Teriflunomide Modulates the PD-1/PD-L1 Axis and Enhances Antitumor Immunity in Colorectal Cancer

Teriflunomide Modulates the PD-1/PD-L1 Axis and Enhances Antitumor Immunity in Colorectal Cancer | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Teriflunomide Modulates the PD-1/PD-L1 Axis and Enhances Antitumor Immunity in Colorectal Cancer Hwan-Suck Chung, Jung Ho Han, Eun-Ji Lee, Young-Hoon Park, Jung-Hye Ha, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7590655/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 18 Mar, 2026 Read the published version in Oncogenesis → Version 1 posted 10 You are reading this latest preprint version Abstract Inhibitors that target the programmed cell death protein 1 (PD-1)/programmed death-ligand 1 (PD-L1) immune checkpoint pathway have revolutionized cancer immunotherapy; however, many patients exhibit a limited response. In this study, we examined the potential of teriflunomide (TER), an FDA-approved drug for multiple sclerosis, as a novel immune checkpoint modulator for treating colorectal cancer (CRC). We determined the effect of TER on PD-L1 expression in human CRC cell lines, its direct binding to PD-1, and its impact on CD8 + T-cell function. Antitumor activity was determined in vivo using a humanized mouse model of hPD-1 knock-in mice implanted with hPD-L1 expressing MC38 tumor cells. TER treatment reduced PD-L1 expression in CRC cells and disrupted the PD-1/PD-L1 interaction directly. In vivo , TER significantly suppressed tumor growth without systemic toxicity, and enhanced the infiltration and activation of CD8 + T cells within tumors, as evidenced by increased granzyme B expression. Moreover, the antitumor efficacy of TER was abolished by the depletion of CD8 + T cells, which indicated its dependency on this cell population. These findings highlight TER as a promising immune checkpoint modulator that targets the PD-1/PD-L1 axis to promote CD8 + T-cell-mediated antitumor immunity. Because of its established safety profile, TER is a readily translatable therapeutic for enhancing cancer immunotherapy in CRC. Biological sciences/Cancer/Tumour immunology Biological sciences/Immunology/Immunotherapy Teriflunomide Colorectal cancer Immunotherapy PD-1/PD-L1 axis CD8+ T cell Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Immune checkpoint inhibitors (ICIs) are a class of cancer immunotherapeutics that target immune checkpoint proteins on T cells, enhancing the body’s ability to mount an antitumor immune response ( 1 ). These agents have significantly improved survival rates and revolutionized treatment for various malignancies ( 2 ). Numerous clinical trials have demonstrated the efficacy of ICIs, leading to their widespread use in cancer therapy ( 3 ). Immunotherapy comprises various strategies, including immune checkpoint blockade targeting PD-1/PD-L1 and CTLA-4, as well as chimeric antigen receptor T (CAR-T) cell therapy ( 4 ). PD-1/PD-L1 inhibitors, such as pembrolizumab and nivolumab, prevent PD-L1 on tumor cells from binding to PD-1 receptors on T cells, thereby restoring immune activity ( 5 ). CTLA-4 inhibitors, such as ipilimumab, enhance T-cell activation by blocking CTLA-4-mediated immunosuppression ( 6 ). CAR-T therapy involves engineering T cells to recognize tumor-specific antigens, resulting in an enhanced cytotoxic response ( 7 ). Despite these advances, the application of immunotherapy remains limited ( 8 ). Many patients develop resistance to checkpoint inhibitors, which reduces their effectiveness across various cancer types ( 9 ). Moreover, immune-related adverse events (irAEs)—including inflammation of the skin, gastrointestinal tract, and endocrine organs—pose significant treatment challenges ( 10 ). CAR-T therapy is also associated with cytokine release syndrome and neurotoxicity, which can be severe ( 11 ). These limitations highlight the need for alternative approaches to enhance immunotherapy while minimizing side effects ( 12 ). Teriflunomide (TER) is an FDA-approved disease-modifying agent that is primarily used for the treatment of relapsed multiple sclerosis (MS) ( 13 ). As the active metabolite of leflunomide, TER functions by inhibiting dihydroorotate dehydrogenase (DHODH), a key enzyme in pyrimidine biosynthesis, reducing the proliferation of activated lymphocytes and attenuating the immune response ( 14 ). Owing to its immunosuppressive properties, TER effectively reduces relapse frequency and slows disease progression in patients with MS ( 15 ). Besides MS, TER has been examined for its potential efficacy in other autoimmune and inflammatory diseases, including rheumatoid arthritis, spondylo arthritis, and polyarteritis nodosa ( 16 , 17 ). In addition, DHODH inhibitors have attracted attention for their anticancer properties, with studies suggesting a role in modulating tumor metabolism and immune function ( 18 ). However, despite its known immunomodulatory effects, the role of TER in regulating immune checkpoint pathways, particularly PD-1/PD-L1 interactions, remains unclear. Checkpoint inhibitors targeting the PD-1/PD-L1 pathway have shown clinical success, resulting in FDA approval of several antibody-based therapies ( 19 ); however, not all patients respond to PD-1/PD-L1 inhibitors, and resistance mechanisms, such as compensatory immune escape pathways and the immunosuppressive tumor microenvironment, can limit their efficacy ( 20 ). In addition, these therapies may cause immune-related toxicities and affect multiple organ systems ( 21 ). This limitation is particularly evident in colorectal cancer (CRC), where PD-1/PD-L1 blockade shows limited efficacy in microsatellite-stable tumors ( 22 ). Thus, there is a need to explore novel checkpoint inhibitors that can effectively target PD-1/PD-L1 pathway in CRC. This study evaluated the potential of TER, an FDA-approved drug for multiple sclerosis, as an immune checkpoint inhibitor for treating CRC. Unlike conventional approaches that primarily target either PD-1 or PD-L1, TER has the unique ability to regulate both PD-1 and PD-L1 simultaneously, which represents a novel strategy for immune checkpoint blockade. Results Inhibition of PD-L1 expression by TER The human CRC cell lines DLD-1, HCT116, and RKO were treated with TER at the indicated concentrations for 24 hours to evaluate its effects on PD-L1 expression. Cell viability was measured to determine the appropriate TER concentrations (Figure 1A), and subsequent experiments were conducted at doses that maintained cell viability. Western blot analysis revealed that PD-L1 expression decreased in a dose-dependent manner in all cell lines (Figure 1B). To determine whether TER counteracts IFN-γ-induced PD-L1 upregulation, cells were pretreated with IFN-γ, which enhances PD-L1 expression in cancer cells (23). As expected, IFN-γ significantly increased PD-L1 expression; however, this induction was reversed by TER treatment (Figure 1C). To elucidate the mechanism underlying this downregulation, we determined whether it occurred at the transcriptional level. Real-time PCR analysis revealed that TER did not significantly alter PD-L1 mRNA levels, either under basal conditions or following IFN-γ stimulation. This suggests that the effect of TER on PD-L1 is not mediated by transcriptional repression (Figure 1D). Therefore, we hypothesized that TER regulates PD-L1 at the post-translational level. We performed a cycloheximide (CHX) pulse-chase assay to assess PD-L1 protein stability. The results indicated that the degradation of PD-L1 was significantly enhanced in cells treated with TER compared with that in untreated cells (Figure 1E). To identify the specific degradation pathway involved, we examined the role of the proteasomal and lysosomal systems. RKO cells were co-treated with TER and either the proteasome inhibitor MG132 or the lysosomal inhibitors, bafilomycin A1 (Baf) and chloroquine (CQ). The increased degradation of PD-L1 by TER was rescued by Baf and CQ, but not by MG132 (Figure 1F). Taken together, these results demonstrate that TER downregulates PD-L1 expression by promoting its lysosome-dependent degradation, thus confirming a post-translational regulatory mechanism. TER inhibits the PD-1/PD-L1 axis by suppressing PD-L1 expression To determine whether TER disrupts the PD-1/PD-L1 axis, we evaluated its effects on cell viability and PD-L1 expression in human CRC cell lines (DLD-1, HCT116, and RKO) and hPD-1 Jurkat-T cells. The