Blocking interaction between SHP2 and PD‐1 denotes a novel opportunity for developing PD‐1 inhibitors

Article11 May 2020Open Access Source Data Blocking interaction between SHP2 and PD-1 denotes a novel opportunity for developing PD-1 inhibitors Zhenzhen Fan Zhenzhen Fan MOE Key Laboratory of Tumor Molecular Biology and Key Laboratory of Functional Protein Research of Guangdong Higher Education Institutes, Institute of Life and Health Engineering, Jinan University, Guangzhou, China Search for more papers by this author Yahui Tian Yahui Tian MOE Key Laboratory of Tumor Molecular Biology and Key Laboratory of Functional Protein Research of Guangdong Higher Education Institutes, Institute of Life and Health Engineering, Jinan University, Guangzhou, China Search for more papers by this author Zhipeng Chen Zhipeng Chen MOE Key Laboratory of Tumor Molecular Biology and Key Laboratory of Functional Protein Research of Guangdong Higher Education Institutes, Institute of Life and Health Engineering, Jinan University, Guangzhou, China Search for more papers by this author Lu Liu Lu Liu MOE Key Laboratory of Tumor Molecular Biology and Key Laboratory of Functional Protein Research of Guangdong Higher Education Institutes, Institute of Life and Health Engineering, Jinan University, Guangzhou, China Search for more papers by this author Qian Zhou Qian Zhou MOE Key Laboratory of Tumor Molecular Biology and Key Laboratory of Functional Protein Research of Guangdong Higher Education Institutes, Institute of Life and Health Engineering, Jinan University, Guangzhou, China Search for more papers by this author Jingjing He Jingjing He Sun Yat-Sen University Cancer Center, Guangzhou, China Search for more papers by this author James Coleman James Coleman orcid.org/0000-0001-7430-2587 Biomedical Research Centre, Norwich Medical School, University of East Anglia, Norwich Research Park, Norwich, UK Search for more papers by this author Changjiang Dong Changjiang Dong orcid.org/0000-0002-7020-5648 Biomedical Research Centre, Norwich Medical School, University of East Anglia, Norwich Research Park, Norwich, UK Search for more papers by this author Nan Li Nan Li MOE Key Laboratory of Tumor Molecular Biology and Key Laboratory of Functional Protein Research of Guangdong Higher Education Institutes, Institute of Life and Health Engineering, Jinan University, Guangzhou, China Search for more papers by this author Junqi Huang Junqi Huang MOE Key Laboratory of Tumor Molecular Biology and Key Laboratory of Functional Protein Research of Guangdong Higher Education Institutes, Institute of Life and Health Engineering, Jinan University, Guangzhou, China Search for more papers by this author Chenqi Xu Chenqi Xu State Key Laboratory of Molecular Biology, Shanghai Science Research Center, CAS Center for Excellence in Molecular Cell Science, Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, University of Chinese Academy of Sciences, Shanghai, China Search for more papers by this author Zhimin Zhang Zhimin Zhang School of Pharmacy, Jinan University, Guangzhou, China Search for more papers by this author Song Gao Song Gao orcid.org/0000-0001-7427-6681 Sun Yat-Sen University Cancer Center, Guangzhou, China Search for more papers by this author Penghui Zhou Corresponding Author Penghui Zhou [email protected] orcid.org/0000-0003-0519-461X Sun Yat-Sen University Cancer Center, Guangzhou, China Search for more papers by this author Ke Ding Corresponding Author Ke Ding [email protected] orcid.org/0000-0001-9016-812X School of Pharmacy, Jinan University, Guangzhou, China Search for more papers by this author Liang Chen Corresponding Author Liang Chen [email protected] orcid.org/0000-0001-7300-6604 MOE Key Laboratory of Tumor Molecular Biology and Key Laboratory of Functional Protein Research of Guangdong Higher Education Institutes, Institute of Life and Health Engineering, Jinan University, Guangzhou, China The First Affiliated Hospital of Jinan University, Guangzhou, China Search for more papers by this author Zhenzhen Fan Zhenzhen Fan MOE Key Laboratory of Tumor Molecular Biology and Key Laboratory of Functional Protein Research of Guangdong Higher Education