The E3 ubiquitin ligase Peli1 regulates the metabolic actions of mTORC1 to suppress antitumor T cell responses

Article20 November 2020free access Source DataTransparent process The E3 ubiquitin ligase Peli1 regulates the metabolic actions of mTORC1 to suppress antitumor T cell responses Chun-Jung Ko Chun-Jung Ko orcid.org/0000-0001-6565-7060 Department of Immunology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA Search for more papers by this author Lingyun Zhang Lingyun Zhang Department of Immunology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA Center for Reproductive Medicine, Henan Key Laboratory of Reproduction and Genetics, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, China Search for more papers by this author Zuliang Jie Zuliang Jie Department of Immunology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA Search for more papers by this author Lele Zhu Lele Zhu Department of Immunology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA Search for more papers by this author Xiaofei Zhou Xiaofei Zhou Department of Immunology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA Search for more papers by this author Xiaoping Xie Xiaoping Xie orcid.org/0000-0001-6616-1059 Department of Immunology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA Search for more papers by this author Tianxiao Gao Tianxiao Gao Department of Immunology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA Search for more papers by this author Jin-Young Yang Jin-Young Yang Department of Immunology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA Department of Biological Sciences, Pusan National University, Busan, South Korea Search for more papers by this author Xuhong Cheng Xuhong Cheng Department of Immunology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA Search for more papers by this author Shao-Cong Sun Corresponding Author Shao-Cong Sun [email protected] Department of Immunology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA MD Anderson Cancer Center UT Health Graduate School of Biomedical Sciences, Houston, TX, USA Search for more papers by this author Chun-Jung Ko Chun-Jung Ko orcid.org/0000-0001-6565-7060 Department of Immunology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA Search for more papers by this author Lingyun Zhang Lingyun Zhang Department of Immunology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA Center for Reproductive Medicine, Henan Key Laboratory of Reproduction and Genetics, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, China Search for more papers by this author Zuliang Jie Zuliang Jie Department of Immunology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA Search for more papers by this author Lele Zhu Lele Zhu Department of Immunology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA Search for more papers by this author Xiaofei Zhou Xiaofei Zhou Department of Immunology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA Search for more papers by this author Xiaoping Xie Xiaoping Xie orcid.org/0000-0001-6616-1059 Department of Immunology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA Search for more papers by this author Tianxiao Gao Tianxiao Gao Department of Immunology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA Search for more papers by this author Jin-Young Yang Jin-Young Yang Department of Immunology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA Department of Biological Sciences, Pusan National University, Busan, South Korea Search for more papers by this author Xuhong Cheng Xuhong Cheng Department of Immunology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA Search for more papers by this author Shao-Cong Sun Corresponding Author Shao-Cong Sun [email protected] Department of Immunology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA MD Anderson Cancer Center UT Health Graduate School of Biomedical Sciences, Houston, TX, USA Search for more papers by this author Author Information Chun-Jung Ko1, Lingyun Zhang1,2, Zuliang Jie1, Lele Zhu1, Xiaofei Zhou1, Xiaoping Xie1, Tianxiao Gao1, Jin-Young Yang1,3, Xuhong Cheng1 and Shao-Cong Sun *,1,4 