cells were treated with indicated doses of TER for 72 h, and cell viability was assessed. TER treatment decreased cell viability in a dose-dependent manner in all of the cell lines, including hPD-1 Jurkat-T cells (Figure 2A). Based on these results, a co-culture system was established using human CRC cells and hPD-1 Jurkat-T cells, which were treated with TER for 72 h. The results revealed a dose-dependent decrease in cell viability (Figure 2B). Western blot analysis was conducted using protein samples from the co-culture system to measure PD-L1 expression. The results indicated that following TER treatment, PD-L1 protein expression significantly decreased in a dose-dependent manner (Figure 2C). Together, these results indicate that TER inhibits the PD-1/PD-L1 axis, primarily through the suppression of PD-L1 expression in cancer cells, suggesting its potential role as an immune checkpoint modulator. TER interferes with PD-1/PD-L1 binding by directly targeting PD-1 The interaction between PD-L1 on cancer cells and PD-1 on T cells suppresses T cell function (24). To determine whether TER inhibits this binding, an immunofluorescence assay was performed using RKO cells treated with TER and recombinant human PD-1 Alexa 647 protein. Fluorescence microscopy revealed a dose-dependent reduction in red fluorescence in the TER-treated cells, indicating that TER interferes with the PD-1/PD-L1 interaction by reducing the binding between PD-L1 on RKO cells and recombinant PD-1 protein (Figure 3A). To determine whether TER selectively inhibits the PD-1/PD-L1 interaction, a competitive ELISA assay was performed to measure the binding between PD-1 and PD-L1. An αPD-L1-blocking antibody was used as a positive control, as it specifically binds to the extracellular domain of PD-L1 and prevents its interaction with PD-1 (25). TER effectively disrupted PD-1/PD-L1 binding in a concentration-dependent manner, similar to the positive control (Figure 3B). Notably, TER showed an IC50 of 136.7 nM, indicating its potent inhibitory activity against PD-1/PD-L1 interaction. To determine whether TER directly targets the PD-1 receptor and disrupts its interaction with PD-L1, a molecular docking analysis was performed using the CB-Dock2 platform. The upper panel of Figure 3C shows the 3D structure of TER, which is the small-molecule ligand used for docking. The lower panel shows the predicted binding pose of TER within the ligand-binding domain of human PD-1, based on its crystal structure (PDB ID: 3RRQ). TER was found to interact with several key residues, including PHE56, SER57, ASN58, THR59, SER62, PHE63, PHE82, PRO83, GLN99, ASN102, GLY103, and ARG104, primarily located in the immunoglobulin-like V-type (Ig-like V-type) domain of PD-1, which is responsible for PD-L1 engagement and is required for ligand recognition, as annotated in UniProt (Entry: Q15116). The predicted binding affinity (Vina score) was –5.9 kcal/mol, indicating a specific interaction. Figure 3D displays the docked complex, where TER is bound within the ligand-binding pocket of PD-1, and the protein is presented in a cartoon format with the binding site highlighted. To validate these computational predictions, SPR analysis was conducted using recombinant human PD-1 protein and TER. The results indicated a strong binding interaction, with an affinity constant (Kd) of 1.47 nM (Figure 3E), thus confirming the high affinity between TER and PD-1. Together, these data indicate that TER directly binds to PD-1 and likely interferes with PD-1/PD-L1 complex formation, supporting its immunomodulatory effects and providing mechanistic insight into its function. TER enhances T-cell activation by blocking PD-1/PD-L1 interaction The PD-1/PD-L1 axis suppresses T-cell activity and hinders their ability to mount an effective immune response. This immunosuppressive mechanism allows cancer cells to evade immune detection and elimination (26). To determine the effects of TER on T-cell function, a co-culture system was established consisting of CHO-K1 cells engineered to stably express hPD-L1 and a TCR agonist, and Jurkat-T cells engineered to stably express hPD-1 and a TCR-inducible NFAT luciferase reporter. Before evaluating T-cell activation, we measured the cytotoxic effects of TER in both cell lines. The cells were treated with TER at the indicated doses for 24 hours, and viability was measured (Figure 4A). The NFAT family of transcription factors plays an important role in T-cell activation (27). To determine whether TER-mediated blockade of the PD-1/PD-L1 axis affects T-cell functional activation, we assessed NFAT-mediated luciferase activity using a PD-1/PD-L1 blockade bioassay. In this model system, co-culturing aAPC/CHO-K1 cells with PD-1-expressing effector cells promoted the PD-1/PD-L1 interaction, resulting in the suppression of TCR signaling and NFAT-mediated luciferase activity. Agents that disrupt PD-1/PD-L1 binding may restore TCR signaling and increase luciferase activity, thus serving as an indicator of T-cell activation. The results indicated that the PD-L1-blocking antibody enhanced TCR signaling, with an EC50 of 0.4733 μg/mL. Moreover, TER induced a dose-dependent increase in luciferase activity, with an EC50 of 345.2 nM, indicating its ability to reverse PD-1/PD-L1-mediated immune suppression (Figure 4B). TER augments CD8 + T-cell cytotoxicity against CRC cells CD8 + T cells play an important role in cytotoxicity, cytokine production, and immune surveillance (28). We hypothesized that blocking the PD-1/PD-L1 interaction with TER would enhance CD8 + T-cell-mediated cytotoxicity against CRC cells. The direct effects of TER on hPD-L1 MC38 cells were first determined. Cells were treated with TER for 72 h, and cell viability was measured, which showed a dose-dependent reduction (Figure 5A). To further examine T-cell-mediated cytotoxicity, tumor formation was induced in hPD-1 knock-in mice using hPD-L1 MC38 cells, and CD8 + T cells were isolated from the tumors. These tumor-infiltrating CD8 + T cells were co-cultured with hPD-L1 MC38 cells as the target cells. The results indicated that TER treatment resulted in a dose-dependent increase in cancer cell cytotoxicity, confirming its role in augmentation of T-cell activity (Figure 5B). Western blot analysis was used to determine whether TER modulates PD-L1 expression in a T-cell co-culture setting. The results indicated a dose-dependent reduction in PD-L1 expression (Figure 5C). Furthermore, analysis of key immune-related factors in the co-culture media, including granzyme B (GrB), interleukin-2 (IL-2), and interferon-γ (IFN-γ), which play important roles in T-cell-mediated cytotoxicity and immune activation, revealed that their levels were significantly increased at higher TER doses (Figure 5D). Overall, these findings indicate that TER enhances CD8 + T-cell cytotoxicity by inhibiting the PD-1/PD-L1 interaction, resulting in increased immune activation and tumor cell elimination. TER promotes CD8 + T cell infiltration and inhibits tumor growth in vivo To determine the effects of TER on tumor suppression, in vivo allograft experiments were carried out using hPD-1 knock-in mice bearing hPD-L1 MC38 tumors. TER was administered at various doses [10 and 30 mpk], and tumor growth was assessed over time. Body and spleen weight remained similar across all groups, indicating that TER does not induce systemic toxicity or remarkable adverse effects (Fig. 6A, B). In contrast, TER treatment significantly reduced tumor volume and weight compared with the vehicle control group (Figure 6C, D). To determine the effects of TER on CD8 + T-cell infiltration, we analyzed the proportion of CD8 + T cells within tumors. Flow cytometry revealed a dose-dependent increase in CD8 + T-cell infiltration in the TER-treated groups (Figure 6E). We then measured PD-L1 expression levels in tumor tissues. Western blot analysis revealed a dose-dependent decrease in PD-L1 expression following TER treatment, indicating that TER downregulates PD-L1 in vivo (Figure 6F). Moreover, immunohistochemistry (IHC) revealed a dose-dependent increase in CD8 + T-cell infiltration and GrB expression in tumor tissues following TER treatment (Figure 6G). CD8 + T cell depletion abrogates the antitumor effects of TER in vivo To determine whether the in vivo antitumor effects of TER are CD8 + T-cell-dependent, we conducted additional allograft experiments using CD8 depletion antibodies. The mice