Translational Cancer Mechanisms and Therapy

Pik3ip1 Is a Negative Immune Regulator thatInhibits Antitumor T-Cell ImmunityYichen Chen1, Jun Wang2, Xi Wang1, Xinye Li1, Jingjing Song1, Juan Fang1,Xiangqi Liu1,Tao Liu3, DikanWang1, Qunxing Li1, ShuqiongWen1, Da Ma1, Juan Xia1,Liqun Luo4, Song Guo Zheng5, Jun Cui3, Gucheng Zeng4, Lieping Chen2,Bin Cheng1, and Zhi Wang1

Abstract

Purpose:Multiple negative regulators restrict the ability of Tcells to attack tumors. Thisworkdemonstrates the role of PI3K-interacting protein 1 (Pik3ip1) in restraining T-cell responsesand antitumor immunity.

Experimental Design: An anti-Pik3ip1 mAb was generatedto identify the Pik3ip1 expression pattern of hematopoieticcells. Pik3ip1�/� mice and a Pik3ip1 fusion protein weregenerated to investigate the effect of Pik3ip1 on T-cell–mediated antitumor immunity in MC38 and B16-F10 tumormodels. Immunoblotting and confocal microscopy were usedto identify inhibitory effects of Pik3ip1 on T-cell receptor(TCR) signaling. Pik3ip1 expression was quantified, and itsimpact on T-cell function in human tumors was measured.

Results:We demonstrated that Pik3ip1 was predominantlyexpressed on T cells and served as an essential rheostat for

T-cell–mediated immunity. A Pik3ip1 genetic deficiency led toenhanced T-cell responsiveness upon immunization with aneoantigen. Pik3ip1�/� mice exhibited a marked increase inantitumor immunity and were resistant to tumor growth.Furthermore, Pik3ip1 extracellular domain fusion proteinenhancedMC38 tumor growthwas observed.Mechanistically,we found that Pik3ip1 inhibited TCR signaling by mediatingthe degradationof SLP76 throughPik3ip1oligomerization viaits extracellular region. Consistent with the results from themouse models, PIK3IP1 expression correlated with T-celldysfunction in human tumors.

Conclusions: Our data reveal a critical role for Pik3ip1as a novel inhibitory immune regulator of T-cell responsesand provide a potential molecular target for cancerimmunotherapy.

IntroductionIn recent years, the field of cancer immunotherapy has made

substantial progress in improving cancer treatment. It has beenwell recognized that multiple negative checkpoint regulators(NCRs) could restrict the ability of T cells to effectively attack

tumors. Releasing these brakes through antibody blockade, firstwith an anti-CTLA-4 antibody and then with anti-PD-1 and anti-PD-L1 antibodies, has emerged as an exciting strategy for cancertherapy (1–3). Numerous studies have also shown the criticalfunctions in maintaining peripheral tolerance and controllingantitumor immune responses by several other inhibitory mole-cules such as T-cell immunoglobulin domain andmucin domain3 (Tim-3; ref. 4), lymphocyte activation gene 3 (LAG-3; ref. 5), andB- and T-lymphocyte attenuator (BTLA; ref. 6). Thus, the identi-fication of additional NCR pathways could have important ther-apeutic implications.

PI3K/Akt/mTOR pathway is one of the key signaling pathwaysinvolved in T-cell activation and function (7). During T-cellactivation, Src homology 2 domain-containing leukocyte proteinof 76 kDa (SLP76) associates with PI3K subunit p85, leading tothe downstream Akt activation (8). Inhibition of PI3K isoformpromoted robust CD8þ T-cell activation, improved antitumorimmunity, and host survival by enhancing the cytokines produc-tion of cytotoxic lymphocytes (9, 10). PI3K/Akt/mTOR complexhas also been shown to be involved in the differentiation ofvarious immunosuppressive T-cell subsets including Foxp3þ Tregcell and tumor-associated macrophages (11, 12). Thus, specificPI3K/Akt/mTOR-targeted therapies could regulate cellularmetab-olismand immune cell function in cancer andother diseases (13).

PI3K-interacting protein 1 (Pik3ip1), a cell-surface proteinwithan intracellular motif homologous to the PI3K regulatory subunitp85, was reported as a negative regulator of the PI3K path-way (14). It is reported that the overexpression of Pik3ip1 inmouse hepatocytes leads to a reduction in PI3K signaling and the

1Guanghua School of Stomatology, Guangdong Provincial Key Laboratory ofStomatology, Stomatological Hospital, Sun Yat-Sen University, Guangzhou, P.R.China. 2Department of Immunobiology and Yale Cancer Center, Yale University,New Haven, Connecticut. 3MOE Key Laboratory of Gene Function and Regula-tion, State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-senUniversity, Guangzhou, P.R. China. 4Zhongshan School of Medicine, Sun Yat-senUniversity, Guangzhou, P.R. China. 5Department of Internal Medicine, The OhioState University College of Medicine and Wexner Medical Center, Columbus,Ohio.

Note: Supplementary data for this article are available at Clinical CancerResearch Online (http://clincancerres.aacrjournals.org/).

Y. Chen, J. Wang, X. Wang, and X. Li contributed equally to this article.

Z. Wang is a lead contact.

Corresponding Authors: Zhi Wang, Guanghua School of Stomatology,Stomatological Hospital, Guangdong Provincial Key Laboratory of Stomatology,Sun Yat-sen University, Guangzhou, Guangdong 510055, China. Phone:8613829784088; E-mail: wangzh75@mail.sysu.edu.cn; and Bin Cheng,chengbin@mail.sysu.edu.cn

Clin Cancer Res 2019;25:6180–94

doi: 10.1158/1078-0432.CCR-18-4134

�2019 American Association for Cancer Research.

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suppression of hepatocyte carcinoma development (15). The LPKane group first reported that Pik3ip1 plays an inhibitory role inT-cell activation. Ectopic expression of Pik3ip1 in Jurkat or D10 Tcell lines inhibited the activity of an NFAT/AP-1 transcriptionalreporter (16, 17). In addition, both the kringle and p85-likedomain were shown to be important for the inhibitory functionof Pik3ip1 in activated T cells. Using an inducible knockout (KO)mouse model, the authors showed that Pik3ip1�/� T cells exhibitmore robust activation and faster clearance of Listeria monocyto-genes infection than T cells fromwild-type (WT)mice through theinhibition of the PI3K/Akt pathway (16, 17). However, whetherPik3ip1 contributes to antitumor T-cell immunity is largelyunknown.

In this study, we found that Pik3ip1�/� mice were resistant toMC38andB16-F10 tumor growth comparedwith littermates. Theectodomain Pik3ip1 fusion protein promoted MC38 tumorgrowth in vivo. Mechanistic studies further suggested that inaddition to inhibiting the PI3K/Akt pathway, Pik3ip1 could alsoform oligomers upon TCR stimulation to degrade SLP76, thusglobally inhibiting the downstream pathways of TCR signaling,including the ERK1/2 and p38 MAPK pathways. Furthermore,PIK3IP1 expression in tumor infiltrating T cells was correlatedwith impaired functionality in patients with hepatocellular car-cinoma (HCC) andoral squamous cell carcinoma (OSCC). Takentogether, our results identified Pik3ip1 as a novel negativeimmune regulator that might be a potential target for futurecancer immunotherapy.