1Department of Immunology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA 2Center for Reproductive Medicine, Henan Key Laboratory of Reproduction and Genetics, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, China 3Department of Biological Sciences, Pusan National University, Busan, South Korea 4MD Anderson Cancer Center UT Health Graduate School of Biomedical Sciences, Houston, TX, USA *Corresponding author. Tel: +1 713 563 3218; E-mail: [email protected] The EMBO Journal (2021)40:e104532https://doi.org/10.15252/embj.2020104532 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Metabolic fitness of T cells is crucial for immune responses against infections and tumorigenesis. Both the T cell receptor (TCR) signal and environmental cues contribute to the induction of T cell metabolic reprogramming, but the underlying mechanism is incompletely understood. Here, we identified the E3 ubiquitin ligase Peli1 as an important regulator of T cell metabolism and antitumor immunity. Peli1 ablation profoundly promotes tumor rejection, associated with increased tumor-infiltrating CD4 and CD8 T cells. The Peli1-deficient T cells display markedly stronger metabolic activities, particularly glycolysis, than wild-type T cells. Peli1 controls the activation of a metabolic kinase, mTORC1, stimulated by both the TCR signal and growth factors, and this function of Peli1 is mediated through regulation of the mTORC1-inhibitory proteins, TSC1 and TSC2. Peli1 mediates non-degradative ubiquitination of TSC1, thereby promoting TSC1-TSC2 dimerization and TSC2 stabilization. These results establish Peli1 as a novel regulator of mTORC1 and downstream mTORC1-mediated actions on T cell metabolism and antitumor immunity. Synopsis The mTORC1 signaling pathway mediates metabolic reprograming and effector function of activated T cells, which is important for antitumor immunity. TSC1 ubiquitination by E3 ubiquitin ligase Peli1 negatively regulates mTORC1 activation and controls glycolytic metabolism and antitumor effector function of T cells. Peli1 negatively regulates activation of the metabolic kinase mTORC1. Peli1 mediates K63 ubiquitination of mTORC1-inhibitory protein TSC1 and promotes TSC1/TSC2 complex stability. Peli1 deletion promotes the metabolic activities of T cells. Peli1 deficiency in mice promotes antitumor immunity. Introduction T cells form a central component of the immune system and are required for host defense against infections and tumorigenesis (Durgeau, Virk et al, 2018). Naïve T cells become activated upon detection of an antigen by the T cell receptor (TCR) and ligation of the costimulatory molecule CD28 (Smith-Garvin, Koretzky et al, 2009; Pollizzi & Powell, 2014). The activated T cells undergo proliferation and subsequently differentiate into effector T cells that participate in the destruction of pathogens or tumor cells. T cell activation involves cascades of signaling events that lead to activation of transcription factors, including NF-κB, NFAT, and AP1 families, and induction of genes involved in T cell proliferation and differentiation (Smith-Garvin et al, 2009; Pollizzi & Powell, 2014). In addition, T cell activation is associated with metabolic reprogramming, which is vital for the proliferation, differentiation, and effector functions of T cells (Aagaard, Lukas et al, 1995; Maciver, Michalek et al, 2013). Resting T cells rely on oxidative phosphorylation (OXPHOS) for energy generation, but activated T cells shift the cellular metabolism toward aerobic glycolysis and also increase the level of OXPHOS (Almeida, Lochner et al, 2016). In tumor microenvironment, T cell metabolism is typically compromised due to nutrient limitation and inhibition by tumor-derived waste products, which is associated with T cell hypofunction (Chang, Curtis et al, 2013; Buck, Sowell et al, 2017; Rivadeneira & Delgoffe, 2018; Li, Wenes et al, 2019). The molecular mechanism that regulates metabolic fitness of T cells is incompletely understood, although the PI3 kinase (PI3K) signaling pathway is known to play an important role (Pollizzi & Powell, 2014). Upon activation by TCR/CD28 costimulation, PI3K activates