were divided into four experimental groups: vehicle–isotype control, vehicle–CD8 depletion antibody, TER–isotype control, and TER–CD8 depletion antibody. TER was administered at a dose of 30 mpk. To elucidate the role of CD8 + T cells in TER-mediated tumor suppression, the mice in the CD8 depletion groups were treated with a CD8-depleting antibody. No significant changes in body or spleen weight were observed in the groups, suggesting that TER treatment does not cause significant toxicity (Fig. 7A, B). In contrast, CD8 depletion abolished the antitumor effects of TER, as evidenced by the loss of tumor growth suppression in the TER–CD8 depletion group compared with the TER–isotype control group (Figure 7C, D). These results indicate that TER's efficacy is dependent on CD8 + T cells. Flow cytometry revealed that TER treatment resulted in an increase in CD8 + T-cell infiltration within the tumor. However, this effect was abolished by CD8 depletion, confirming the dependency of its antitumor effects on CD8 + T cells (Figure 7E). In addition, IHC analysis revealed an increase in CD8 + T-cell infiltration and GrB expression in tumor tissues following TER treatment; however, these effects were reversed by CD8 + T-cell depletion, thus confirming the essential role of CD8 + T cells in TER-mediated tumor suppression (Figure 7F). Discussion We demonstrated that TER, an FDA-approved drug for multiple sclerosis, acts as a dual inhibitor of both PD-1 and PD-L1, promoting CD8 + T-cell-mediated antitumor immunity in CRC models. TER directly binds to PD-1, preventing its interaction with PD-L1, and also downregulates PD-L1 expression in tumor cells. These results highlight the potential of TER as a novel immune checkpoint modulator with dual functionality targeting both PD-1 and PD-L1 to enhance the antitumor immune response in CRC. CRC is a leading cause of cancer-related mortality worldwide, and ICIs exhibit significant efficacy in a subset of patients ( 29 ). PD-1/PD-L1 blockade has shown clinical benefit primarily in microsatellite instability-high (MSI-H) or mismatch repair-deficient (dMMR) CRC, which exhibits a high tumor mutational burden and increased immune cell infiltration ( 30 ). However, the majority of CRC cases are MSS, which are generally unresponsive to PD-1/PD-L1 inhibitors because of the immunosuppressive tumor microenvironment ( 31 ). This emphasizes the need for alternative or complementary strategies to enhance antitumor immunity in CRC, particularly in MSS subtypes ( 32 ). TER may enhance antitumor immunity by modulating the immune response, including enhancing CD8 + T-cell activation and modifying the tumor microenvironment. This may overcome some of the resistance mechanisms observed with traditional immune checkpoint therapies. Because of its dual ability to block PD-1 and reduce PD-L1 expression, TER represents a promising candidate for addressing these limitations. Previous studies have primarily focused on PD-1 or PD-L1 inhibitors, with several monoclonal antibodies, such as pembrolizumab and nivolumab, showing efficacy in various cancers ( 33 ). However, many patients exhibit limited response because of immune resistance mechanisms and insufficient T-cell activation ( 34 ). In contrast, TER uniquely targets both PD-1 and PD-L1 simultaneously, offering a more comprehensive strategy for enhancing the immune response. Our study builds on previous work showing the importance of PD-1/PD-L1 blockade in overcoming tumor-induced immune suppression and also introduces TER as a dual-action immune modulator. Our results suggest that TER enhances CD8 + T-cell infiltration and activation within tumors, likely through its direct inhibition of PD-1 and a reduction of PD-L1 expression. Moreover, TER may induce apoptosis in tumor cells, which further contributes to its antitumor effects ( 35 ). Flow cytometry and IHC analyses confirmed that TER significantly increases CD8 + T-cell infiltration and GrB expression, indicating enhanced cytotoxic activity against cancer cells. CD8 depletion experiments further confirmed the essential role of CD8 + T cells in mediating the antitumor effects of TER. These results are consistent with those of other studies showing that CD8 + T-cell activation is important for the positive effects of ICIs ( 36 ). In the present study, we examined the potential of TER as a dual inhibitor targeting both PD-1 and PD-L1. Although TER demonstrated a strong binding affinity for PD-1, as confirmed by SPR analysis, no binding was observed between TER and PD-L1 (data not shown). This suggests that TER exerts its immune checkpoint inhibition primarily through a direct interaction with PD-1, rather than directly affecting PD-L1. This finding aligns with that of previous studies indicating that many PD-1 inhibitors function by disrupting the PD-1/PD-L1 interaction without binding directly to PD-L1 ( 37 ). Interestingly, our data clearly demonstrate that TER effectively downregulates PD-L1 expression via the lysosomal degradation pathway. Nevertheless, a direct interaction between TER and PD-L1 was not detected in our experiments, suggesting that the upstream mechanism initiating this process remains unclear. These findings underscore a current limitation of our study and highlight the complexity of TER’s mode of action. We speculate that TER may function through an indirect mechanism, such as by interacting with an unknown intermediary protein or by modulating an upstream signaling pathway that targets PD-L1 for degradation. Therefore, future studies are warranted to precisely identify the upstream mediators of this effect. Overall, these results underscore the complex nature of TER’s immunomodulatory effects, in which direct PD-1 blockade enhances CD8 + T-cell activation, while a distinct, indirect mechanism of PD-L1 downregulation contributes to a broader antitumor immune response. TER is approved for the treatment of multiple sclerosis, in which it acts as an immunomodulator by inhibiting dihydroorotate dehydrogenase (DHODH) and limiting the proliferation of activated lymphocytes ( 38 ). This immunoregulatory function of TER suggests that its effects on the PD-1/PD-L1 axis may extend beyond direct checkpoint inhibition. The ability of TER to influence T-cell responses and immune regulation in an inflammatory disease setting may translate into enhanced antitumor immunity, which warrants further examination into its broader immunomodulatory effects. An important aspect of our findings is the apparent paradox that TER, a known immunosuppressant, induces potent antitumor immunity within the tumor microenvironment. Although TER suppresses the immune system during multiple sclerosis by inhibiting the proliferation of activated lymphocytes ( 14 ), we demonstrated its function as a CD8 + T-cell activator through a PD-1/PD-L1 axis blockade. This dual functionality may be explained by the unique cellular dynamics of the TME. For example, the primary mechanism of TER, which is DHODH inhibition, targets highly proliferative cells. This may affect not only effector T cells, but also proliferative immunosuppressive populations, such as regulatory T cells (Tregs), which are abundant in the TME ( 39 ). Consequently, the suppression of the Treg proliferation within the TME may synergize with the direct blockade of PD-1 by TER. This results in a net positive effect that enhances CD8 + T-cell-mediated antitumor immunity, and suggests that TER is not merely an immunosuppressant, but a sophisticated immunomodulator whose function is contingent on a specific immunological context. Although the use of murine models presents inherent limitations in terms of translational relevance to human cancers, we used humanized models involving human PD-1 knock-in mice and hPD-L1 MC38 cells. These models provide a more accurate representation of human tumor–immune interactions compared with traditional murine models ( 40 ). Although this approach is not fully representative of human physiology, the use of these humanized models enables the evaluation of TER’s effects on the PD-1/PD-L1 axis and CD8 + T-cell-mediated immunity, thus enhancing the reliability of our findings. In addition to its potential efficacy as a monotherapy, TER may also exhibit synergy when combined with existing PD-1/PD-L1 inhibitors. Monoclonal antibodies targeting PD-1 or PD-L1, such as pembrolizumab and nivolumab, have demonstrated remarkable clinical success; however, a