Materials and MethodsMice

C57BL/6, Rag-1�/� and OT-1 mice were purchased from theModel Animal Research Center of Nanjing University (Nanjing,China). Pik3ip1�/� mice was created in C57BL/6 mice by micro-injection of TALENs in fertilized eggs from Cyagen BiosciencesInc. The mouse Pik3ip1 gene (GenBank accession number:NM_178149.4; Ensembl: ENSMUSG00000034614) is locatedon mouse chromosome 11. Exon 4 was selected as target site.TALENmRNAs (listed in Table S1 in SupplementaryMaterial andMethod) generatedby in vitro transcriptionwere then injected intofertilized eggs for KO mouse productions. The founders weregenotyped by PCR followed DNA sequencing analysis. The pos-

itive founders were breeding to the next generation. Pik3ip1homozygous (þ/þ) C57BL/6 mice (littermates, LT) generatedfrom Pik3ip1 heterozygotes were bred and maintained underconditions identical to those of thePik3ip1�/�mice andwere usedas controls for experiments. All animal experiments wereapproved by the Institutional Animal Care and Use Committeeof the Sun Yat-sen university and performed following local rules.

Tumor modelsThe MC38 murine colon adenocarcinoma cell line and B16-

F10melanoma cell line used in the tumor model were purchasedfrom the ATCC. Cell were passaged a maximum of 4 times, testedfor mycoplasma contamination, and identified by STR before theexperiments. MC38 tumor cells (1,000,000) or B16-F10 tumorcells (1,000,000) were inoculated on the right flank of Pik3ip1�/�

mice and littermates. For protein treatment MC38 tumor model,200 mg Pik3ip1 fusion protein ormouse IgGproteinwere injectedperitoneally starting from day 0 and every 3 days throughout thewhole experiment. To generate chimerism, Rag-1�/� mice werelethally irradiated with a total dose of 10 Gy radiation followedby bone marrow (BM) reconstitution with 2 � 107 cells fromeither Pik3ip1�/� mice and littermates via tail vein approxi-mately 8 hours later. Recipient mice were bled at 7 weeks toanalyze the T-cell constitution. At 8 weeks, MC38 (1,000,000)was inoculated on the right flank of the recipient mice. Alltumors were measured with calipers every 2 days. The tumorvolume was estimated by the formula: (L�W2)/2. Themice wereeuthanized right before the tumors were dissected.

Patients and specimensFresh paired samples of blood, tumor tissue, adjacent normal

tissues were obtained from patients with oral squamous carcino-ma (OSCC) and hepatic cellular cancer (HCC) who underwentcurative resection at the Stomatological Hospital and CancerCenter of Sun Yat-sen University. None of the patients hadreceived anticancer therapy before sampling. All samples wereanonymously coded with local ethical guidelines. The study wasapproved by the institutional review board of StomatologicalHospital and Cancer Center of Sun Yat-sen University and wasconducted in agreement with the Helsinki Declaration. Writteninformed consent was provided by all participants at baseline andduring follow-up.

Production of the Pik3ip1–Ig fusion proteinPik3ip1–mIg was prepared by fusing the coding region of

extracellular domain of Pik3ip1 to the Fc constant region ofmouse IgG2a. The construct was transfected into 293T cells bythe polyethyleneimines (Invitrogen) transfection method andcultured in serum-free DMEM. The supernatant was collected at5 days and the fusion proteinwas purified byHiTrap Protein AHP(GE Healthcare) and dialyzed in LPS-free PBS. The purity andexpected molecular size of fusion protein were confirmed byelectrophoresis on polyacrylamide gels and by MS analysis.

Generation of anti-Pik3ip1 mAbsWistar rats were immunized with purifiedmouse Pik3ip1–mIg

mixed with complete Freud's adjuvant (Sigma Aldrich) andboosted three times with mouse Pik3ip1-mIgG2a in incompleteFreud's adjuvant. Sera from the mice were collected, and theirspecific binding to Pik3ip1was determined by ELISA andby FACSanalysis on Pik3ip1/CHO cells. The splenocytes from mice with

Translational Relevance

The identification of novel negative checkpoint regulatorpathways should have important therapeutic implications.Here we identified a candidate gene named PI3K-interactingprotein 1 (Pik3ip1) as a novel check point regulator in tumorimmunology. Pik3ip1 genetic deficiency leads to enhanced T-cell responsiveness. Pik3ip1�/�mice exhibit a marked increasein antitumor immunity and are resistant to tumor growth.Consistent with results from mouse model, Pik3ip1 expres-sion correlates with T cells dysfunction in tumor patients. Thisstudy provides compelling evidence to uncover a critical rolefor Pik3ip1 as a novel immune inhibitory regulator of T-cellresponses against antigen-specific immune challenges andtumor, thus provide a potential molecular target for cancerimmunotherapy.

Pik3ip1 Is a Negative Immune Regulator

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highest titer of antisera were fused with YB2/0 myeloma cells toproduce hybridoma cells. After several rounds of selection byELISA and FACS, 5 clones, 17C9, 19G3, 22A11, 23G3, 25H3,which consistently stain Pik3ip1/CHO cells, were selected. Theisotype of these clones is rat IgG2a. The culture supernatant ofhybridoma was concentrated and purified by a protein G-Sepharose column (Pierce) and dialyzed in LPS-free PBS.

Peptide immunizationOVA257–264: SIINFEKL and OVA323-339: ISQAVHAAHAEINEA

peptides were synthesized by GenScript. Peptides (100 mg) weredissolved in PBS, mixed with the TLR3 agonist polyI:C (100 mgi.p.; Sigma-Aldrich). Splenocytes were harvested on day 7 post-immunization and restimulated with the relevant peptides incomplete IMDM. IFNg and TNFa producing CD4þ or CD8þ Tcells were analyzed by FACS. IFNg and TNFa production wasanalyzed by ELISA (Invitrogen).

Isolation of mononuclear cells from blood and tissuesPBMCs were isolated by Ficoll density gradient centrifugation

as manufacturer's instruction (TBD). Tumor-infiltrating leuko-cytes (TILs) were obtained as manufacturer's instruction (Milte-nyi). Briefly, fresh tumor tissue were digested in 200 mg/mLcollagenase IV and 50 mg/mL DNase (Sigma-Aldrich) solutionand disassociated in C tube using GentleMACS dissociator(Miltenyi). Cell suspension were performed by a density gradientcentrifugation step to remove erythrocytes and dead cells.

Plasmids transfectionPlasmids forHA- or FLAGorGFP-tagged SLP76, LAT,mPik3ip1

and their fragment were cloned into the pcDNA3.1 vector fortransient expression, and transfected using Lipofectamine 2000according to the manufacturer's instructions (Invitrogen).

In vitro and in vivo T-cell proliferationFor in vitro studies, purified CD3þ T cells (100,000 cells per

well) were cultured in 96-well flat-bottom plates coated with1.25 mg/mL anti-CD3 and 1.25 mg/mL anti-CD28 antibody.Cultures were analyzed on day 3 for Carboxyfluorescein succini-midyl ester (CFSE; Invitrogen) profiles or according to a timecourse as indicated. For in vivo studies, 3 � 106 splenocytes wereseparated from OT-1 transgenic mice, labeled with 10 mmol/LCFSE, were injected to female C57BL/6mice and Pik3ip1�/�micevia tail vein injection (day 0). On day 1, host mice were immu-nized with OVA257–264 peptides (100 mg) emulsified in the rightflank. On day 2, draining lymph node (dLN) cells were harvestedand the percentages of donor T cells were determined by congenicmarker (Va2 and Vb5.1/5.2). Donor CD8þ T-cell proliferationwere analyzed by flow cytometry.