Institutes, Institute of Life and Health Engineering, Jinan University, Guangzhou, China Search for more papers by this author Yahui Tian Yahui Tian MOE Key Laboratory of Tumor Molecular Biology and Key Laboratory of Functional Protein Research of Guangdong Higher Education Institutes, Institute of Life and Health Engineering, Jinan University, Guangzhou, China Search for more papers by this author Zhipeng Chen Zhipeng Chen MOE Key Laboratory of Tumor Molecular Biology and Key Laboratory of Functional Protein Research of Guangdong Higher Education Institutes, Institute of Life and Health Engineering, Jinan University, Guangzhou, China Search for more papers by this author Lu Liu Lu Liu MOE Key Laboratory of Tumor Molecular Biology and Key Laboratory of Functional Protein Research of Guangdong Higher Education Institutes, Institute of Life and Health Engineering, Jinan University, Guangzhou, China Search for more papers by this author Qian Zhou Qian Zhou MOE Key Laboratory of Tumor Molecular Biology and Key Laboratory of Functional Protein Research of Guangdong Higher Education Institutes, Institute of Life and Health Engineering, Jinan University, Guangzhou, China Search for more papers by this author Jingjing He Jingjing He Sun Yat-Sen University Cancer Center, Guangzhou, China Search for more papers by this author James Coleman James Coleman orcid.org/0000-0001-7430-2587 Biomedical Research Centre, Norwich Medical School, University of East Anglia, Norwich Research Park, Norwich, UK Search for more papers by this author Changjiang Dong Changjiang Dong orcid.org/0000-0002-7020-5648 Biomedical Research Centre, Norwich Medical School, University of East Anglia, Norwich Research Park, Norwich, UK Search for more papers by this author Nan Li Nan Li MOE Key Laboratory of Tumor Molecular Biology and Key Laboratory of Functional Protein Research of Guangdong Higher Education Institutes, Institute of Life and Health Engineering, Jinan University, Guangzhou, China Search for more papers by this author Junqi Huang Junqi Huang MOE Key Laboratory of Tumor Molecular Biology and Key Laboratory of Functional Protein Research of Guangdong Higher Education Institutes, Institute of Life and Health Engineering, Jinan University, Guangzhou, China Search for more papers by this author Chenqi Xu Chenqi Xu State Key Laboratory of Molecular Biology, Shanghai Science Research Center, CAS Center for Excellence in Molecular Cell Science, Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, University of Chinese Academy of Sciences, Shanghai, China Search for more papers by this author Zhimin Zhang Zhimin Zhang School of Pharmacy, Jinan University, Guangzhou, China Search for more papers by this author Song Gao Song Gao orcid.org/0000-0001-7427-6681 Sun Yat-Sen University Cancer Center, Guangzhou, China Search for more papers by this author Penghui Zhou Corresponding Author Penghui Zhou [email protected] orcid.org/0000-0003-0519-461X Sun Yat-Sen University Cancer Center, Guangzhou, China Search for more papers by this author Ke Ding Corresponding Author Ke Ding [email protected] orcid.org/0000-0001-9016-812X School of Pharmacy, Jinan University, Guangzhou, China Search for more papers by this author Liang Chen Corresponding Author Liang Chen [email protected] orcid.org/0000-0001-7300-6604 MOE Key Laboratory of Tumor Molecular Biology and Key Laboratory of Functional Protein Research of Guangdong Higher Education Institutes, Institute of Life and Health Engineering, Jinan University, Guangzhou, China The First Affiliated Hospital of Jinan University, Guangzhou, China Search for more papers by this author Author Information Zhenzhen Fan1,‡, Yahui Tian1,‡, Zhipeng Chen1, Lu Liu1, Qian Zhou1, Jingjing He2, James Coleman3, Changjiang Dong3, Nan Li1, Junqi Huang1, Chenqi Xu4, Zhimin Zhang5, Song Gao2, Penghui Zhou *,2, Ke Ding *,5 and Liang Chen *,1,6 1MOE Key Laboratory of Tumor Molecular Biology and Key Laboratory of Functional Protein Research of Guangdong Higher Education Institutes, Institute of Life and Health Engineering, Jinan University, Guangzhou, China 2Sun Yat-Sen University Cancer Center, Guangzhou, China 3Biomedical Research Centre, Norwich Medical