significant proportion of patients do not respond or develop resistance to these agents ( 41 ). The ability of TER to block PD-1 while simultaneously downregulating PD-L1 suggests its potential to enhance the efficacy of these antibody-based therapies by providing an additional checkpoint inhibition mechanism. Moreover, as a small-molecule inhibitor, TER offers advantages such as enhanced tissue penetration and suitability for oral delivery, which may improve patient compliance and expand therapeutic accessibility. Future studies should examine the efficacy of TER combined with PD-1/PD-L1 antibodies for enhancing the immune response and overcoming resistance in non-responding patients. As TER is already an FDA-approved drug, the barrier for clinical translation is lower compared with novel drug candidates ( 42 ). Most small-molecule inhibitors or monoclonal antibodies targeting the PD-1/PD-L1 axis require extensive safety and pharmacokinetic evaluation before reaching clinical trials ( 43 ). Because TER has already been approved for treating multiple sclerosis, its established safety profile, pharmacodynamics, and clinical tolerability provide a strong foundation for its potential repurposing in cancer immunotherapy ( 44 ). Repositioning an existing drug for oncological applications offers several advantages, including reduced time and cost associated with drug development, a faster regulatory approval process, and improved accessibility for patients. Given its demonstrated efficacy in enhancing CD8 + T-cell-mediated antitumor responses, further clinical studies are needed to evaluate the efficacy of TER as an immune checkpoint modulator for cancer treatment. In conclusion, TER shows potential as a dual PD-1/PD-L1 inhibitor, enhancing CD8 + T-cell-mediated antitumor immunity in CRC. Because of its established safety profile as an FDA-approved drug, TER represents a promising therapeutic drug for enhancing cancer immunotherapy, particularly in patients who are unresponsive to traditional PD-1/PD-L1 inhibitors. Materials and Methods Reagents TER was purchased from Targetmol (Boston, MA, USA). The PD-1/PD-L1 Blockade Bioassay kit was obtained from Promega (Madison, WI, USA). The cell counting kit-8 was purchased from Dojindo Molecular Technologies (Rockville, MD, USA). For cell dissociation and purification, a tumor dissociation kit, MACSmix tube rotator, gentleMACS C tubes, and gentleMACS dissociator were obtained from Miltenyi Biotec (Auburn, CA, USA). The anti-PD-L1 antibodies were purchased from BPS Bioscience (San Diego, CA, USA). Anti-PD-1 antibodies were purchased from Selleck Chemicals (Houston, TX, USA). A magnet for column-free cell separation, a Mouse CD8 + T Cell Isolation Kit, and polystyrene round-bottom tubes were purchased from STEMCELL Technologies (Vancouver, BC, Canada). Mouse IL-2 and IFN-gamma ELISAs were purchased from BD Biosciences (San Diego, CA, USA), and a Mouse GrB ELISA was purchased from Thermo Fisher Scientific (Waltham, MA, USA). Anti-CD8 antibodies were obtained from Cell Signaling Technology (Danvers, MA, USA). Cell culture Humanized PD-L1-expressing MC38 cells, derived from C57BL/6 mouse CRC, were obtained from the Shanghai Model Organisms Center (Shanghai, China). The cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM) containing 10% heat-inactivated fetal bovine serum (FBS) and antibiotics (100 units/ml penicillin). Recombinant Jurkat-T cells expressing human PD-1 and an NFAT reporter gene (hPD-1/NFAT Jurkat-T cells, #60535) and recombinant CHO-K1 cells expressing human PD-L1 and a T-cell receptor (TCR) activator (hPD-L1/TCR CHO-K1 cells, #60536) were obtained from BPS Bioscience. Jurkat and CHO-K1 cells were infected with human PD-1- and PD-L1-expressing lentiviruses (GeneChem, Shanghai). The cells were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium containing 10% heat-inactivated FBS and antibiotics (100 units/ml penicillin). Human CRC cells (DLD-1, HCT116, and RKO) were obtained from the Korean Cell Line Bank (Seoul, Korea) and cultured in DMEM containing 10% heat-inactivated FBS and antibiotics (100 units/ml penicillin). All cell culture solutions were obtained from Hyclone Laboratories Inc. (Chicago, IL, USA). The cells were cultured at 37°C in a humidified 5% CO 2 incubator. Cell viability assay Cell viability was measured using the CCK assay (#CK04, Dojindo Molecular Technologies, Inc., Rockville, MD, USA) based on the manufacturer's instructions. Briefly, cells were seeded into 96-well plates at a density of 1 × 10 4 cells per well and incubated overnight, followed by treatment with TER. A range of TER concentrations was added to the wells. After the designated incubation period, 10 µL of CCK solution was added to each well, and the plates were incubated for 2 h at 37°C. The absorbance at 450 nm was measured using a microplate reader (Molecular Devices i3, San Jose, CA, USA), and cell viability was calculated. Western blotting analysis The cells were washed with 1× PBS, and total protein lysates were prepared using RIPA buffer containing 1% NP-40 lysis buffer and protease inhibitor cocktail tablets (Roche, Basel, Switzerland). Protein concentration was measured using the Bio-Rad protein assay. Equivalent amounts of protein were separated by 8–15% SDS-PAGE and transferred to nitrocellulose membranes (GE Healthcare, Munich, Germany). The membranes were blocked with 5% nonfat dry milk at room temperature (20–25°C) for 1 h and incubated overnight at 4°C with primary antibodies. The membranes were washed three times with 1× Tris-buffered saline for 10 min per wash, followed by incubation with the corresponding secondary antibody. After washing, protein detection was performed using the ChemiDoc Touch Imaging System (Bio-Rad, Hercules, USA). GAPDH was used as a loading control for protein normalization. The original uncropped images for all western blots are provided in the Supplementary Information (Supplementary Figures S1 –S6). Quantitative reverse transcription–polymerase chain reaction (qRT–PCR) Total RNA was isolated using the RiboEx Total RNA Extraction Kit (GeneAll Biotechnology, Seoul, Korea). Complementary DNA (cDNA) was synthesized using the AccuPower CycleScript RT premix (dT20) (Bioneer, Daejeon, Korea). For the reverse transcription reaction, 1 µg of total RNA was used in a final cDNA reaction volume of 20 µL. Quantitative PCR was performed using a QuantStudio 3D Digital PCR System (Thermo Fisher Scientific, Waltham, MA, USA) along with the AccuPower 2X Greenstar qPCR master mix (Bioneer). The protocol consisted of 40 cycles of denaturation at 95°C for 15 sec, followed by annealing and extension at 60°C for 30 sec per cycle. All reagents were used according to the manufacturer’s instructions. The relative expression of target mRNA were normalized to GAPDH expression as an endogenous control. Competitive ELISA A competitive ELISA was performed using the PD-1/PD-L1 inhibitor ELISA screening kit (BPS Bioscience, #72005, San Diego, CA, USA), following the manufacturer's instructions. The αPD-L1 antibody (#71213) served as the positive control. Recombinant hPD-L1 (#71104, BPS Bioscience) was coated onto 96-well plates (Corning Inc., New York, NY, USA) at a concentration of 1 mg/mL in PBS and incubated overnight. The plates were then washed with PBS containing 0.1% Tween (PBS-T), blocked with 2% BSA in PBS for 1 h at room temperature, and re-washed. Next, 5 µL of 0.5 mg/mL biotinylated hPD-1 (#71109, BPS Bioscience) was added, and the plates were incubated for 2 h at room temperature. After three washes in PBS-T, 50 µL of 0.2 mg/mL HRP-conjugated streptavidin was added and the plates were incubated for 1 h. Finally, the plates were washed three times with PBS-T, and chemiluminescence was measured using a SpectraMax L Luminometer (Molecular Devices, San Jose, CA, USA). PD-1/PD-L1 blockade bioassay To determine the effectiveness of TER in blocking the PD-1/PD-L1 interaction, a cell-based PD-1/PD-L1 blockade bioassay was performed using the PD-1/PD-L1 blockade bioassay kit (#J4011, Promega, Madison, WI, USA), with slight modifications to the manufacturer’s instructions. In this assay, 5 × 10 4 PD-L1 aAPC/CHO-K1 cells were seeded into 96-well plates containing DMEM supplemented with 10% FBS. On the experiment day, the medium was removed, and PD-L1-blocking antibody or TER (as indicated) was added along with 1 × 10 5 PD-1 effector cells. Luminescence was