic Medicine, University of California, San Diego, La Jolla, 92093, California, USA
Roman Sasik
Department of Medicine, University of California, San Diego, La Jolla, 92093, California, USA
Ezra E. W. Cohen & Judith A. Varner
Department of Pathology, University of California, San Diego, La Jolla, 92093, California, USA
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 forFig.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 InformationNature 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 ofIL12A (P = 0.026),IL12B (P = 0.039),IFNG (P = 0.002),CD8A (P = 0.001) andIL6 (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) andPik3cg−/− mice inoculated with LPS (endotoxin).P = 0.05, log-rank test.j, Pro-inflammatory cytokine mRNA expression in bone marrow from wild-type andPik3cg−/− 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 inPik3cg−/− 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 onin vitro MEER cell viability.n, Spontaneous PyMT lung metastases per high-power field (200×) in wild-type andPik3cg−/− 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 andPik3cg−/− 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.
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 fromc.e, Flow cytometry analysis of Ly6G, CCR2, CX3CR1, CD206, CD11c, F4/80 and CD45 expression in myeloid cell populations fromc (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 andPik3cg−/− LLC, PyMT and MEER tumours (n = 3 biological replicates).h, Quantification of CD11b+ myeloid cell populations (P = 0.001;t-test) fromg.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 fromi. All experiments were performed two or more times.b,d,h,j, Data are shown as mean ± s.e.m.
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 andPik3cg−/− 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 fromPik3cg−/− 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 inPik3cg−/− 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 andPik3cg−/− mice.n = 4 biological replicates; ***P < 0.0003;t-test.f, Protein concentration of cytokines in LLC tumours and TAMs from wild-type andPik3cg−/− 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.
Extended Data Figure 4 Effect of PI3Kγ deletion onin vitro macrophage mRNA expression.
a, Relative immune response mRNA expression inPik3cg−/− 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 inin vitro polarized mouse macrophages.n = 3 biological replicates;P = 0.00001.d, Heat map of immune response transcripts in mCSF-, IL4- and IFNγ/LPS-stimulatedPik3cg−/− 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 polarizedPik3cg−/− 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-polarizedPik3cg−/− mouse macrophages.n = 3 biological replicates;P = 0.00001.g, Heat map of differentially expressed chemokine and chemokine receptor mRNAs in polarizedPik3cg−/− mouse macrophages.n = 3 biological replicates;P = 0.00001. All experiments were performed two or more times.
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 asFizz1) andChil3 (also known asYm1) in wild-type andPik3cg−/− IL4-stimulated macrophages (n = 3 biological replicates;P = 0.05;t-test).d, Relative RNA expression of MHC family members in wild-type andPik3cg−/− 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 orPik3cg−/− (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 andPik3cg−/− macrophages.n = 3 biological replicates;P = 0.01;t-test. All experiments were performed two or more times.
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 andPik3cg−/− 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, RelativeArg1 mRNA expression in myeloid cells transfected with constitutively active, membrane-targeted PI3Kγ (Pik3cgCAAX) andMtor,S6ka,Cebpb or control siRNA.n = 3 biological replicates;P = 0.001,t-test.d, Validation of mRNA expression in macrophages expressing siRNAs fromc (n = 3 biological replicates).e, Effect on cytokine mRNA expression in wild-type macrophages transfected withCebpb,Mtor orS6ka 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 inFig. 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 andPik3cg−/− 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.
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 froma.n = 3 biological replicates;P = 0.02; one-sided ANOVA with Tukey’s post-hoc test.c, Quantification of F4/80+ macrophages in livers froma.n = 3 biological replicates;P < 0.005; one-sided ANOVA with Tukey’s post-hoc test.d, Quantification of T cells in tumours froma.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 frome.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 andPik3cg−/− 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 andPik3cg−/− 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 andPik3cg−/− mice.n = 3 biological replicates;P = 0.001;t-test.j, Volumes of LLC lung tumours from wild-type,Pik3cg−/−,CD8−/− andCD8−/−,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 andPik3cg−/− 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 andPik3cg−/− mice (n = 3 biological replicates).m, IFNγ and granzyme B protein expression in T cells froml (n = 3 biological replicates). All data are shown as mean ± s.e.m. and all experiments were performed two or more times.
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 orPik3cg−/− 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 andPik3cg−/− (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 inFig. 3h.f, Quantification of TUNEL+ cells in tumour sections frome.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 orPik3cg−/− 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 andPik3cg−/− 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 andTgfb1 mRNA expression in T cells isolated from LLC tumours grown in wild-type andPik3cg−/− or control- and PI3Kγ-inhibitor-treated mice.n = 3 biological replicates;P = 0.05,t-test).k, Relative mRNA expression ofCd4,Cd8,Gzmb andIfng in control- and PI3Kγ-inhibitor-treated PyMT tumours.n = 3 biological replicates;P = 0.05,t-test.l, Relative mRNA expression ofCd4, Cd8,Gzmb andIfng in wild-type andPik3cg−/− 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.
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 andPik3cg−/− LLC tumours (a) and wild-type andPik3cg−/−in vitro cultured IFNγ/LPS− and IL4-stimulated macrophages (b) (n = 3 biological replicates).c, HPV+ HNSCC tumour volume in female wild-type orPik3cg−/− 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 fromFig. 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.
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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Kaneda, M., Messer, K., Ralainirina, N.et al. PI3Kγ is a molecular switch that controls immune suppression.Nature539, 437–442 (2016). https://doi.org/10.1038/nature19834
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