School, University of East Anglia, Norwich Research Park, Norwich, UK 4State Key Laboratory of Molecular Biology, Shanghai Science Research Center, CAS Center for Excellence in Molecular Cell Science, Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, University of Chinese Academy of Sciences, Shanghai, China 5School of Pharmacy, Jinan University, Guangzhou, China 6The First Affiliated Hospital of Jinan University, Guangzhou, China ‡These authors contributed equally to this work *Corresponding author. Tel: +86 20 20 8734 3392; Fax: +86 20 20 8734 3392; E-mail: [email protected] *Corresponding author. Tel: +86 020 8522 0850; Fax: +86 020 8522 4766; E-mail: [email protected] *Corresponding author. Tel: +86 20 20 8522 2875; Fax: +86 20 8522 7039; E-mail: [email protected] EMBO Mol Med (2020)12:e11571https://doi.org/10.15252/emmm.201911571 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 Small molecular PD-1 inhibitors are lacking in current immuno-oncology clinic. PD-1/PD-L1 antibody inhibitors currently approved for clinical usage block interaction between PD-L1 and PD-1 to enhance cytotoxicity of CD8+ cytotoxic T lymphocyte (CTL). Whether other steps along the PD-1 signaling pathway can be targeted remains to be determined. Here, we report that methylene blue (MB), an FDA-approved chemical for treating methemoglobinemia, potently inhibits PD-1 signaling. MB enhances the cytotoxicity, activation, cell proliferation, and cytokine-secreting activity of CTL inhibited by PD-1. Mechanistically, MB blocks interaction between Y248-phosphorylated immunoreceptor tyrosine-based switch motif (ITSM) of human PD-1 and SHP2. MB enables activated CTL to shrink PD-L1 expressing tumor allografts and autochthonous lung cancers in a transgenic mouse model. MB also effectively counteracts the PD-1 signaling on human T cells isolated from peripheral blood of healthy donors. Thus, we identify an FDA-approved chemical capable of potently inhibiting the function of PD-1. Equally important, our work sheds light on a novel strategy to develop inhibitors targeting PD-1 signaling axis. Synopsis PD-1 inhibitors that are currently used in the clinic exhibit toxicity and limited patient response rate. This study identifies methylene blue (MB), an FDA-approved chemical for treating methemoglobinemia, as a new potent PD-1 inhibitor. MB activates T-cell functions through inhibiting the recruitment of SHP2 to PD-1. MB treatment effectively shrinks tumors in both an allograft mouse model and an autochthonous mouse model for lung cancer. MB activates human CD8+ T cells that are otherwise suppressed by PD-1 signaling. The paper explained Problem PD-1/PD-L1 inhibitory antibodies have been successfully used in immuno-oncology clinics. However, three major problems remain to be explored: (i) Alternative drugs other than antibody need to be explored. Toxicity has been reported for antibody drugs in clinic. Lines of evidence suggest that the toxicity issues may not be the results of PD-1 inhibition per se, but from Fc part of antibodies. (ii) Most of the current antibody drugs used in clinic inhibit PD-1 function by blocking interaction between PD-1 and its ligands. Other strategies for developing PD-1 inhibitors remain to be explored. (iii) Small molecular PD-1 inhibitors targeting steps other than PD-1/PD-L1 interaction remain to be explored. Results We set up a high-throughput screening system consisting of PD-1 expressing T cell and PD-L1 expressing antigen-presenting cell. Using this system, we screened small chemical library for PD-1 inhibitors and identified methylene blue (MB), an FDA-approved drug, as a potent PD-1 inhibitor. Mechanistically, MB blocked recruitment of SHP2 by PD-1 of T cell in response to stimulation by PD-L1 and thus inhibited PD-1 signaling. MB potently restored proliferation, cytokine expression, and cytotoxicity of cytotoxic T lymphocytes (CTL) in the context of PD-1 signaling. MB effectively shrank tumor allografts or autochthonous lung cancer in vivo. MB was also effective to enhance human CTL function in vitro. Impact We have identified an FDA-approved chemical as a potent PD-1 inhibitor, implicating immediate clinical application. Our data