measured using the GloMax® Explorer Multimode Microplate Reader (Promega) after adding Bio-Glo™ Reagent (Promega, #G7940). The results are expressed as the mean ± standard error of the mean from four independent experiments. Tumor-infiltrating t cells (TILs) isolation A humanized PD-1 mouse model (C57BL/6J background, genetically engineered to express the full-length human PD-1 protein) was obtained from the Shanghai Model Organisms Center (Shanghai, China). The animal experiments were conducted in compliance with the guidelines established by the Institutional Animal Care and Use Committee (IACUC) of the Korea Institute of Oriental Medicine (KIOM) and approved by the IACUC (approval number: KIOM-D-23-099). To extract tumor-infiltrating lymphocytes (TILs, CD8 + T cells), MC38 tumor tissues were collected and digested with collagenase IV (0.5 mg/mL) at 37°C for 1 h. The resulting cell suspension was strained twice using 100-µm and 40-µm cell strainers (SPL) to isolate single cells. TILs were isolated from the single-cell suspension using negative selection beads according to the manufacturer’s instructions (STEMCELL Technologies Inc., Vancouver, Canada). Co-culture experiments Tumor-infiltrating CD8 + T cells (1 × 10 6 cells) were used as effector cells and activated with Dynabeads T Activator CD3/CD28 (Life Technologies, Carlsbad, CA, USA) for 72 h at 37°C. Murine hPD-L1 MC38 cells, which served as target cells, were labeled using the CellTrace™ Far Red Cell Proliferation Kit (Thermo Fisher Scientific, Waltham, MA, USA) and treated with IFN-γ (10 ng/mL) for 24 h at 37°C to induce PD-L1 expression. hPD-L1 MC38 cells (5 × 10 4 cells) were co-cultured with the activated CD8 + T cells (2.5 × 10 5 cells) at an effector-to-target cell ratio of 5:1 for 72 h at 37°C in the presence of different concentrations of TER. Then, the plates were washed with PBS, and the remaining viable cancer cells were stained with crystal violet solution and quantified using a SpectraMax i3 microplate reader at 540 nm. In vivo analysis The experimental mice (male, n = 6) were housed in a specific pathogen-free facility and anesthetized using a 2% isoflurane–oxygen mixture administered through an Avesko inhalation system. To induce subcutaneous tumor formation, hPD-L1 MC38 cells were injected in the right flank of humanized PD-1 mice. Each mouse received 200 µL of cell suspension (3.0 × 10 6 cells/mL, equivalent to 1.5 × 10 5 cells/mouse). Tumor progression was assessed biweekly using a digital caliper (Hi-Tech Diamond, Westmont, IL, USA). Tumor volume was calculated using the formula: (W 2 × L)/2, where W represents the short diameter and L represents the long diameter. Once the tumors reached a volume of 100 mm3, the mice were divided into several experimental groups. The control group received vehicle [PBS, 10 mL/kg, orally (q.d., i.g.)], whereas the other groups were treated with either low or high doses of TER [10 or 30 mg/kg (mpk), administered intraperitoneally (q.d., i.p.)], or a CD8 depletion antibody (#BP0061, Bio Cell, West Lebanon, NH, USA). TER was administered continuously for 16 consecutive days. In contrast, the CD8 depletion antibody was administered twice a week through intragastric injections beginning after tumor implantation. On day 16 following treatment, the animals were euthanized for further analysis. Tissue dissociation for cell isolation and flow cytometry Tumor tissues were collected in RPMI Medium 1640 containing 10% heat-inactivated FBS, 1% penicillin and streptomycin (all from HyClone™), minced, and incubated for 30 min in DPBS (Well-gene) with 0.5 mg/mL Collagenase IV (Sigma) at 37°C. The tissues were squeezed through a 100 µm cell strainer (#93100, SPL), re-filtered through a 70 µm cell strainer (#93070, SPL), and incubated for 5 min in Red Blood Cell Lysis Buffer (#10-548E, Lonza). Flow cytometry was conducted to determine cell surface antigen expression by a 30-min incubation on ice with the following antibodies: mouse-specific monoclonal antibodies PerCP/Cyanine5.5 anti-mouse CD45 (#103131, BioLegend), APC anti-mouse CD8a (#100712, BioLegend), corresponding isotype control mAbs PerCP/Cy5.5 Rat IgG2b (#400632, BioLegend), and APC Rat IgG2a (#400512, BioLegend). The cells were washed three times with PBS containing 2% FBS, fixed in suspension with 4% paraformaldehyde, and stored at 4°C until subsequent analysis with a CytoFLEX flow cell counter (Beckman). The data were analyzed using Kaluza analysis software (Beckman Coulter). Surface plasmon resonance analysis SPR was used to measure binding affinity between PD-L1 and PD-1 proteins using a Biacore T200 biosensor (GE HealthCare Technologies, Inc., Chicago, IL, USA) with a nitrilotriacetic acid (NTA) sensor chip (GE HealthCare). The NTA surfaces were washed with 350 mM EDTA and loaded with 0.5 mM NiCl2. The hPD-1 protein (#10377-H08H, Sino Biologicals) was bound to the NTA sensor chip using an affinity capture method. Various concentrations of analytes were prepared by diluting in HBS-P buffer (10 mM HEPES, pH 7.4, 150 mM NaCl, and 0.005% surfactant P20). The equilibrium dissociation constants (KD; kd/ka) were calculated using sensorgrams. Molecular docking analysis To examine the interaction between human PD-1 and TER, molecular docking was conducted using the CB-Dock2 platform, an automated docking tool capable of cavity detection and AutoDock Vina-based docking ( 45 ). The target protein structure was obtained from the Protein Data Bank (PDB ID: 3RRQ) ( 46 ) and used in its native form without any mutations or modifications. TER was used as a ligand, and its 3D structure file (.sdf) was downloaded from the PubChem database (CID: 54684141) ( 47 ). CB-Dock2 automatically detected potential binding cavities within the PD-1 structure, and the ligand was docked using AutoDock Vina. Subsequently, the interaction between PD-1 and Teriflunomide was visualized with the built-in visualization tools of the CB-Dock2 platform. Statistical analysis The data are presented as ratios relative to their respective control values. The results are shown as means ± standard error of the mean (SEM) based on three independent experimental replicates. Differences between the mean values within each group were evaluated using a Student's t-test, whereas comparisons across multiple groups were done using a one-way analysis of variance, followed by Tukey’s post hoc test. The statistical analyses were performed using GraphPad Prism version 8.0 (GraphPad Software, San Diego, CA, USA). Declarations Acknowledgments The authors would like to thank Enago (www.enago.co.kr) for English language editing. This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Ministry of Science and ICT (NRF-2022R1A2C2092834), and by the Korea Institute of Oriental Medicine (KIOM) grant (KSN2412013), also provided by the Ministry of Science and ICT, Korea. Author Contributions Jung Ho Han : Data curation, Investigation, Visualization, Writing – original draft, Writing – review & editing. Eun-Ji Lee : Conceptualization, Data curation, Investigation, Writing – original draft. Young-Hoon Park : Investigation. Jung-Hye Ha : Investigation. Kazi Rejvee Ahmed : Investigation. Jang-Gi Choi : Conceptualization, Funding acquisition, Investigation, Writing – review & editing. 