Flow cytometry. Staining cell surface or intracellular targets wasdescribed previously (18). Briefly, single-cell preparations werestained for dead cells using a Zombie NIR Fixable Viability Kit(Biolegend). For intracellular staining, cells were stimulated with50 ng/mL PMA (Sigma-Aldrich) and 5 mg/mL Ionomycin (Sigma-Aldrich) in the presence of GolgiPlug (BD Bioscience). After4 hours, cells were stained for dead cells and surface markersthen fixed prior to intracellular staining for cytokines. Data wereanalyzed using a FACSVerse flow cytometer (BD Biosciences) andusing FlowJo software (TreeStar). All antibodies for FACS werelisted in Table S1 in Supplementary Materials and Methods.

Confocal microscopy. For confocal analysis, purified mouse CD3þ

T cell were stained with rabbit anti-p-SLP76, SLP76, p-LAT,LAT plus rat antimouse Pik3ip1, followed by Alexa Fluor 594–conjugated anti-rat IgGor Alexa Fluor 488–conjugated anti-rabbitIgG. For oligomerization analysis, purified CD3þ T cell from KOor LT were stained with rat antimouse monoclonal Pik3ip1antibody, followed by Alexa Fluor 594–conjugated anti-rat IgG.Then the T cells were incubated with either Pik3ip1 ecto-domainfusion protein or control protein tagged with biotin, followed byAlexa Fluor 488–conjugated streptavidin. Positive cells werequantified using Zen black software or detected by LSM 780confocal microscopy (Zeiss). All antibodies for immunofluores-cence and IHC stainingwere listed in Table S1 and SupplementaryMaterials and Methods.

Immunoprecipitation and immunoblot analysis. For immunoblotanalysis, cell extracts were prepared after transfection or stimu-lation with appropriate ligands and resolved by SDS-PAGE. Forimmunoprecipitation, whole-cell extracts were prepared aftertransfection, followed by incubation overnight with anti-FLAGbeads (Sigma-Aldrich). Beads were washed five times withlow-salt lysis buffer, and immunoprecipitates were eluted with3� SDS Loading Buffer (Cell Signaling Technology) and resolvedby SDS-PAGE. Proteins were transferred to polyvinylidene fluo-ride (PVDF) membranes (Bio-Rad) and further incubated withindicated antibodies. The LumiGlo Chemiluminescent SubstrateSystem (KPL) was used for protein detection. The densitometrywas measured by Image J (NIH) software.

Statistical methodsAll statistical analyses were performed by GraphPad Prism 7.0.

All tests were two-sided and a P value of less than 0.05 wasconsidered significant and represented as �, P < 0.05; ��, P < 0.01;���, P < 0.001, and ����, P < 0.0001.

ResultsPik3ip1 was predominantly expressed on T cells with dynamicchanges in vitro and in vivo

To study protein expression, amouse Pik3ip1-specificmAbwasproduced. Antibody specificity was confirmed by staining CHOcells stably transfected with a plasmid encoding full-lengthPik3ip1 and by loss of staining when Pik3ip1–Ig blocked theantibody (Supplementary Fig. S1).

Using this mAb, Pik3ip1 expression on hematopoietic cellswas analyzed in four central and peripheral mouse hemato-poietic organs (Supplementary Fig. S2). In BM, spleen, lymphnode, and peripheral blood cells, relatively higher expressionof Pik3ip1 was observed on CD4þ and CD8þ T cells, butmarkedly lower expression was detected on natural killer(NK) cells, B cells, CD11bþ monocytes, and CD11cþ dendriticcells (DC; Fig. 1A). Moreover, in spleen, the surface expressionof Pik3ip1 on na€�ve and central memory CD4þ and CD8þ T-cellpopulations was far more obvious than that on Tregs andCD62Llo CD44hi effector memory CD4þ and CD8þ T cells(Fig. 1B).

In addition to assessing freshly isolated cells, we also deter-mined the Pik3ip1 expression on splenic CD4þ and CD8þ T cells,NK cells, B cells, CD11bþ monocytes, and CD11cþ DCs uponin vitro culture with or without activation. Splenocytes werecultured with medium, anti-CD3 (for activating T cells), ConA

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(for activating B cells), or IFNg and lipopolysaccharide (LPS; foractivating CD11bþ monocytes, CD11cþ DCs, and NK cells).Strikingly, Pik3ip1 expression was quickly lost on all CD4þ andCD8þ T cells, including CD62Lhi CD44lo na€�ve T cells, CD62Lhi

CD44hi centralmemory T cells, CD62Llo CD44hi effectormemoryT cells and Tregs, upon in vitro stimulation (Fig. 1B). Pik3ip1

expression did not fluctuate in B cells, NK cells, CD11bþ mono-cytes, or CD11cþ DCs with stimulation (Fig. 1B).

This phenomenon was further confirmed in vivo. OT-1 TCRtransgenic mice were immunized with the cognate antigenOVA257–264 peptide emulsified in complete Freund's adjuvant(CFA). At 24 hours after immunization, cells from the dLN were

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Figure 1.

The expression pattern of Pik3ip1 on hematopoietic cells with and without activation in vitro and in vivo. A, Pik3ip1 expression onmouse hematopoietic cell types,including CD4þ and CD8þ T cells, CD19þ B cells, CD11bþmonocytes, CD11cþDCs, NK cells. Pik3ip1 expression in mouse BM, spleen, lymph node, and peripheralblood was analyzed by flow cytometry. Overlays showed Pik3ip1 (red) or isotype control (black) live singlet-gated events. Means of five samples areshown� SEM. One representative experiment of two is depicted. B, Comparison of in vitro Pik3ip1 expression on hematopoietic cell types with or without (w/o)activation. CD4þ and CD8þ T cells (CD62Lhi CD44lo naive T cells, CD62Lhi CD44hi central memory T cells, CD62Llo CD44hi effector memory T cells, Tregs),CD19þ B cells, CD11bþmonocytes, CD11cþDCs, NK cells, freshly isolated or in vitro cultured for 24 hours with or without activation. CD4þ and CD8þ T cells wereactivated with 1.25 mg/mL plate-bound a-CD3; CD19þ B cells were activated with ConA; and CD11bþmonocytes, CD11cþ DCs, and NK cells were activated with20 ng/mL IFNg and 200 ng/mL LPS. C, Comparison of in vivo expression patterns of Pik3ip1. OT-1 TCR transgenic mice were immunized with 100 mg ofOVA257-264 emulsified in CFA or CFA alone on the flank. Draining LN were collected after 24 hours and analyzed for the expression of Pik3ip1 on CD19þ B cells,CD11bþmonocytes, CD11cþ DCs, NK cells, CD4þ and CD8þ T cells. D, The expression pattern of Pik3ip1 on CD4þ and CD8þ T cells. The line connecting thePBMC, spleen, nLN, dLN, and TILs were from the samemice. The five separate data were from five tumor-bearing mice. One representative experiment of threeis depicted (� , P < 0.05; �� , P < 0.01; ���, P < 0.001 by Student t test).