showed that targeting PD-1/SHP2 interaction is a reasonable strategy for developing PD-1 inhibitor, which sheds light on novel strategies to develop PD-1 inhibitors. Introduction Antibodies against PD-1 or PD-L1 prevent interaction between PD-1 and its ligand, block the activation of PD-1, and thereby restore the activity of CTL to lyse PD-L1 expressing target cells and shrink tumor in vivo (Hirano et al, 2005). Some of these antibodies have been approved for clinical trial or usage (Dolan & Gupta, 2014). Impressive clinical benefit was seen in a portion of cancer patients (Brahmer et al, 2010; Topalian et al, 2012; Powles et al, 2014). Unfortunately, PD-1 inhibitors currently used in clinic show severe, sometimes fatal, side effects (Johnson et al, 2016; Moslehi et al, 2018). In contrast, PD-1-deficient mice of C57BL/6 background develop and grow apparently normally, with autoimmune disease observed in late stage of their life (Nishimura et al, 1998, 1999). Lines of evidence suggest that the fragment crystallizable regions (Fc region) of these antibodies play a role in mediating treatment effect (Dahan et al, 2015). These data suggest that it is antibodies, rather than PD-1 inhibition per se, that cause the side effects currently seen in clinic. Small molecular PD-1 inhibitors are, therefore, expected to achieve therapeutic effect comparable to antibody drugs while eliminating the toxicity issues. Unfortunately, small molecular PD-1 inhibitors are lacking in the current immuno-oncology clinic. Moreover, significant portion of the current PD-1 drugs inhibit PD-1 function through blocking interaction between PD-1 and PD-L1. Whether other steps along the PD-1 signaling pathway can be targeted to enhance cytotoxicity of CD8+ T cells remains to be determined. Signaling pathways leading from binding of PD-L1/2 to PD-1 down to inhibition of T-cell activation and cytokine expression have been relatively clear (Riley, 2009; Xia et al, 2016). Although SHP1 and SHP2 were earlier reported to be recruited by PD-1 to exert immunoinhibitory function, it is been confirmed that SHP2 is the main effecter (Yokosuka et al, 2012). Upon stimulation by PD-L1, immunoreceptor tyrosine-based switch motif (ITSM) in the cytoplasmic tail of human PD-1 is phosphorylated on Y248, whereby providing a docking site for Src Homology 2 (SH2) domain of SHP2, which is essential for interaction between PD-1 and SHP2 (Yokosuka et al, 2012). Reports have shown that phosphorylation of CD28 in T cells is critical for mediating treatment effect of PD-1 inhibition (Hui et al, 2017; Kamphorst et al, 2017). Protein–protein interactions (PPI) were previously considered “undruggable” by small molecular chemicals. Recent mutational study, however, revealed “hot spots” anchor residues that contributed the most to the binding free energy of the protein–protein complex (Jin et al, 2014). By placing molecules at these sites, orthosteric (i.e., competitive) small molecular inhibitors can effectively block interaction of the targeted two proteins. Currently, PPIs are targeted by an increasingly larger number of small molecular chemicals (Ran & Gestwicki, 2018). Impressive successes have been reported with inhibitors of protein–protein interactions (iPPIs) in clinic as exemplified by Bcl-2 inhibitor, Venclexta (ABT-199) (Mihalyova et al, 2018). Methylene blue (MB) is an FDA-approved small molecular drug, used to treat patients with methemoglobin levels greater than 30% or those who have symptoms despite oxygen therapy (Committee, 2015). It has previously been used for cyanide poisoning and urinary tract infections, which is no longer recommended. MB is known for its highly favorable safety profile as reflected in a recent study showing that MB can be safely administered to reach a serum concentration up to 6 μM (Baddeley et al, 2015). In our current study, we report that MB effectively counteracted the suppressive activity of PD-1 on CTLs and restored their cytotoxicity, activation, proliferation, and cytokine-secreting activity. Mechanistically, MB blocked interaction between SHP2 and Y248-phosphorylated ITSM motif of human PD-1 and thus potently inhibited the recruitment of SHP2 by