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Supplementary Files 3.SupplementaryInformation.pdf Supplementary Information qPCRHct116RKO.zip qPCR Hct116, RKO Raw data qPCRDLD1.zip qPCR DLD-1 Raw data qPCRDLD1Hct116RKOdatamerge.xlsx qPCR DLD-1, Hct116, RKO data merge Cite Share Download PDF Status: Published Journal Publication published 18 Mar, 2026 Read the published version in Oncogenesis → Version 1 posted Editorial decision: revise 09 Oct, 2025 Review # 2 received at journal 09 Oct, 2025 Review # 1 received at journal 27 Sep, 2025 Reviewer # 2 agreed at journal 23 Sep, 2025 Reviewer # 1 agreed at journal 22 Sep, 2025 Reviewers invited by journal 16 Sep, 2025 Submission checks completed at journal 15 Sep, 2025 First submitted to journal 14 Sep, 2025 Unknown event 12 Sep, 2025 Editor assigned by journal 11 Sep, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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12:08:07","extension":"png","order_by":24,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":103128,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-7590655/v1/a10da8d4c3ed6d4465c0fa76.png"},{"id":92172038,"identity":"c8743711-1071-4f61-9784-b45e5da76eb1","added_by":"auto","created_at":"2025-09-25 12:08:07","extension":"xml","order_by":25,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":128391,"visible":true,"origin":"","legend":"","description":"","filename":"ONCSIS2505520structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-7590655/v1/82ea68b424abf5152a0bd472.xml"},{"id":92172044,"identity":"151856b1-8917-4a13-b1fb-b18578f04031","added_by":"auto","created_at":"2025-09-25 12:08:08","extension":"html","order_by":26,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":137807,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7590655/v1/29f534de3a6024dec8433851.html"},{"id":92172009,"identity":"d0fe9989-4ac3-406a-95f6-e5da06b1a656","added_by":"auto","created_at":"2025-09-25 12:08:07","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":444946,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of TER on PD-L1 Expression in CRC. (A) DLD-1, HCT116, and RKO cells were treated with the indicated concentrations of TER for 24 h. Cell viability was measured using a CCK assay. (B) PD-L1 expression levels in CRC cells treated with the indicated doses of TER for one day, as evaluated by western blot analysis. (C) The combined effects of TER and IFN-γ on PD-L1 expression levels in CRC cells, as examined by western blot analysis. (D) mRNA levels of PD-L1 in CRC cells treated with TER, as assessed by quantitative PCR. (E) PD-L1 expression in CRC cells treated with TER and CHX, as measured by western blot analysis. (F) The mechanism of PD-L1 degradation was evaluated in RKO cells using inhibitors for the lysosome (Baf and CQ) or proteasome (MG132) in the presence of TER. The results are shown as the mean ± SEM. * \u0026lt; 0.05, ** p \u0026lt; 0.01, *** p \u0026lt; 0.001, and **** p \u0026lt; 0.0001; # \u0026lt; 0.05, ## p \u0026lt; 0.01, *** p \u0026lt; 0.001, ### p \u0026lt; 0.001, and #### p \u0026lt; 0.0010, compared with the respective control.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7590655/v1/264aba461801d0f3e05197d7.png"},{"id":92172011,"identity":"ca19f75b-4675-4143-a255-1ac233a43a89","added_by":"auto","created_at":"2025-09-25 12:08:07","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":243463,"visible":true,"origin":"","legend":"\u003cp\u003eTER Reduces Cell Viability and Inhibits PD-L1 Expression in CRC Cells. (A) CRC cell lines were treated with the indicated concentrations of TER for 72 h. Cell viability assessed using the CCK assay is shown. (B) Human CRC cell lines were co-cultured with hPD-1 Jurkat-T cells and treated with the indicated concentrations of TER for 72 h. (C) PD-L1 protein expression in CRC cells co-cultured with hPD-1 Jurkat-T cells and treated with TER for 72 h. GAPDH was used as a loading control. The results are shown as the mean ± SEM. * \u0026lt; 0.05, ** p \u0026lt; 0.01, *** p \u0026lt; 0.001, and **** p \u0026lt; 0.0001 compared with the respective control.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7590655/v1/695b645209dfd8e04afdaa6d.png"},{"id":92172015,"identity":"5a4dceab-28d7-4c02-8037-6704aef67e9f","added_by":"auto","created_at":"2025-09-25 12:08:07","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":431751,"visible":true,"origin":"","legend":"\u003cp\u003eTER Directly Binds to PD-1 and Inhibits the PD-1/PD-L1 Interaction. (A) Immunofluorescence analysis of PD-1 binding to PD-L1 on RKO cells treated with TER. RKO cells were incubated with recombinant human PD-1 Alexa 647 protein and treated with the indicated concentrations of TER. Red fluorescence represents PD-1 binding; the nuclei were stained with Hoechst (blue). (B) Competitive ELISA assay measuring the inhibition of PD-1/PD-L1 interaction by TER. The binding between PD-1 and PD-L1 in the presence of increasing concentrations of TER was assessed. (C) The 3D structure of TER (upper panel) and its predicted binding pose within the active site of PD-1 (PDB ID: 3RRQ, lower panel). The key interacting residues include PHE56, SER57, ASN58, THR59, SER62, PHE63, PHE82, PRO83, GLN99, ASN102, GLY103, and ARG104. (D) Protein–ligand complex showing TER docked to PD-1, with the binding pocket highlighted. (E) Surface plasmon resonance (SPR) analysis showing the binding of TER to PD-1. αPD-L1 was used as a positive control. *** p \u0026lt; 0.001 and **** p \u0026lt; 0.0001 compared with the respective control.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7590655/v1/46a847433f23d5ad373e1072.png"},{"id":92172991,"identity":"52442d64-dccc-41c9-adc3-77e5a8a5164c","added_by":"auto","created_at":"2025-09-25 12:16:07","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":135525,"visible":true,"origin":"","legend":"\u003cp\u003eTER Enhances T-Cell Activation by Blocking the PD-1/PD-L1 Interaction. (A) The viability of hPD-1 Jurkat-T cells and hPD-L1 CHO cells following treatment with the indicated concentrations of TER for 24 h. (B) Luciferase activity measured using a PD-1/PD-L1 blockade bioassay. hPD-1 Jurkat-T cells (effector cells) were co-cultured with hPD-L1-expressing aAPC/CHO-K1 cells (target cells) in the presence of indicated concentrations of TER. The luminescence signal indicates the level of TCR signaling activation. αPD-L1 was used as a positive control. * \u0026lt; 0.05, ** p \u0026lt; 0.01, *** p \u0026lt; 0.001, and **** p \u0026lt; 0.0001 compared with the respective control.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-7590655/v1/be3547cf398baf5fd68ee6c6.png"},{"id":92172017,"identity":"91e77afb-2aad-489a-bada-abe258181dd4","added_by":"auto","created_at":"2025-09-25 12:08:07","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":160790,"visible":true,"origin":"","legend":"\u003cp\u003eTER Enhances CD8\u003csup\u003e+\u003c/sup\u003e T-Cell-Mediated Cytotoxicity Against hPD-L1 MC38 Cells. (A) The viability of hPD-L1 MC38 cells following treatment with the indicated concentrations of TER for 72 h. (B) CD8\u003csup\u003e+\u003c/sup\u003e T cells were isolated from tumors of hPD-1 knock-in mice bearing hPD-L1 MC38 tumors. These tumor-infiltrating CD8\u003csup\u003e+\u003c/sup\u003e T cells were co-cultured with hPD-L1 MC38 cells as target cells in the presence of TER for 72 h. Cell viability measured using the CCK assay is depicted. (C) PD-L1 expression in hPD-L1 MC38 cells, as assessed by western blot analysis using protein lysates from co-culture conditions. GAPDH was used as a loading control. (D) The levels of immune-related factors, including GrB, IL-2, and IFN-γ, measured in the co-culture supernatant by ELISA. * \u0026lt; 0.05, ** p \u0026lt; 0.01, *** p \u0026lt; 0.001, and **** p \u0026lt; 0.0001 compared with the respective control.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-7590655/v1/c4c03d185044bf692afb1439.png"},{"id":92172997,"identity":"c87e9643-b002-436e-8f32-9518d1feeaad","added_by":"auto","created_at":"2025-09-25 12:16:07","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":366763,"visible":true,"origin":"","legend":"\u003cp\u003eTER Suppresses Tumor Growth and Modulates the Tumor Immune Environment \u003cem\u003ein vivo\u003c/em\u003e. (A) Body weight of hPD-1 knock-in mice during the treatment period. The mice were treated with vehicle or TER (10 or 30 mpk) for the indicated time. (B) Spleen weight of mice at the endpoint of the experiment. (C) Tumor volume was measured over time in hPD-1 knock-in mice bearing hPD-L1 MC38 tumors treated with vehicle or TER (10 or 30 mpk). Representative images of excised tumors from each group are shown. (D) Tumor weight at the endpoint of the experiment. (E) Flow cytometry analysis of CD8\u003csup\u003e+\u003c/sup\u003e T-cell populations in tumors from each treatment group. (F) PD-L1 expression in tumors from each group, as assessed by western blot analysis. GAPDH was used as a loading control. (G) IHC staining of tumor sections for immune-related markers, including CD8\u003csup\u003e+\u003c/sup\u003e T cells and GrB. Representative images from each group are shown, and the quantitation of marker-positive cells per field is presented. * \u0026lt; 0.05, ** p \u0026lt; 0.01, *** p \u0026lt; 0.001, and **** p \u0026lt; 0.0001 compared with the respective control.