Pik3ip1 Is a Negative Immune Regulator

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analyzed for Pik3ip1 expression. Pik3ip1 expression on CD4þ

and CD8þ T cells decreased drastically upon immunization withantigen (CFA/OVA) but not immunization with adjuvant alone(Fig. 1C).

Because Pik3ip1 expression was downregulated after stimula-tion, we wondered how the expression levels of Pik3ip1 werealtered under continuous tumor antigen stimulation conditions.We compared the expression levels of Pik3ip1 on CD4þ andCD8þ T cells in samples of leukocytes freshly isolated from thespleen, dLN, nondraining lymph node (nLN), and tumor of axenograft tumor model. Pik3ip1 was expressed in significantlyhigher proportions of CD4þ and CD8þ T cells in the spleen,peripheral bloodmononuclear cell (PBMC) population andnLN,whereas themean percentages of Pik3ip1-expressing T cells in thedLN and TIL population were significantly lower. The expressionpattern of Pik3ip1 on CD4þ and CD8þ T cells followed thepattern of PBMCs, spleen, and nLN > dLN > TILs (Fig. 1D).

Collectively, these data strongly suggest that Pik3ip1 is differ-entially expressed on T cells with a dynamic feature that mightcontribute to the control of antitumor T-cell responses.

Pik3ip1 deficiency enhanced T-cell responses to antigen-specific immune challenges

We first evaluated whether Pik3ip1 deficiency affected T-cellresponses to an acute immune challenge with a neoantigen. Wecompared the cell-mediated immune responses of littermates andPik3ip1�/� mice to two soluble peptides (100 mg), OVA257–264

(MHC class I restricted) or OVA323–339 (MHC class II restricted),mixed with the toll-like receptor (TLR) agonist polyI:C as anadjuvant. On day 7 after immunization, splenocytes were exam-ined for the production of IFNg and TNFa upon restimulationwith the peptides. Significantly increased levels of IFNg and TNFacytokine secretion were observed in cells derived from thePik3ip1�/� mice in response to OVA257–264 and OVA323–339

peptide immunization (Fig. 2A).Furthermore, T-cell proliferation in response to peptide immu-

nization was also analyzed in Pik3ip1�/� mice in vivo. CFSE-labeled na€�ve splenocytes from OT-1 TCR transgenic mice wereadoptively transferred into Pik3ip1�/� mice and littermates andsubsequently activatedwith theOVA257–264 peptide.Weobservedthat the percentages of CD8þ OT-1 cells were significantlyincreased in the Pik3ip1�/� mice compared with the littermates(Fig. 2B).

T-cell proliferation in response to tumor-associated antigenswas then measured in littermates and Pik3ip1�/� mice. Interest-ingly, the proliferation of the Pik3ip1�/� T cells was substantiallyhigher than Pik3ipþ/þ T cells in the presence of the anti-CD3/anti-CD28mAbs after tumor vaccination. This enhanced responsewasalso sustained, as indicated by time-course studies. Culturedsupernatants from Pik3ip1�/� T cells contained increased levelsof IFNg and TNFa from24 to 72 hours after stimulation (Fig. 2C).Therefore, Pik3ip1 expressed on T cells appears to function as aninhibitor of antigen-specific polyclonal TCR stimulation.

Pik3ip1 deficiency promoted antitumor T-cell functions toinhibit tumor growth in vivo

To address whether T-cell dysfunction can be influenced byPik3ip1 in the setting of antitumor immunity, MC38 tumor cellswere injected subcutaneously into littermates or Pik3ip1�/�mice,and their growth was monitored. There was better tumor controlin the Pik3ip1�/� mice than in the littermates, as indicated by

slower growth in the Pik3ip1�/� mice. Additionally, 40% of thePik3ip1�/� mice were tumor free, whereas all littermates hadtumors (Fig. 3A and B). Tumor-infiltrating CD8þ T cells and theiractivation evidenced by Ki67 expression were significantlyincreased in the Pik3ip1�/� mice compared with the littermates(Fig. 3C and D; Supplementary Fig. S3). We also observedsignificantly greater frequencies of multifunctional CD8þ T cellsthat produced IFNg and TNFa or expressed CD107a on the cellsurface in the Pik3ip1�/�mice than in the littermates (Fig. 3E). Tocheck the cytotoxic T-cell (CTL) activity of the Pik3ip1�/� T cells,purified CD8þ T cells isolated from tumor-bearing mouse sple-nocytes were used in a CTL assay against MC38 tumor cells. Asshown in Fig. 3F, MC38-specific killing was significantly promot-ed in the Pik3ip1�/� mice. Although there was a trend towards adecrease in Treg frequency and an increase in myeloid-derivedsuppressor cell (MDSC) and IL17þ Th17 frequency in the tumorsof the Pik3ip1�/� mice, there was significant within-group het-erogeneity, and differences were not statistically significant(Fig. 3G). In contrast to CD8þ T cell, we were unable to detectdifferences in the activation status and effector molecule produc-tion of the tumor-infiltrating CD4þ T cells from the Pik3ip1�/�

and littermate mice (Fig. 3C–E). Moreover, there were no differ-ences in the CD4þ and CD8þ T-cell populations (SupplementaryFig. S4A), activation phenotype (Supplementary Fig. S4B), orcytokine production (Supplementary Fig. S4C) in the spleen,dLN, or peripheral blood from the Pik3ip1�/� and littermatetumor-bearing mice. Moreover, we observed a similar antitumorT-cell effect in Pik3ip1�/� mice using a B16-F10 tumor model(Supplementary Fig. S5A and S5B). More robust T-cell effectorfunctions were observed in Pik3ip1�/� mice than in littermates(Supplementary Fig. S5C and S5D). Collectively, these datasuggest that Pik3ip1 deficiency enhances antitumor responses bypromoting T-cell activation and effector functions.

Pik3ip1 deficiency in hematopoietic cells was sufficient tocontrol tumor growth in vivo

Given that Pik3ip1 is a critical regulator of PI3K, amajor familyof intracellular signal-transducing enzymes involved in a multi-tude of cellular processes (19), although Pik3ip1 expression wasobserved on CD45þ host immune cells and not on CD45� cells,including tumor cells and fibroblasts (Supplementary Fig. S6), itis still necessary to carefully identify the role of Pik3ip1 inimmune regulation to exclude the possibility that a deficiencyin Pik3ip1 expression in endothelial or epithelial cells can impairT-cell trafficking. To evaluate the contributions of Pik3ip1 tohematopoietic and nonhematopoietic cells in MC38 tumorgrowth, we used BM chimeric models in which lethally irradiatedRag-1�/� mice (recipients) were reconstituted with BM fromeither Pik3ip1�/� (BM�/�) or littermate (BMþ/þ) donors. Com-plete myeloablation is accomplished by giving a lethal dose ofradiation followed by the rescue of the immune system with BMfrom the donor mice to prevent lethality and replacement of theimmune system. In this model, Pik3ip1 expression was deficientin the hematopoietic cells of the mice reconstituted with BM�/�,whereas the somatic cells and radiation-resistant cells remainedPik3ip1 sufficient. After the BM transfer, themice were allowed toreconstitute for 8 weeks. The mice were bled at 7 weeks, and theBM�/� and BMþ/þ mice were found to have similar and reason-able levels of CD4þ and CD8þ T cells. At 8 weeks after reconsti-tution, we injected MC38 tumor cells subcutaneously into theBM�/� and BMþ/þ mice and monitored tumor growth (Fig. 4A).