PD-1 in CTLs when stimulated by PD-L1. Impressive antitumor effect of MB was seen in allograft and genetically engineered mouse tumor models. MB also recovered proliferation and cytokine expression by human CD8+ T cells. Our work therefore not only identified a potent small molecular iPPI for blocking interaction between PD-1 and SHP2, but shed light on a novel strategy for developing inhibitors targeting PD-L1/PD-1 signaling axis. Results MB enhances cytotoxicity of activated CTL against PD-L1 expressing target cells We took advantage of engineered T-cell systems in which luciferase gene was placed under control of NFAT binding sequence, such that luciferase activity could serve as surrogate IL-2 mRNA transcription (Chow et al, 1999). To identify a chemical PD-1 inhibitor through a high-throughput screening, we generated a system composed of a stable Jurkat T-cell line expressing human PD-1 and harboring NFAT-luciferase reporter (designated JP-luc for Jurkat-PD-1-NFAT-luciferase) and a stable Raji cell line (an antigen-presenting cell) expressing human PD-L1 (designated Raji-L1) (Fig EV1A, Appendix Fig S1A and B). When co-incubated with superantigen SEE-loaded parental Raji, JP-luc exhibited robust luciferase activity. In contrast, JP-luc showed limited luciferase activity when co-cultured with SEE-loaded Raji-L1 (Appendix Fig S1C). Treatment with PD-1 antibody (designated aPD1) recovered the luciferase activity in JP-luc in the presence of SEE-loaded Raji-L1. We thus established an efficient system to assay the intensity of PD-1 signaling by monitoring luciferase activity of JP-luc (Appendix Fig S1C) and whereby screened our chemical library. We identified 3,7-bis(dimethylamino)-5-phenothiazinium chloride (also called methylene blue; designated MB) as the best hit (Figs 1A and EV1B). Click here to expand this figure. Figure EV1. MB enhances cytotoxicity of activated CTL against PD-L1 expressing target cells A. Schematic diagram for compounds screening on Jurkat and Raji stable lines. B. Scatterplot of relative luciferase activity of JP-luc stimulated with SEE-loaded Raji-L1 in the presence of 1 μM chemicals. Y axis denotes the ratio of luciferase of chemical treated well against that of DMSO treated well. JP-luc: Jurkat cell harboring NFAT-luciferase transgene and overexpressing PD-1; Raji-L1: Raji overexpressing PD-L1. C. FACS analysis of the cytotoxic efficiency of OT-I CTLs against EG7-L1 in the presence of MB. aPD1 antibody served as positive control. Splenocytes from OT-I mice in culture were stimulated with 10 nM of SINFEEKL peptide for 3 days to generate mature CTLs. CTLs were incubated with CFSE-labeled EG7-L1 cells in the presence of MB at indicated concentrations. Cytotoxicity was determined by flow cytometry. Data are representative of three independent experiments (effector-to-target ratio = 10:1, 5:1, 2:1, unpaired t-test, killing time: 5 h). EG7-L1: EG7 overexpressing PD-L1. D. Statistical results of (Fig EV1C). E. MB enhanced cytotoxicity of OT-I CTLs against IFNγ-treated B16-F10-OVA. Cytotoxicity was determined by the relative area unoccupied by crystal violet stained cells examined under microphotograph. Data information: Data are representative of three independent experiments and were analyzed by unpaired t-test. Error bars denote SEM. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. Download figure Download PowerPoint Figure 1. MB enhances cytotoxicity of activated CTL against PD-L1 expressing target cells A. Structural formula of 3,7-bis(dimethylamino)-5-phenothiazinium chloride (MB). B. Impact of MB on cytotoxicity of OT-1 CTLs against EG7 or EG7-L1. Splenocytes from OT-1 mice in culture were stimulated with 10 nM of OVA257–264 (SIINFEKL) for 3 days to generate mature CTLs. CTLs were incubated with CFSE-labeled EG7-L1 cells in the presence of MB at indicated concentrations. Cytotoxicity was determined by flow cytometry analysis. Data are representative of three independent experiments (effector-to-target ratio = 5:1, unpaired t-test). EG7-L1: EG7 overexpressing PD-L1. C. Statistical results of (B). D. ELISA measured of LDH release to assess the cytotoxicity of OT-I CTLs on EG7-L1 in the presence of