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-7590655/v1/abfb468b8b0532b3f0569fd5.png"},{"id":92172045,"identity":"eec6fd3a-24ec-4d45-a726-24be9352d8bf","added_by":"auto","created_at":"2025-09-25 12:08:08","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":445404,"visible":true,"origin":"","legend":"\u003cp\u003eCD8\u003csup\u003e+\u003c/sup\u003e T-Cell Depletion Abolishes the Antitumor Effects of TER \u003cem\u003ein vivo\u003c/em\u003e. (A) Body weight of hPD-1 knock-in mice during the treatment period. The mice were treated with vehicle or TER (30 mpk) and received either an isotype control or a CD8 depletion antibody. (B) Spleen weight of mice at the endpoint of the experiment. (C) Tumor volume was measured in hPD-1 knock-in mice bearing hPD-L1 MC38 tumors over time following treatment with vehicle or TER (30 mpk) with or without CD8 depletion. Representative images of excised tumors from each group are shown. (D) Tumor weight at the endpoint of the experiment. (E) Flow cytometry analysis confirming CD8\u003csup\u003e+\u003c/sup\u003e T-cell depletion in tumors from each treatment group. The proportion of CD8\u003csup\u003e+\u003c/sup\u003e cells among total live cells was quantified. (F) IHC staining of tumor sections for CD8\u003csup\u003e+\u003c/sup\u003e T cells and GrB. Representative images from each treatment group are shown, and the quantitation of marker-positive cells per field is presented. * \u0026lt; 0.05, ** p \u0026lt; 0.01, and **** p \u0026lt; 0.0001 compared with the respective control.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-7590655/v1/96079f1ec810f7be34943396.png"},{"id":105039489,"identity":"03003d1e-4d8d-408b-9d61-38e70d4d3a59","added_by":"auto","created_at":"2026-03-20 07:46:25","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2771570,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7590655/v1/bad682af-fcb1-4682-8a8c-97049d173235.pdf"},{"id":92172018,"identity":"c0faf144-a9aa-4e04-a666-a9f9c3762917","added_by":"auto","created_at":"2025-09-25 12:08:07","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":213592,"visible":true,"origin":"","legend":"Supplementary Information","description":"","filename":"3.SupplementaryInformation.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7590655/v1/e3c912a0f86d9bb86b3428c6.pdf"},{"id":92172993,"identity":"d3f5944a-b1ec-4239-80a7-66b7b5c5aa82","added_by":"auto","created_at":"2025-09-25 12:16:07","extension":"zip","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":1474524,"visible":true,"origin":"","legend":"qPCR Hct116, RKO Raw data","description":"","filename":"qPCRHct116RKO.zip","url":"https://assets-eu.researchsquare.com/files/rs-7590655/v1/922f63b0e619b15e9c716c9a.zip"},{"id":92173950,"identity":"afc3468e-8a22-40b1-8bc8-3d5542df013e","added_by":"auto","created_at":"2025-09-25 12:24:07","extension":"zip","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":1435917,"visible":true,"origin":"","legend":"qPCR DLD-1 Raw data","description":"","filename":"qPCRDLD1.zip","url":"https://assets-eu.researchsquare.com/files/rs-7590655/v1/34a6eba7112226ff61c9b63c.zip"},{"id":92172014,"identity":"2b935708-f7a1-4521-9f1e-13124738aaed","added_by":"auto","created_at":"2025-09-25 12:08:07","extension":"xlsx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":22608,"visible":true,"origin":"","legend":"qPCR DLD-1, Hct116, RKO data merge","description":"","filename":"qPCRDLD1Hct116RKOdatamerge.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7590655/v1/c5fb600a28e587c1d459d74c.xlsx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e conflict of interest to disclose.","formattedTitle":"Teriflunomide Modulates the PD-1/PD-L1 Axis and Enhances Antitumor Immunity in Colorectal Cancer","fulltext":[{"header":"Introduction","content":"\u003cp\u003eImmune checkpoint inhibitors (ICIs) are a class of cancer immunotherapeutics that target immune checkpoint proteins on T cells, enhancing the body\u0026rsquo;s ability to mount an antitumor immune response (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). These agents have significantly improved survival rates and revolutionized treatment for various malignancies (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). Numerous clinical trials have demonstrated the efficacy of ICIs, leading to their widespread use in cancer therapy (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eImmunotherapy comprises various strategies, including immune checkpoint blockade targeting PD-1/PD-L1 and CTLA-4, as well as chimeric antigen receptor T (CAR-T) cell therapy (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e). PD-1/PD-L1 inhibitors, such as pembrolizumab and nivolumab, prevent PD-L1 on tumor cells from binding to PD-1 receptors on T cells, thereby restoring immune activity (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e). CTLA-4 inhibitors, such as ipilimumab, enhance T-cell activation by blocking CTLA-4-mediated immunosuppression (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e). CAR-T therapy involves engineering T cells to recognize tumor-specific antigens, resulting in an enhanced cytotoxic response (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eDespite these advances, the application of immunotherapy remains limited (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e). Many patients develop resistance to checkpoint inhibitors, which reduces their effectiveness across various cancer types (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e). Moreover, immune-related adverse events (irAEs)\u0026mdash;including inflammation of the skin, gastrointestinal tract, and endocrine organs\u0026mdash;pose significant treatment challenges (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e). CAR-T therapy is also associated with cytokine release syndrome and neurotoxicity, which can be severe (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e). These limitations highlight the need for alternative approaches to enhance immunotherapy while minimizing side effects (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eTeriflunomide (TER) is an FDA-approved disease-modifying agent that is primarily used for the treatment of relapsed multiple sclerosis (MS) (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e). As the active metabolite of leflunomide, TER functions by inhibiting dihydroorotate dehydrogenase (DHODH), a key enzyme in pyrimidine biosynthesis, reducing the proliferation of activated lymphocytes and attenuating the immune response (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e). Owing to its immunosuppressive properties, TER effectively reduces relapse frequency and slows disease progression in patients with MS (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eBesides MS, TER has been examined for its potential efficacy in other autoimmune and inflammatory diseases, including rheumatoid arthritis, spondylo arthritis, and polyarteritis nodosa (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e). In addition, DHODH inhibitors have attracted attention for their anticancer properties, with studies suggesting a role in modulating tumor metabolism and immune function (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e). However, despite its known immunomodulatory effects, the role of TER in regulating immune checkpoint pathways, particularly PD-1/PD-L1 interactions, remains unclear.\u003c/p\u003e\u003cp\u003eCheckpoint inhibitors targeting the PD-1/PD-L1 pathway have shown clinical success, resulting in FDA approval of several antibody-based therapies (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e); however, not all patients respond to PD-1/PD-L1 inhibitors, and resistance mechanisms, such as compensatory immune escape pathways and the immunosuppressive tumor microenvironment, can limit their efficacy (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e). In addition, these therapies may cause immune-related toxicities and affect multiple organ systems (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e). This limitation is particularly evident in colorectal cancer (CRC), where PD-1/PD-L1 blockade shows limited efficacy in microsatellite-stable tumors (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e). Thus, there is a need to explore novel checkpoint inhibitors that can effectively target PD-1/PD-L1 pathway in CRC.\u003c/p\u003e\u003cp\u003eThis study evaluated the potential of TER, an FDA-approved drug for multiple sclerosis, as an immune checkpoint inhibitor for treating CRC. Unlike conventional approaches that primarily target either PD-1 or PD-L1, TER has the unique ability to regulate both PD-1 and PD-L1 simultaneously, which represents a novel strategy for immune checkpoint blockade.