Chen et al.

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We found better tumor control in the mice reconstituted withBM�/� than in those reconstitutedwithBMþ/þ, as indicatedby theslower growth in the BM�/� mice, which is consistent with ourdata from the conventional Pik3ip1�/�mousemodel (Fig. 4B andC). The percentages of tumor-infiltrating CD4þ and CD8þ T cellsand their effector phenotype (CD44hi CD62Llo)were significantlyincreased in the BM�/� mice compared with the BMþ/þ mice(Fig. 4D and E). CD8þ TILs displayed higher baseline surfaceexpression of CD107a and produced more IFNg and TNFa uponPMA and ionomycin stimulation than BMþ/þ cells (Fig. 4F).

Together, these results confirm that the observed deceleration oftumor progression by Pik3ip1 deficiency is solely an immune cell-associated effect.

Pik3ip1 inhibited T-cell activation by mediating thedegradation of SLP76 through Pik3ip1 oligomerizationvia its extracellular region

Two studies previously demonstrated that Pik3ip1 inhibitsT-cell activation by dampening distal NFAT/AP-1 and Akt signal-ing (16, 17). From our results, we found that Pik3ip1 could not

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Genomic ablation of Pik3ip1 enhanced T-cell responses to antigen-specific immune challenges. A, Pik3ip1 deficiency enhanced T-cell responses against acuteimmune challenges. Pik3ip1�/�mice (KO) and littermates (LT; 6 weeks old) were immunized with soluble OVA257–264 and OVA323–339 peptides (100 mg) mixedwith TLR3 agonist polyI:C (100 mg) as an adjuvant. Splenocytes were harvested on day 7 postimmunization and restimulated with the relevant peptides. Theproduction of IFNg and TNFa by CD4þ and CD8þ T cells were analyzed by FACS and the production of IFNg and TNFa by splenocytes were analyzed by ELISA.B, CFSE-labeled OT1 spleen cells were transferred into Pik3ip1�/�mice and littermates that were immunized with OVA257–264 (100 mg) mixed with TLR3 agonistpolyI:C (100 mg) as an adjuvant. Two days later, LN cells were harvested for flow cytometry analysis to determine the proliferation of CD8þ T cells. The resultsshowed that the absence of Pik3ip1 promoted the proliferation of CD8þ T cells in Pik3ip1�/�mice. C,MC38 tumor cells (1,000,000) were inoculated into the flankof Pik3ip1�/� mice and littermates on day 0. On day 14, Bulk purified littermates and Pik3ip1�/� T cells from spleen were harvested to determine the proliferationof CD4þ and CD8þ T cells. The results showed that the proliferation of Pik3ip1-deficient T cells in response to anti-CD3/CD28 were substantially higher than that ofthe littermates. This enhanced response was also sustained compared with that of littermate CD4þ and CD8þ T cells, as indicated by time-course studies. Culturedsupernatants from anti-CD3/CD28 stimulated Pik3ip1�/� T cells contained increased levels of IFNg , TNFa from 24 to 72 hours. One representative experiment oftwo is depicted (�, P < 0.05; �� , P < 0.01; ��� , P < 0.001; ���� , P < 0.0 001 by Student t test).

Pik3ip1 Is a Negative Immune Regulator

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Knockout of Pik3ip1 inhibited tumor growth in mouse MC38model. MC38 tumor cells (1,000,000) were inoculated into the flank of Pik3ip1�/�mice andlittermates on day 0. Tumor size was measured by a caliper every 3 days. Tumors were harvested on day 25. Representative in situ images and gross appearanceof tumor mass (A) and kinetics of tumor volumes (B) of Pik3ip1�/�mice and littermates postinjection were shown. C and D, Tumor-infiltrating CD4þ and CD8þ

T cells populations and activation status evidenced by Ki67 expression were quantified. E, Tumor-infiltrating CD4þ and CD8þ T cells were stimulated with PMA(10 ng/mL) and ionomycin (0.5 mg/mL) for 5 hours. Cytokine production (IFNg , TNFa) and surface mobilization of CD107a were quantified. F, To check the CTLactivity in Pik3ip1 deficient T cells, purified CD8þ T from the tumor bearing mice splenocytes were used in CTL assay against MC38 tumor cells. MC38 specifickilling were significantly promoted in Pik3ip1�/�mice.G, Tumor-infiltrating Th17, Tregs, and MDSCwere identified by flow cytometry. The data are representativeof three independent experiments. Means of five samples are shown� SEM. One representative experiment of two is depicted (� , P < 0.05; ��, P < 0.01 byStudent t test).

Chen et al.

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Pik3ip1 deficiency on hematopoietic cells is sufficient to control tumor growth in MC38model. A, Experimental schema detailing the BM chimeric constructionand tumor inoculation protocol. Lethally irradiated Rag-1�/�mice (recipients) were reconstituted with BM from either Pik3ip1�/�mice (BM�/�) or littermates(BMþ/þ) donors. After BM transfer, mice were allowed to reconstitute for 8 weeks. At 8 weeks after reconstitution, we injected MC38 tumor cells (1,000,000)subcutaneously into BM�/� and BMþ/þmice andmonitored their growth. Tumor size was measured by a caliper every 2 or 3 days. Tumors were harvested onday 30. Representative in situ images and gross appearance of tumor mass (B) and kinetics of tumor volumes (C) of BM�/� and BMþ/þmice postinjection wereshown. D and E, Tumor-infiltrating CD4þ and CD8þ T cells populations and activation status evidenced by CD44 CD62L expression were quantified. F, Tumor-infiltrating CD4þ and CD8þ T cells were stimulated with PMA (10 ng/mL) and ionomycin (0.5 mg/mL) for 5 hours. Cytokine production (IFNg , TNFa) and surfacemobilization of CD107awere quantified. One representative experiment of two is depicted (� , P < 0.05; �� , P < 0.01 by Student t test).

Pik3ip1 Is a Negative Immune Regulator

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Pik3ip1 inhibited T cells activation by mediating degradation of SLP76 through oligomerization in a proteasome-dependent manner. A, Splenic T cells wereincubated on ice with a-CD3/CD28, and were then stimulated for 2, 5, and 15 minutes at 37� with hamster IgG. The total protein and phosphorylation status ofSLP76, LAT, and Akt was examined by immunoblot analysis. B, The protein densitometry analysis for three independent experiments. (Continued on thefollowing page.)

Chen et al.

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only inhibit Akt signaling but also inhibit other downstream TCRsignaling pathways including ERK1/2 and p38 MAPK (Fig. 5Aand B). To determine whether and how Pik3ip1 could impactproximal upstream TCR signaling in primary T cells, we purifiedsplenic T cells from Pik3ip1�/� and littermate tumor-bearingmice, stimulated these cells with anti-CD3/anti-CD28 mAbsand examined the upstream adaptors LAT and SLP76. LAT is aproximal signaling adaptor that is phosphorylated upon TCRstimulation and forms a complex with multiple signalingmolecules, including SLP76 and phospholipase C (PLC)-g1 (20).Compared with that in the Pik3ip1þ/þ T cells, the phosphorylationof SLP76 in the Pik3ip1�/� T cells was markedly enhanced,which was maximally apparent at 5 minutes after stimulation(Fig. 5A and B).