MB. Anti-PD1 antibody (aPD-1) served as positive control. E. Impact of MB on cytotoxicity of PD-1−/− (PD-1KO) OT-1 CTLs against EG7 or PD-L1 expressing EG7 stable cell line. Data are representative of three independent experiments (unpaired t-test). F. Statistical results of (E). G. Cytotoxicity of OT-I CTLs against B16-F10-OVA cells (designated B16-OVA) in the presence of 1 μM of MB or DMSO. Cytotoxicity was determined by the relative area unoccupied by cells examined under microscope (scale bar = 50 μm). Data are representative of three independent experiments and were analyzed by unpaired t-test. H. Statistical results of (G). Data information: Data are representative of three independent experiments. Unpaired t-test; error bars denote SEM. **P < 0.01; ***P < 0.001; ****P < 0.0001. Download figure Download PowerPoint We then assayed MB's ability to enhance cytotoxicity of PD-1 expressing CTL using in vitro cellular system. E.G7-OVA (designated EG-7) is a cell line derived from spontaneous mouse thymoma cell, EL-4, through stably transfecting with the complementary DNA of chicken ovalbumin (OVA). This cell line presents OVA with an H-2Kb-restricted CTL epitope (SIINFEKL) that is recognized by OT-1 transgenic TCR (Moore et al, 1988). We first generated an EG-7 cell clone stably expressing PD-L1 (referred hereafter to as EG7-L1) (Appendix Fig S1D). SIINFEKL peptide stimulation rapidly activated splenic cells of OT-1 mice and expanded CD8+ T cells population to a purity of almost 100% within 3 days (Appendix Fig S1E). OT-1 CTL rapidly upregulated PD-1 expression during this activation (Appendix Fig S1F). We found that OT-1 CTL exhibited limited cytotoxicity against EG7-L1 target cells, which was significantly enhanced by administration of aPD1 (Appendix Fig S1G, Fig EV1C and D). FACS analysis showed that MB potently recovered cytotoxicity of CTL in a dose-dependent manner (Fig 1B and C). Moreover, this cytotoxicity was repeated at various effector-to-target ratios (Appendix Fig S1H and I). Of note, concentrations of MB in these experiments are well below clinically achievable serum concentration. Moreover, we saw no cytotoxicity of MB at these concentrations toward EG-7 cells (Appendix Fig S1J). Measuring lactate dehydrogenase (LDH) released by target tumor cells into medium is another accurate assay to determine CTL cytotoxicity (Decker & Lohmann-Matthes, 1988). We quantified the LDH level in media and confirmed the effectiveness of MB in enhancing cytotoxicity of PD-1-positive OT-1 CTL against EG7-L1 (Fig 1D). To unequivocally show that PD-1 was a critical target mediating the cytotoxicity enhancing effect of MB, we repeated this experiment with OT-1 CTLs of PD-1−/− (PD-1KO) background (Nishimura et al, 1998) (Appendix Fig S1K and L). Critically, we found that MB treatment did not further significantly enhance cytotoxicity of PD-1KO OT-1 CTLs against EG7-L1 (Fig 1E and F). We went on to validate the effectiveness of MB to enhance cytotoxicity of OT-1 CTL against mouse melanoma cell B16-F10 (designated B16 cells). We confirmed the ability of IFNγ to induce PD-L1 expression in B16 cells (Appendix Fig S1M). Earlier reports also revealed that surface expression of MHC class I by B16 cells was strikingly upregulated by IFNγ (Bohm et al, 1998). Hence, OT-1 CTL could only recognize and effectively kill IFNγ-treated B16-OVA, but not B16 cell or B16-OVA untreated with IFNγ. Consistently, OT-1 CTL exhibited no obvious cytotoxicity against B16 or B16-OVA and limited cytotoxicity against IFNγ-treated B16-OVA while aPD1 treatment significantly enhanced OT-1 CTL's cytotoxicity against IFNγ-treated B16-OVA (Appendix Fig S1N and O). Using this system, we found that administration of MB potently enhanced cytotoxicity of OT-1 CTL against IFNγ treated B16-OVA (Figs 1G and H, and EV1E). Taken together, MB enhanced cytotoxicity of PD-1-positive CTL against PD-L1 expressing target cells. MB enhances proliferation and activation of CTL Proliferation and activation are critical for function of CTLs. We asked whether MB enhances both aspects of CTLs. We first checked the ability of MB to promote proliferation of T cells in the presence of PD-L1. CFSE-labeled