the downstream kinase AKT, which in turn promotes glucose uptake by inducing translocation of glucose transporter 1 (Glut1) to the plasma membrane (Maciver et al, 2013). In addition, AKT activates the metabolic kinase mammalian target of rapamycin complex 1 (mTORC1) through phosphorylation of the mTORC1 inhibitor TSC2(Chi, 2012). Upon activation, mTORC1 activates two major glycolysis-regulatory transcription factors, c-Myc and HIF1α, thereby inducing the expression of genes encoding various glycolytic enzymes (Lunt & Vander Heiden, 2011; Zeng & Chi, 2014). The mechanism that negatively regulates TCR/CD28-stimulated metabolic pathway is less well understood. Peli1 (also called Pellino 1) is a member of the Pellino family of E3 ubiquitin ligases known to interact with different E2 ubiquitin-conjugating enzymes and conjugate both K63- and K48-linked polyubiquitin chains (Jin, Chang et al, 2012; Moynagh, 2014). While Pellino family members were initially discovered as signaling mediators of IL-1 receptor (IL-1R) and Toll-like receptors (TLRs) in innate immune cells, recent work has demonstrated a crucial role for Peli1 in regulating the activation and tolerance of T cells (Chang, Jin et al, 2011). Peli1-deficient mice display aberrant T cell activation and autoimmune symptoms at the age of 6 months or older, although younger mice do not display obvious autoimmune diseases (Chang et al, 2011). In the present study, we demonstrate that Peli1 has a T cell-intrinsic role in regulating antitumor CD8 T cell responses. Peli1 deletion greatly promotes antitumor immunity in different mouse tumor models. Peli1 negatively regulates the metabolic reprogramming of CD8 T cells by controlling the activation of mTORC1. Our data suggest that Peli1 mediates K63 ubiquitination of the mTORC1 inhibitor TSC1, which in turn facilitates the binding of TSC1 to its partner protein TSC2. Peli1 deficiency impairs TSC1 K63 ubiquitination and attenuates TSC1/TSC2 dimerization, thereby sensitizing these mTORC1 inhibitors for degradation. Results Peli1 deficiency promotes antitumor immunity For studying the in vivo function of Peli1, we employed both germline Peli1 knockout (KO) mice and conditional KO mice (Appendix Fig S1). The Peli1 conditional KO mice were generated by crossing Peli1-flox mice with different types of Cre mice (Appendix Fig S1). To determine the role of Peli1 in regulating antitumor immunity, we challenged the Peli1-KO and wild-type control mice with B16F10 melanoma cells, a poorly immunogenic tumor model extensively used in cancer immunotherapy studies (Wang, Saffold et al, 1998). Remarkably, tumor growth was profoundly reduced in the Peli1-deficient mice as compared with the wild-type control mice (Fig 1A). Similar results were obtained when the mice were challenged with B16 melanoma cells expressing the surrogate antigen chicken ovalbumin (B16-OVA) or the EG7 thymoma tumor cells (Fig 1B and Appendix Fig S2). Figure 1. Peli1 deficiency promotes antitumor immunity A, B. Tumor growth curve (left) and summary of end-point tumor masses (right) of 6-8 week-old wild-type (WT) and Peli1-KO (KO) mice inoculated s.c. with B16F10 (A; WT, n = 7; KO, n = 6) or E.G7 (B; WT, n = 5; KO, n = 5) tumor cells. C, D. Flow cytometry analysis of CD4+ and CD8+ T cells in CD45.2+ TILs and dLN cells from B16F10 tumor-bearing wild-type and Peli1-KO mice, presented as a representative plot (C) and summary graph based on multiple mice (D; WT, n = 6; KO, n = 6). E, F. Flow cytometry analysis of IFNγ-producing CD4+ and CD8+ T cells in TILs, and dLN cells of B16F10 tumor-bearing (day 19) wild-type and Peli1-KO mice, presented as a representative plot (E) and summary graph (F). (WT, n = 6; KO, n = 6). G, H. Flow cytometry analysis of IFNγ+CD8+ T cells in TILs of E.G7 tumor-bearing (day 24) wild-type and Peli1-KO mice, presented as a representative plot (G) and summary graph (H). (WT, n = 3; KO, n = 3) Data information: Data are representative of 3 independent experiments, and bar graphs are presented as mean ± SEM with P values being determined by a two-way ANOVA with Bonferroni correction (left panel of A and B) and two-tailed