And we also noticed that the phosphorylation and theprotein amount of total SLP76 but not LAT increased consid-erably in the Pik3ip1�/� T cells upon stimulation with anti-CD3/anti-CD28 mAbs (Fig. 5A and B). We next observed thatPik3ip1 interacted with both LAT and SLP76 in mouse T cellsthrough confocal microscopy analysis (Fig. 5C). These resultsindicated that Pik3ip1 might inhibit TCR signaling by medi-ating the degradation of SLP76. To test this hypothesis, weoverexpressed SLP76 or LAT with increasing amounts ofPik3ip1 and found that Pik3ip1 specifically degraded SLP76but not LAT (Fig. 5D; Supplementary Fig. S7A). We nextinvestigated the molecular mechanisms underlying Pik3ip1-mediated SLP76 degradation. Two major systems that euka-ryotic cells use for protein clearance are the ubiquitin–proteasome and autophagy–lysosome pathways (21, 22). Toidentify which degradation system dominantly regulates thedegradation of SLP76, we examined the protein stability ofSLP76 using pharmacologic approaches. We observed that theproteasome inhibitor MG132 but not the autophagic-sequestration inhibitor 3-methyladenine (3-MA) or the lyso-somal-acidification inhibitor chloroquine (CQ) enhanced theprotein level of SLP76 (Fig. 5E; Supplementary Fig. S7B),indicating that the protein stability of SLP76 was controlledby Pik3ip1 through a proteasome-dependent pathway.

Interestingly, we found that the extracellular region ofPik3ip1, not its intracellular region, led to the degradationof SLP76 (Fig. 5F; Supplementary Fig. S7C). Because manytransmembrane proteins, such as TLR4, and some membraneproteins undergo oligomerization to achieve their activeform (23–25), we wondered whether Pik3ip1 also needs toform oligomers via its extracellular region to degrade SLP76.Indeed, we found that Pik3ip1 extracellular region could formoligomers (Fig. 5G). In addition, we observed that Pik3ip1extracellular regions could induce the oligomerization ofPik3ip1 (Fig. 5H), suggesting that the interaction betweenPik3ip1 extracellular regions plays a critical role in affectingthe activation and function of Pik3ip1.

A Pik3ip1 ecto-domain fusion protein altered the cellularcomposition of the tumor-immune microenvironment andenhanced MC38 tumor growth

Based on our mechanistic studies, we hypothesized that aPik3ip1 ecto-domain fusion protein (Pik3ip1 extracellularregion) could be an antagonist of T-cell activity. Therefore, weassessed the antitumor response after generating a Pik3ip1 fusionprotein consisting of the extracellular domain of Pik3ip1 fusedwith the Fc region of a mouse IgG molecule. MC38 tumor cellswere injected subcutaneously into littermates, and tumor growthwas monitored. The tumor microenvironment (TME) plays acrucial role in suppressing tumor-specificT-cell responses (26,27).Pik3ip1 fusion protein treatment significantly promoted tumorgrowth in the MC38 model (Fig. 6A and B) and altered the TILcomposition. Pik3ip1-Ig administration decreased the percentageof tumor-infiltrating CD4þ T cells (Fig. 6C; SupplementaryFig. S8). Within the CD4þ TIL population, decreased frequenciesof Foxp3þ Tregs and IL-17þ Th17 cells were found in the Pik3ip1–Ig treatment group (Fig. 6D). The development of activatedphenotypes by tumor-infiltrating CD8þ T cells and the produc-tion of effector molecules by both CD4þ and CD8þ tumor-infiltrating T cells were strongly suppressed by Pik3ip1–Ig, asevidenced by the decreased numbers of CD44hi CD62Llo andIFNgþ TNFaþ effector T cells in the Pik3ip1–Ig treatment group(Fig. 6E and F). Our data indicate that the Pik3ip1–Ig fusionprotein can alter the cellular composition of the TMEand functionas an antagonist of antitumor immunity by decreasing T-cellactivation and effector molecule production. As T-cell exhaus-tion is linked to the expression of multiple inhibitorymolecules (28–30), we examined PD-1, and LAG-3 expressionon T cells in tumor tissue. PD-1 and LAG-3 expression wassignificantly increased in the Pik3ip1–Ig group compared withthe control group (Fig. 6G). Although the percentages of bothTh17 cells and Tregs in the CD4þ T-cell population were found tobe decreased in the TME in the Pik3ip1–Ig treatment group, morestudies are needed to determine the roles of these cells in theantitumor T-cell response.

PIK3IP1 marked a dysfunctional T-cell phenotype in patientswith OSCC and HCC

To further define the phenotypic and functional features of thephenotype of T cellswith differentially expressed PIK3IP1, PBMCsand paired TILs from patients with OSCC and HCC were stainedwith a panel of activation markers. We observed that PIK3IP1hi

T cells exhibited a PD-1hi HLA-DRhi CD62Lhi CD28hi phenotype(Fig. 7A and B; Supplementary Fig. S9). Furthermore, whenstimulating these T cells from the peripheral blood and tumortissue, the PIK3IP1hi T cells were obviously incapable of produc-ing TNFa and IFNg (Fig. 7C).Moreover, the PIK3IP1hi T cells fromtheperipheral blood showed a lower proliferative capacity in bothsets of patients (Fig. 7D). Together, these data indicate that

(Continued.) C, Confocal microscopy of mouse T cells showed colocalization of SLP76, p-SLP76, LAT, p-LAT, and Pik3ip1 in the indicated samples (scale bar,5 mm). D and E, Immunoblot analysis of extracts of 293T cells transfected with plasmids for HA-SLP76 and Flag-mPik3ip1 and treated with MG-132 (10 mmol/L),3-MA (10 mmol/L), or CQ (50mmol/L) for 6 hours. F, HEK293T cells were transfected with plasmids expressing FLAG-mPik3ip1 WT or mutant forms togetherwith HA-SLP76 and cell extracts were collected for immunoblot (the full-length, Flag-mPik3ip1-CDS; the extracellular fragment, Flag-mPik3ip1-Ext; theintracellular fragment, Flag-mPik3ip1-N). G, HEK293T cells were transfected with plasmids expressing FLAG-mPik and GFP-mPik. Whole-cell lysates wereharvested for immunoprecipitation (IP) with anti-FLAG beads and immunoblot analysis with anti-GFP. WCL, immunoblot analysis of whole-cell lysates without IP(throughout). H, Live confocal microscopy image of CD3þ T cells from Pik3ip1�/�mice and littermates were stained with Pik3ip1 mAb before incubating withPik3ip1 ecto-domain fusion protein (mPik-Ext) or control-Ig tagged with biotin. Arrow indicates the colocalization of fusion protein and Pik oligomers. Green:mPik-Ext-biotin; red: membrane pik3ip1 on T cells; blue: DAPI. Scale bar, 5 mm (� , P < 0.05; �� , P < 0.01 by Student t test).