splenic cells were stimulated with aCD3/aCD28 and proliferation, as indicated by dilution of CFSE, of T cells of wild type (WT), and PD-1KO background was analyzed 48 h after stimulation through FACS analysis by gating on CD8+ population. Data showed that proliferation of WT T cells was significantly suppressed by PD-L1 treatment and this suppression was eliminated by administration of aPD1 or MB (Fig EV2A and B, Appendix Fig S2A). In contrast, neither MB nor aPD1 significantly enhanced proliferation of PD-1KO CTLs, arguing that MB, just like aPD1, enhanced proliferation of T cell through inhibiting PD-1 (Fig 2A and B). Click here to expand this figure. Figure EV2. MB enhanced activation and effector function of CTL A. Effect of PD-1 antibody on the proliferation of OT-I CTLs. Splenocytes from OT-I mice were labeled with CFSE and seeded in a 96-well plate. Media were supplemented with 10 nM of SINFEEKL peptide, 10 ng/ml of human IL-2 and 10 μg/ml of mouse PD-L1 protein in the presence of 10 μg/ml of PD-1 antibody. Cell proliferation was measured by FACS. B. Bar graph of (Fig EV2A). C. MB enhancing production of cytokine and cytolytic granule by OT-1 CTLs. CTLs were co-incubated with CFSE-labeled EG7-L1 cells in the presence of protein transport inhibitor (PTI) and MB at indicated concentrations. Expression of cytokine and cytolytic granule was determined by flow cytometry. aPD-1 antibody served as positive control. EG7-L1: EG7 overexpressing PD-L1. D. Bar graph of IFNγ production of OT-1 CTLs in (Fig EV2C). E. Bar graph of GZMB production of OT-1 CTLs in (Fig EV2C). Data information: Data are representative of three independent experiments and were analyzed by unpaired t-test. Error bars denote SEM. *P < 0.05; **P < 0.01; ***P < 0.001. Download figure Download PowerPoint Figure 2. MB enhanced activation and effector function of CTL A. Effect of MB on the proliferation of WT CTLs and PD-1KO CTLs. Splenic cells were stained with 5 μM of CFSE and seeded into aCD3/aCD28-coated 96-well plates. 10 μg/ml of PD-L1 was administered in media. Cell proliferation was checked by monitoring dilution of CFSE 48 h after CD3/CD28 stimulation by gating on CD8-positive population through FACS analysis. WT: splenic cell from wild-type C57BL/J mice. PD-1KO: splenic cell from PD-1 knockout mice. B. Statistical results of (A). C. FACS analysis of the effect of MB on the activation of OT-I CTLs. OT-I CTLs were stimulated with precoated aCD3/aCD28 and 10 μg/ml of mouse PD-L1 protein in the presence of 100 nM MB or 10 μg/ml aPD1 (served as positive control). After 24 h, surface expression of CD25 and CD69 on OT-1 CTLs was determined through FACS analysis. D. Statistical results of (C). E. Effect of MB on the activation status of Jurkat T cells. Luciferase activity is suppressed in JP-luc by co-culture with Raji-L1 preloaded with 1 μg/ml superantigen (SEE). Treatment with MB enhanced luciferase expression (SEE-loaded Raji (PD-L1 negative) served as positive control). IC50 is calculated to be 117 nM. JP-luc: Jurkat cell harboring NFAT-luciferase transgene and overexpressing PD-1; Raji-L1: Raji overexpressing PD-L1. F. Impact of MB on luciferase activity of various engineered Jurkat T cells. J-luc: Jurkat cell harboring NFAT-luciferase transgene; J-luc-sgPD-1: J-luc cells treated with lentivirus expressing sgPD-1/CAS9 simultaneously. G. qRT–PCR analysis of IL-2 mRNA level in JP-luc cells stimulated with precoated aCD3/aCD28 (10 μg/ml) in the presence of 10 μg/ml of PD-L1 and MB at indicated concentrations. H. MB enhancing IL-2 expression by JP-luc stimulated with Raji-L1 for 24 h quantified through ELISA analysis (aPD1 served as positive control). I. MB enhancing production of cytokine and cytolytic granule by OT-I CTLs. CTLs were incubated with EG7-L1 cells in the presence of protein transport inhibitor (PTI) and MB at indicated concentrations. Expression of cytokine and cytolytic granule was determined by flow cytometry. aPD1 served as positive control. EG7-L1: EG7 overexpressing PD-L1. J. Statistical results of (I). Data information: Data are representative of three independent experiments. Unpaired t-test; error bars denote SEM. *P < 0.05; **P < 0.0