unpaired Student's t-test (right panel of A and B; D, F, H). *P < 0.05; **P < 0.01; ***P < 0.001. Download figure Download PowerPoint Analysis of immune cells in the B16F10 melanoma model revealed significantly increased frequency and absolute numbers of tumor-infiltrating CD4 and CD8 T cells in the Peli1-KO mice, although the frequency of T cells in draining lymph node was comparable between the Peli1-KO and wild-type mice (Fig 1C and D). Moreover, both the draining lymph node and tumor of Peli1-KO mice contained increased frequencies and absolute numbers of CD8 T cells producing the effector cytokine IFNγ (Fig 1E and F). The tumor of Peli1-KO mice also had an increased number, although not frequency, of IFNγ-producing CD4 T cells (Fig 1E and F). Similar results were obtained with the EG7 tumor model (Fig 1G and H). To assure that the phenotype of the Peli1-KO mice was not due to developmental effect, we repeated the experiments using an inducible KO (iKO) model, in which Peli1 was inducibly deleted in adult mice. To this end, we crossed Peli1-flox mice with CreER mice to generate Peli1fl/flCreER and control Peli1+/+CreER mice, which were then injected with tamoxifen for creating Peli1-iKO and wild-type control mice (Fig EV1A). Compared to the wild-type control mice, the iKO mice displayed a much stronger tumor-suppressing function (Fig EV1B and C). This phenotype was associated with increased numbers of tumor-infiltrating CD4 and CD8 T cells (Fig EV1D). Both the frequency and absolute numbers of IFNγ-producing CD4 and CD8 T cells were also drastically increased in the tumor of Peli1-iKO mice (Fig EV1E). Together, these results suggest an important role for Peli1 in regulating antitumor responses of T cells. Click here to expand this figure. Figure EV1. Inducible deletion of Peli1 in adult mice promotes antitumor immunity A, B. Schematic of experimental design for generating B16F10-bearing Peli1-iKO (iKO) and wild-type (WT) control mice (A) and immunoblot analysis of Peli1 in CD8 T cells of Peli1-iKO and wild-type mice (B). C. Tumor growth curve (left) and summary graph of end-point tumor masses (right) of wild-type (n = 4) and Peli1-iKO (n = 5) mice inoculated s.c. with B16F10 melanoma cells. D, E. Flow cytometry analysis of total (D, WT: n = 4; iKO: n = 4) or IFNγ-producing (E, WT: n = 4; iKO: n = 5) CD4+ and CD8+ T cells in TILs of B16F10 tumor-bearing (day 19) wild-type and iKO mice, presented as a representative plot (left) and summary graph (right). Data information:: Data are representative of 3 independent experiments, and summary data are presented as mean ± SEM with P values being determined by a two-way ANOVA analysis with Bonferroni correction (left panel of C) and two-tailed unpaired Student's t-test (right panel of C; D,E). *P < 0.05; ***P < 0.001. Download figure Download PowerPoint Peli1 functions in T cells to regulate antitumor immunity To determine the T cell-intrinsic function of Peli1, we generated T cell-conditional Peli1 KO (TKO) mice and challenged these mice with B16-OVA melanoma and EG7 thymoma cells. Compared with wild-type control mice, the Peli1-TKO mice displayed a much stronger ability to suppress the growth of B16 tumor (Fig 2A), and similar results were obtained with the EG7 tumor model (Fig 2B). Consistently, the draining lymph node and tumor of the B16-bearing Peli1-TKO mice had a markedly increased frequency and absolute number of CD8 T cells producing IFNγ and granzyme B (Fig 2C and D). Similar results were obtained with the EG7 tumor model (Fig EV2). Furthermore, the Peli1 deficiency also promoted the generation of IFNγ-producing CD4 T cells, although this result was less prominent than that of CD8 T cells (Fig 2C). These results demonstrated a T cell-intrinsic function of Peli1 in the regulation of antitumor immunity and suggested a predominant role for Peli1 in regulating CD8 T cell function. Figure 2. T cell-specific Peli1 deficiency promotes antitumor immunity A, B. Tumor growth curve (left) and summary graph of end-point tumor masses (right) of wild-type (WT) and Peli1-TKO (TKO) mice inoculated s.c. with B16-OVA (A; WT, n = 5; KO, n = 5) or E.G7 (B; WT, n = 6; KO, n = 4) tumor cells. C. Flow cytometry analysis of IFNγ-producing CD4+ and CD8+ T cells in TILs and dLN cells from B16OVA tumor-bearing wild-type and Peli1-TKO mice, presented as a representative plot (upper) and summary graph (lower). (WT, n = 5; KO, n = 5) D. Flow cytometry analysis of GZMB+CD8+ T cells in TILs from B16-OVA tumor-bearing wild-type and Peli1-TKO mice, presented as a representative plot (upper) and summary graph (lower). (WT, n = 5; KO, n= 5) E. Tumor growth curve (left) and summary graph of end-point tumor masses (right) of B16-OVA tumor-bearing wild-type and the indicated Peli1 conditional KO mice. (Treg KO model: WT, n= 5; Treg KO, n = 6; MKO model: WT, n= 3; MKO, n = 5; BKO model: WT, n = 9; BKO, n = 9) Data information: Data are representative of 2 (E) or 3 (A-D) independent experiments, and bar graphs are presented as mean ± SEM. P values are determined by a two-way ANOVA analysis with Bonferroni correction (left panel of A, B, E) and two-tailed unpaired Student's t-test (C, D and right panel of A, B, E). *P < 0.05; ***P < 0.001. Download figure Download PowerPoint Click here to expand this figure. Figure EV2. T cell-specific Peli1 deficiency promotes antitumor immunity in E.G7 tumor model A, B. Flow cytometry analysis of the frequency of IFNγ-producing CD8 effector T cells in tumor-infiltrating lymphocytes of E.G7 tumor-bearing (day 24) wild-type (WT; n = 3) and Peli1-KO (TKO; n = 3) mice, presented as a representative plot (A) and summary graph (B). Data are representative of 3 independent experiments, and summary data are presented as mean ± SEM with P values being determined by two-tailed unpaired Student's t-test. *P < 0.05. Download figure Download PowerPoint To examine whether Peli1 also functions in other cell types, we generated Treg cell-conditional Peli1 KO (Treg KO), myeloid cell-conditional Peli1 KO (MKO), and B cell-conditional Peli1 KO (BKO) mice by crossing the Peli1-flox mice with Foxp3-Cre, Lyz2-Cre, and Cd19-Cre mice, respectively (Appendix Fig S1). In contrast to the T cell-specific Peli1 deficiency, deletion of Peli1 specifically in Treg cells only had a moderate effect on tumor growth (Fig 2E). Furthermore, deletion of Peli1 in myeloid cells had no significant effect on tumor growth and deletion of Peli1 in B cells even moderately promoted tumor growth (Fig 2E). These results suggest that T cells likely represent the major cell type, in which Peli1 functions to mediate antitumor immunity. Peli1 regulates T cell metabolism We have previously shown that Peli1 negatively regulates TCR/CD28-stimulated T cell proliferation and cytokine production (Chang et al, 2011). Peli1 deficiency did not significantly influence the apoptosis of CD8 T cells (Appendix Fig S3). Our previous work suggests that Peli1 plays a role in regulating the stability of an NF-κB family member, c-Rel; however, it has remained unclear whether additional mechanisms are involved in Peli1 function in T cells. In this regard, metabolic reprogramming occurs along with T cell activation and serves as a pivotal mechanism that mediates effector T cell generation and function (Almeida et al, 2016). Naïve T cells mainly use oxidative phosphorylation (OXPHOS) for energy generation; upon activation, they rapidly shift the cellular metabolism toward aerobic glycolysis and also increases OXPHOS (Almeida et al, 2016). Notably, glycolysis promotes the production of T cell cytokines, including IL-2 and IFNγ (Chang et al, 2013; Menk, Scharping et al, 2018). To examine the role of Peli1 in regulating aerobic glycolysis, we performed Seahorse extracellular flux analyses to measure extracellular acidification rate (ECAR) and oxygen consumption rate (OCR), indicators of aerobic glycolysis and oxidative phosphorylation (OXPHOS), respectively (Pearce, Poffenberger et al, 2013). Activation of wild-type OT-I CD8 T cells led to the induction of both ECAR and OCR (Fig 3A and B). Remarkably, under the activation conditions, the Peli1-deficient OT-I CD8 T cells displayed a drastic increase in both the baseline ECAR and the stressed ECAR (maximum glycolytic capacity), suggesting heightened