Pik3ip1 Is a Negative Immune Regulator

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L26D

C

CD44

Pik3ip1-Ig Control-Ig

14.2

0 103

104

105

0

-102

102

103

104

105

27.8

0 103

104

105

0

-102

102

103

104

105

3pxoF

CD4

Pik3ip1-Ig Control-Ig

2.46

0 103

104

105

0

102

103

104

105

5.09

0 103

104

105

0

102

103

104

105

a71LI

CD4

Pik3ip1-Ig Control-Ig

5.00 12.5

22.560.0

0 103

104

105

0

102

103

104

105 3.30 46.4

13.436.9

0 103

104

105

0

102

103

104

105

NFI

Pik3ip1-Ig Control-Ig

30.9 22.1

3.9543.1

0 103

104

105

0

102

103

104

105 10.1 58.6

7.3824.0

0 103

104

105

0

102

103

104

105

NFI

Pik3ip1-Ig Control-Ig

A B

C

D E

F

0 5 10 15 200

200

400

600

800

Days

Tum

orvo

lum

e(m

m3 ) Pik3ip1-IgControl-Ig

***

Pik3ip1

-Ig

Contro

l-Ig0

10

20

30

40

50

CD

4+am

ong

CD

3(%

) **

Pik3ip1

-Ig

Contro

l-Ig0

20

40

60

80

CD

8+am

ong

CD

3(%

) N.S.

Pik3ip1

-Ig

Contro

l-Ig0

10

20

30

Foxp

3+

amon

gC

D4

(%)

*

Pik3ip1

-Ig

Contro

l-Ig0

2

4

6

8

IL17

+am

ong

CD

4(%

)

*

Pik3ip1

-Ig

Contro

l-Ig40

50

60

70

80

90

CD

44hi

cd62

lloam

ong

CD

4(%

)

N.S.

Pik3ip1

-Ig

Contro

l-Ig0

20

40

60

80

100

CD

44hi

cd62

lloam

ong

CD

8(%

)

**

Pik3ip1

-Ig

Contro

l-Ig0

20

40

60

80

IFN

+TN

F+

amon

gC

D8

(%)

*

Pik3ip1

-Ig

Contro

l-Ig0

20

40

60

IFN

+TN

F+

amon

gC

D4

(%)

*G

Pik3ip1

-Ig

Contro

l-Ig0

10

20

30

40

50

LAG

-3+

amon

gC

D4

(%)

***

Pik3ip1

-Ig

Contro

l-Ig0

20

40

60

80

100

LAG

-3+

amon

gC

D8

(%)

**

Pik3ip1

-Ig

Contro

l-Ig0

20

40

60

80

100

PD-1

+am

ong

CD

8(%

) **

Pik3ip1

-Ig

Contro

l-Ig0

20

40

60

PD-1

+am

ong

CD

4(%

) ***

Figure 6.

Pik3ip1 ecto-domain fusion protein altered the TME and promoted the tumor progression in MC38model. A,A Pik3ip1 fusion protein consisting of theextracellular domain of Pik3ip1 fused with the Fc region of the mouse IgG was generated. MC38 tumor-bearing mice (n¼ 8) were treated every 3 days from day 0throughout the duration of the experiment. Tumor size was measured with a caliper every 2 days. Representative in situ images and gross appearance of tumormass were shown. B, Kinetics of tumor volumes in mice with treatment of indicated proteins. Pik3ip1 fusion protein altered the TME. Tumors were harvested andCD45þ TILs were analyzed for (C) Tumor-infiltrating CD4þ and CD8þ T cells populations. (D) Tumor-infiltrating Foxp3þ Tregs and Th17 by flow cytometry. E, Theactivation status of CD4þ and CD8þ T cells evidenced by CD44hi CD62Llo were identified. F, Cytokine production (IFNg , TNFa) were quantified. G, Exhaustionreceptors PD-1 and LAG-3 were examined on tumor-infiltrating T cells. The data are representative of three independent experiments. Data are represented asmean� SEM (� , P < 0.05; �� , P < 0.01; ���� , P < 0.0001 by Student t test).

Chen et al.

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A

PIK3IP1

HCC Blood HCC TIL

C

D

OSCC Blood OSCC TIL

PIK3IP1

CD

28C

D62

LH

LA-D

RP

D-1

PIK3IP1

HCC Blood HCC TIL

OSCC Blood OSCC TIL

B

IL10GZMB

IL2IL17IL22IL6

IFNγTNFαCD1aCD25

CD107aCD27CD54CCR7CD1b

HLA-DRCD28CD44

CD62LCD80PD-1

HLA-ABCTIM3

OSCC Blood OSCC TIL

20

40

60

80

PIK3IP1- PIK3IP1dim PIK3IP1hi

IL10GZMB

IL2IL17IL22

IL6IFNγ

TNFαCD1aCD25

CD107aCD27CD54CCR7CD1b

HLA-DRCD28CD44

CD62LCD80PD-1

HLA-ABCTIM3

HCC Blood

PIK3IP1- PIK3IP1dim PIK3IP1hi

HCC TIL

OSCC Blood OSCC TIL HCC Blood HCC TIL

0 200 400 600 800 1,00030

50

70

90

OSCC CD8

PIK3IP1 MFI

CFS

Elo

w%

0 200 400 600 800 1,00030

50

70

90

OSCC CD4

PIK3IP1 MFI

CFS

Elo

w%

200 250 300 350 40060

70

80

90

100

HCC CD4

PIK3IP1 MFI

CFS

Elo

w%

200 250 300 350 400 45060

70

80

90

100

HCC CD8

R2 = 0.4492P = 0.0171

R2 = 0.7707P = 0.0002

R2 = 0.3358P = 0.0483

R2 = 0.7072P = 0.0006

PIK3IP1 MFI

CFS

Elo

w%

Figure 7.

PIK3IP1 correlated with impaired T-cell antitumor function in patients with HCC and OSCC. A, Flow cytometry analysis was performed to determine thephenotypic characteristics of the T cells isolated fromOSCC and HCC samples (paired blood and tumor tissue). Data represent mean of at least five independentexperiments (N¼ 8), and triangles showed surface markers which differentially expressed by PIK3IP1. B, Representative dot plots for four surface markers whichstrongly expressed by PIK3IP1hi T cell in blood and TILs (N¼ 8). C, Representative dot plots for TNFa and IFNg production expressed by T cells from peripheralblood and TIL (N¼ 8).D, The correlation analysis between CFSE-low T cell and PIK3IP1 expression (MFI). Linear regression was used to determine the associationbetween PIK3IP1 expression and CFSE-low percentages in more than five independent experiments (N¼ 10–12).

Pik3ip1 Is a Negative Immune Regulator

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PIK3IP1 positivity identifies a less activated T-cell phenotypeand is correlatedwith impaired antitumor T-cell effector functionsand proliferation in patients with cancer.

DiscussionThe results presented here are the first study ever to describe the

function and expression of Pik3ip1 as a negative regulator ofantitumor T-cell response. Pik3ip1 exerts immunosuppressiveeffects on T cells in vitro and in vivo and could be an importantmediator in controlling T-cell responses against acute challengesand cancer. Thus, we propose that Pik3ip1 is a promising newtarget for cancer immunotherapy.