glycolysis (Fig 3A and B). Peli1 deficiency also increased baseline OCR and stressed OCR (maximum respiratory capacity) (Fig 3A and B). These results demonstrate a crucial role for Peli1 in regulating T cell metabolism. Figure 3. Peli1 regulates metabolic reprograming of CD8 T cells A, B. Seahorse analysis of basal (glucose injection) and maximal (oligomycin injection) ECAR or basal (no treatment) and maximal (FCCP injection) OCR in WT and Peli1-KO OT-I CD8 T cells that were either untreated (naïve) or activated with plate-bound anti-CD3 (1 μg/ml) plus anti-CD28 (1 μg/ml) for 16 h. Data are presented as a representative plot (A) or summary graph based on 6 wild-type and 6 KO mice (B). C, D. qRT–PCR analysis of the indicated genes using RNAs isolated from wild-type or Peli1-KO OT-I CD8 T cells stimulated with anti-CD3 and anti-CD28 for 6 h. E. Immunoblot analysis of HK2 and Glut1 in wild-type or Peli1-KO OT-I CD8 T cells that were stimulated with plate-bound anti-CD3 and anti-CD28 for 24 h. Data information: Data are representative of 3 independent experiments, and bar graphs are presented as mean ± SEM with P values being determined by two-tailed unpaired Student's t-test. *P < 0.05; **P < 0.01; ***P < 0.001. Source data are available online for this figure. Source Data for Figure 3 [embj2020104532-sup-0003-SDataFig3.pdf] Download figure Download PowerPoint To examine the mechanism by which Peli1 regulates T cell metabolism, we analyzed the expression of glycolysis-related genes. qRT–PCR analysis revealed that the Peli1-deficient CD8 T cells had a significant increase in the expression level of several glycolysis genes, including glucose transporter 1 (Glut1), hexokinase 2 (Hk2), Pgk1, Eno1, Pkm, Hif1a, and Myc under in vitro activation conditions (Fig 3C and D). Immunoblot analyses also detected upregulated Glut1 and HK2 proteins in the activated Peli1-deficient CD8 T cells (Fig 3E). These results suggest an important role for Peli1 in regulating T cell metabolism. Peli1 negatively regulates mTORC1 activation by TCR/CD28 stimuli The mTORC1 signaling axis is crucial for mediating TCR/CD28-stimulated glycolytic gene expression (Lunt & Vander Heiden, 2011; Yang & Chi, 2012). Interestingly, we found that under homeostatic conditions, the Peli1-deficient CD8 T cells displayed increased mTORC1 activity, as revealed by stronger phosphorylation of mTORC1 target proteins, S6 kinase (S6K), S6, and 4EBP1, in freshly isolated Peli1 KO CD8 T cells compared to the wild-type CD8 T cells (Fig 4A). We next examined the effect of Peli1 deficiency on mTORC1 activation by TCR/CD28 agonistic antibodies. For these studies, we first rested the cells on ice to reduce the steady-state mTORC1 activation (Fig 4B). The Peli1 deficiency promoted mTORC1 activation stimulated by anti-CD3 plus anti-CD28 (Fig 4B and C). Notably, although TCR/CD28-stimulated mTORC1 activation requires AKT, the Peli1 deficiency did not affect AKT activation, as indicated by the comparable AKT phosphorylation at both S473 and T308 in Peli1 KO and WT control T cells (Fig 4B and Appendix Fig S4). These results suggest that Peli1 may regulate mTORC1 activation via an AKT-independent mechanism. Since S473 of AKT is a major target of mTORC2, these findings also suggest that Peli1 is dispensable for regulation of mTORC2. Figure 4. Peli1 deficiency promotes mTORC1 signaling in CD8 T cells A. Immunoblot analysis of the indicated phosphorylated (p-) and total proteins in whole-cell lysates of freshly isolated CD8 T cells from wild-type (WT) or Peli1 KO (KO) mice (6 weeks old). Data are presented as a representative blot (left) and summary graphs of densitometric quantifications of the indicated proteins (presented as phosphorylated/total protein ratio) based on three independent experiments. B, C. Immunoblot analysis of the indicated phosphorylated (p-) and total proteins in whole-cell lysates of wild-type or Peli1-KO OT-I CD8 T cells that were first rested on ice for 15 min and then stimulated in low-serum (1% FBS) medium with anti-CD3 and anti-CD28 for the indicated time periods. D, E. Seahorse analysis of basal and maximal ECAR (D) or OCR (E), as