Pik3ip1 was predominantly expressed on all T-cell subpopula-tions, especially na€�ve and central memory CD4þ and CD8þ

T cells, indicating that Pik3ip1 appears to track with the maturityof T cells (Fig. 1). The expression pattern of Pik3ip1 distinguishesit from other NCR molecules, such as PD-1, PD-L1, and PD-1H (31–33). The steady-state expression of Pik3ip1 was largelyrestricted to T cells, whereas that of other NCRs, such as PD-1H, islargely restricted to hematopoietic cells and is highest on bothantigen-presenting cells (APCs; macrophages and myeloid DCs)and CD4þ T lymphocytes (33). In contrast to CTLA-4 and PD-1,Pik3ip1 exhibited downregulated expression on resting T cellsafter short-term in vitro culture. Our observation agrees with aprevious study by the LP Kane group (16), which showed thatPik3ip1 surface expression is downmodulated in T cells to allowT-cell activation in an in vitro D10 T-cell activation model. Thus,we further explored the expression pattern of Pik3ip1 in differentT-cell subsets in an in vivo antigen-specific activationmodel aswellas a mouse tumor model, and checked the expression statuses ofother hematopoietic cell compartments. The antigen-dependentdecreasing expression pattern by Pik3ip1might reflect a necessaryrole for the lymphoid tissuemicroenvironment inmaintaining orregulating Pik3ip1 expression, but not an outcome of a wide-spectrum T-cell activation. However, when continuous tumorantigen stimulationwas encountered, a pattern of Pik3ip1 expres-sion on T cells following decrease from the peripheral lymphorgans to the TME (Fig. 1D), which could reflect the necessity andcomplexity of the regulation by transcription factors or cytokinesin maintaining Pik3ip1 expression in the TME.

Although it is shown that the infiltration of a large number ofTregs into a tumor is often associated with a poor prognosis.However, the role of Tregs in the tumor-immune microenviron-ment is associated with their subgroups. It has been reported thatthe presence of CD45RA� Foxp3lo CD25lo Treg cells may indicatebetter prognosis in tumors (34). In Fig. 6D, althoughwe observeda decreased number of Tregs in Pik3ip1 fusion protein treatmentgroup, we also reportedmore effector T cells and amore activatedT-cell phenotype. We speculate whether Pik3ip1 affecting thespecific Tregs differentiation. To elucidate the function of Pik3ip1in those Tregs, future studies using chimericmice, Foxp3-cremice,or a Treg adoptive transfer model are required.

PI3K/Akt/mTOR pathway has been shown to regulate tumorimmunosuppressive environment through tumor-intrinsic andimmune-intrinsic effects. Within the TME, PI3K isoform inhibi-tors have been shown to promote CTL infiltration through lim-iting immunosuppressive cytokines/chemokines or immunecheckpoint ligands expression by tumor cell (35, 36). On theother hand, PI3K inhibition could attenuate na€�ve T-cell differ-entiation into Th effector lineages or promote Treg expansion.

Inhibitors of PI3K reduced tumor burden through limitingrecruitment of immunosuppressive components includingMDSCs and Treg cells in tumors (11, 37, 38). In this study, werevealed that Pik3ip1, a critical regulator of PI3K, serves as anessential rheostat for T-cell–mediated immunity, which sets thethreshold for T-cell responses under homeostatic conditions andneoantigen exposure, especially tumor antigen stimulation. Asimilar subtle mechanism has been reported for other moleculessuch as Itk and TIPE2, which serve as modulators to critically finetune the T-cell response (39, 40). Furthermore, given that PI3K is amajor family of intracellular signal-transducing enzymesinvolved in amultitude of cellular processes, BM chimericmodelswere used to carefully select the role of Pik3ip1 in immuneregulation. These results confirmed that Pik3ip1 deficiency decel-erated tumor progression via solely an immune cell-associatedeffect, which could provide a new target for immunotherapeuticstrategies.

A previous study by the LP Kane group showed enhancedactivation of Akt and pS6 in Pik3ip1�/� mice compared withlittermates following TCR activation (16). pS6 is downstream ofthe PI3K/Akt/mTOR pathway, which is often related to cellproliferation and survival. Interestingly, we found several otherdownstream pathways, such as the ERK1/2 and p38 MAPK path-ways, in addition to the Akt signaling pathway could inhibited byPi3kip1. Suggesting that Pik3ip1 may have a global effect on theregulation of TCR signaling by interacting with certain upstreamproximal adaptors including SLP76. SLP76 has been reported tohave a remarkable function in the development or function ofT cells. Sixty percent of SLP76-deficient mice die during theperinatal period (41), and transgenic expression of SLP76 in theT-cell compartment rescues T-cell development (42). Our datademonstrated that Pik3ip1 can directly interact with SLP76 tomediate SLP76 degradation, thus acting as a brake for TCRsignaling. Intriguingly, we found that the inhibitory function ofPik3ip1 required Pik3ip1 oligomerization. Upon interactingthrough its extracellular regions, Pik3ip1 formed oligomers todegrade SLP76. This working model further validated that aPik3ip1 extracellular domain–IgG fusion protein can modulatethe cellular composition of the tumor-immune microenviron-ment and enhance MC38 tumor growth. Because Pik3ip1 can beexpressed in many types of tumor cells and tumor-infiltratingT cells, tumor cells might inhibit T-cell functions through aphysical interaction among Pik3ip1 molecules on both cell sur-faces. Thus, developing an anti-Pik3ip1 antibodymight be anoth-er potentialmethod for future cancer treatment. Correspondingly,PIK3IP1 expression also correlated with T-cell dysfunction inhuman tumors.

Collectively, our findings reveal a critical negative regulatoryrole for Pik3ip1 in antitumor T-cell responses by degrading SLP76via oligomerization. Our findings identify Pik3ip1 as a potentialmolecular target for cancer diagnosis or immunotherapy.

Disclosure of Potential Conflicts of InterestL. Chen reports receiving commercial research grants from and holds

ownership interest (including patents) in NextCure and Tayu Biotech, is aconsultant/advisory board member for NextCure, Vcanbio, GenomiCare, andZai Lab. No potential conflicts of interest were disclosed by the other authors.

Authors' ContributionsConception and design: J. Wang, J. Cui, L. Chen, B. Cheng, Z. WangDevelopment of methodology: Y. Chen, J. Wang, J. Song, J. Fang, X. Liu, Q. Li,S. Wen, J. Xia, L. Luo

Chen et al.

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Acquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): Y. Chen, J. Song, J. Fang, X. Liu, T. LiuAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): Y. Chen, J. Wang, X. Wang, X. Li, J. Song, J. Fang,D. Wang, D. Ma, L. ChenWriting, review, and/or revision of the manuscript: J. Wang, J. Cui, B. Cheng,Z. WangAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): J. Wang, X. Wang, Q. Li, S. Wen, S.G. Zheng,J. CuiStudy supervision: J. Wang,Other (performed the experiments and analyzed data): X. WangOther (performed survival data analysis): X. LiuOther (contributed resources): G. Zeng

AcknowledgmentsThis project was supported by grants from National Natural Science

Foundations of China (Nos. 81772896, 81630025, 81602383) and Scienceand Technology Planning Project of Guangzhou City of China (No.2017004020102).

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received December 26, 2018; revised April 19, 2019; accepted July 24, 2019;published first July 26, 2019.

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