described in Fig 3, using wild-type or Peli1-KO OT-I CD8 T cells that were activated for 16 h with plate-bound anti-CD3 (1 μg/ml) plus anti-CD28 (1 μg/ml) in the presence of solvent control (DMSO), rapamycin (Rapa, 10 nM), or Torin 1 (100 nM). Data are presented as a representative plot (left) and summary graphs (right). F, G. Immunoblot analysis of the indicated phosphorylated (p-) and total proteins in whole-cell lysates of wild-type or Peli1-KO OT-I CD8 T cells that were either not treated (–) or stimulated (+) for 1 h (F) or 24 h (G) with anti-CD3 (1 μg/ml) and anti-CD28 (1 μg/ml) in the presence (+) or absence (–) of rapamycin (10 nM) and Torin 1 (100 nM). H. qRT–PCR analysis of Ifng gene expression in naïve wild-type or Peli1-KO OT-I CD8 T cells that were either not treated (–) or stimulated for 6 h (+) with anti-CD3 plus anti-CD28 in the presence (+) or absence (–) or rapamycin. I–K. Schematic of experimental design (I), tumor growth curve (J), and summary of day 15 tumor weight (K) of B6.SJL mice inoculated with B16-OVA melanoma cells (2 x 105) and adoptively transferred on day 7 with in vitro activated and rapamycin- or DMSO-treated wild-type or Peli1-KO OT-I CD8 T cells (4 × 105). Data information: Data are representative of 2 (J, K) or 3 (A-H) independent experiments and bar graphs are presented as mean ± SEM with P values being determined by two-tailed unpaired Student's t-test (A, D, E, H, K) or two-way ANOVA with Bonferroni correction (J). *P < 0.05; **P < 0.01; ***P < 0.001. Source data are available online for this figure. Source Data for Figure 4 [embj2020104532-sup-0004-SDataFig4.pdf] Download figure Download PowerPoint To determine the functional significance of Peli1-mediated mTORC1 regulation, we tested the involvement of mTORC1 in the hyper-induction of glycolysis in Peli1-deficient CD8 T cells. While the Peli1 KO T cells displayed drastically higher level of ECAR than wild-type control T cells, treatment of T cells with two different mTORC1 inhibitors, rapamycin and Torin, largely blocked the ECAR induction in Peli1 KO T cells and erased the differences between the Peli1 KO T cells and wild-type control T cells (Fig 4D). The mTORC1 inhibitors also attenuated TCR/CD28-stimulated OCR in Peli1-deficient CD8 T cells (Fig 4E). Consistent with the role of metabolism in regulating T cell effector function, inhibition of mTORC1 attenuated the induction of IFNγ and gran-zyme B in activated CD8 T cells (Fig EV3). Parallel immunoblot analyses revealed that the mTOR inhibitor treatment efficiently inhibited TCR/CD28-stimulated phosphorylation of mTORC1 target proteins (Fig 4F). Consistently, the mTOR inhibitors also largely prevented the hyper-induction of metabolism-related proteins, including Glut1, HK1, HK2, Hif1α, and c-Myc, by the TCR/CD28 signals (Fig 4G). Moreover, the TCR/CD28-stimulated hyper-induction of IFNγ in Peli1-deficient T cells was also diminished in the presence of the mTORC1 inhibitor (Fig 4H). We next examined the potential involvement of mTORC1 in regulating the antitumor function of Peli1-deficient CD8 T cells. Since rapamycin regulates mTORC1 signaling and metabolism in both immune cells and tumor cells, we employed an adoptive T cell transfer model using wild-type or Peli1-KO OT1 CD8 T cells treated with rapamycin or solvent control DMSO (Fig 4I). As expected, the Peli1-KO OT1 CD8 T cells were much more potent than wild-type OT1 CD8 T cells in suppressing tumor growth (Fig 4J and K). Moreover, while rapamycin inhibited the antitumor function of both wild-type and Peli1-KO CD8 T cells, this effect was much more profound for the Peli1-KO CD8 T cells (Fig 4J). These results suggest that Peli1 negatively regulates T cell metabolism through controlling mTORC1 activation, thereby modulating the antitumor function of CD8 T cells. Click here to expand this figure. Figure EV3. Hyper-induction of IFNγ and granzyme B in Peli1-deficient CD8 T cells is dependent on mTORC1 A, B. Flow cytometry analysis of intracellular IFNγ (A) and granzyme B (B) in OT-I naïve T cells that were either not treated (NT) or stimulated for 16 h with plate-coated anti-CD3 (1 μg/ml) and anti-CD