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eInhibition of PD-L1 expression by TER\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe human CRC cell lines DLD-1, HCT116, and RKO were treated with TER at the indicated concentrations for 24 hours to evaluate its effects on PD-L1 expression. Cell viability was measured to determine the appropriate TER concentrations (Figure 1A), and subsequent experiments were conducted at doses that maintained cell viability. Western blot analysis revealed that PD-L1 expression decreased in a dose-dependent manner in all cell lines (Figure 1B). To determine whether TER counteracts IFN-\u0026gamma;-induced PD-L1 upregulation, cells were pretreated with IFN-\u0026gamma;, which enhances PD-L1 expression in cancer cells (23). As expected, IFN-\u0026gamma; significantly increased PD-L1 expression; however, this induction was reversed by TER treatment (Figure 1C).\u003c/p\u003e\n\u003cp\u003eTo elucidate the mechanism underlying this downregulation, we determined whether it occurred at the transcriptional level. Real-time PCR analysis revealed that TER did not significantly alter PD-L1 mRNA levels, either under basal conditions or following IFN-\u0026gamma; stimulation. This suggests that the effect of TER on PD-L1 is not mediated by transcriptional repression (Figure 1D). Therefore, we hypothesized that TER regulates PD-L1 at the post-translational level. We performed a cycloheximide (CHX) pulse-chase assay to assess PD-L1 protein stability. The results indicated that the degradation of PD-L1 was significantly enhanced in cells treated with TER compared with that in untreated cells (Figure 1E).\u003c/p\u003e\n\u003cp\u003eTo identify the specific degradation pathway involved, we examined the role of the proteasomal and lysosomal systems. RKO cells were co-treated with TER and either the proteasome inhibitor MG132 or the lysosomal inhibitors, bafilomycin A1 (Baf) and chloroquine (CQ). The increased degradation of PD-L1 by TER was rescued by Baf and CQ, but not by MG132 (Figure 1F). Taken together, these results demonstrate that TER downregulates PD-L1 expression by promoting its lysosome-dependent degradation, thus confirming a post-translational regulatory mechanism.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTER inhibits the PD-1/PD-L1 axis by suppressing PD-L1 expression\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo determine whether TER disrupts the PD-1/PD-L1 axis, we evaluated its effects on cell viability and PD-L1 expression in human CRC cell lines (DLD-1, HCT116, and RKO) and hPD-1 Jurkat-T cells. The cells were treated with indicated doses of TER for 72 h, and cell viability was assessed. TER treatment decreased cell viability in a dose-dependent manner in all of the cell lines, including hPD-1 Jurkat-T cells (Figure 2A). Based on these results, a co-culture system was established using human CRC cells and hPD-1 Jurkat-T cells, which were treated with TER for 72 h. The results revealed a dose-dependent decrease in cell viability (Figure 2B). Western blot analysis was conducted using protein samples from the co-culture system to measure PD-L1 expression. The results indicated that following TER treatment, PD-L1 protein expression significantly decreased in a dose-dependent manner (Figure 2C). Together, these results indicate that TER inhibits the PD-1/PD-L1 axis, primarily through the suppression of PD-L1 expression in cancer cells, suggesting its potential role as an immune checkpoint modulator.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTER interferes with PD-1/PD-L1 binding by directly targeting PD-1\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe interaction between PD-L1 on cancer cells and PD-1 on T cells suppresses T cell function (24). To determine whether TER inhibits this binding, an immunofluorescence assay was performed using RKO cells treated with TER and recombinant human PD-1 Alexa 647 protein. Fluorescence microscopy revealed a dose-dependent reduction in red fluorescence in the TER-treated cells, indicating that TER interferes with the PD-1/PD-L1 interaction by reducing the binding between PD-L1 on RKO cells and recombinant PD-1 protein (Figure 3A).\u003c/p\u003e\n\u003cp\u003eTo determine whether TER selectively inhibits the PD-1/PD-L1 interaction, a competitive ELISA assay was performed to measure the binding between PD-1 and PD-L1. An \u0026alpha;PD-L1-blocking antibody was used as a positive control, as it specifically binds to the extracellular domain of PD-L1 and prevents its interaction with PD-1 (25). TER effectively disrupted PD-1/PD-L1 binding in a concentration-dependent manner, similar to the positive control (Figure 3B). Notably, TER showed an IC50 of 136.7 nM, indicating its potent inhibitory activity against PD-1/PD-L1 interaction.\u003c/p\u003e\n\u003cp\u003eTo determine whether TER directly targets the PD-1 receptor and disrupts its interaction with PD-L1, a molecular docking analysis was performed using the CB-Dock2 platform. The upper panel of Figure 3C shows the 3D structure of TER, which is the small-molecule ligand used for docking. The lower panel shows the predicted binding pose of TER within the ligand-binding domain of human PD-1, based on its crystal structure (PDB ID: 3RRQ). TER was found to interact with several key residues, including PHE56, SER57, ASN58, THR59, SER62, PHE63, PHE82, PRO83, GLN99, ASN102, GLY103, and ARG104, primarily located in the immunoglobulin-like V-type (Ig-like V-type) domain of PD-1, which is responsible for PD-L1 engagement and is required for ligand recognition, as annotated in UniProt (Entry: Q15116). The predicted binding affinity (Vina score) was \u0026ndash;5.9 kcal/mol, indicating a specific interaction. Figure 3D displays the docked complex, where TER is bound within the ligand-binding pocket of PD-1, and the protein is presented in a cartoon format with the binding site highlighted. To validate these computational predictions, SPR analysis was conducted using recombinant human PD-1 protein and TER. The results indicated a strong binding interaction, with an affinity constant (Kd) of 1.47 nM (Figure 3E), thus confirming the high affinity between TER and PD-1.\u003c/p\u003e\n\u003cp\u003eTogether, these data indicate that TER directly binds to PD-1 and likely interferes with PD-1/PD-L1 complex formation, supporting its immunomodulatory effects and providing mechanistic insight into its function.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTER enhances T-cell activation by blocking PD-1/PD-L1 interaction\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe PD-1/PD-L1 axis suppresses T-cell activity and hinders their ability to mount an effective immune response. This immunosuppressive mechanism allows cancer cells to evade immune detection and elimination (26). To determine the effects of TER on T-cell function, a co-culture system was established consisting of CHO-K1 cells engineered to stably express hPD-L1 and a TCR agonist, and Jurkat-T cells engineered to stably express hPD-1 and a TCR-inducible NFAT luciferase reporter. Before evaluating T-cell activation, we measured the cytotoxic effects of TER in both cell lines. The cells were treated with TER at the indicated doses for 24 hours, and viability was measured (Figure 4A).\u003c/p\u003e\n\u003cp\u003eThe NFAT family of transcription factors plays an important role in T-cell activation (27). To determine whether TER-mediated blockade of the PD-1/PD-L1 axis affects T-cell functional activation, we assessed NFAT-mediated luciferase activity using a PD-1/PD-L1 blockade bioassay. In this model system, co-culturing aAPC/CHO-K1 cells with PD-1-expressing effector cells promoted the PD-1/PD-L1 interaction, resulting in the suppression of TCR signaling and NFAT-mediated luciferase activity. Agents that disrupt PD-1/PD-L1 binding may restore TCR signaling and increase luciferase activity, thus serving as an indicator of T-cell activation. The results indicated that the PD-L1-blocking antibody enhanced TCR signaling, with an EC50 of 0.4733 \u0026mu;g/mL. Moreover, TER induced a dose-dependent increase in luciferase activity, with an EC50 of 345.2 nM, indicating its ability to reverse PD-1/PD-L1-mediated immune suppression (Figure 4B).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTER augments CD8\u003csup\u003e+\u003c/sup\u003e T-cell cytotoxicity against CRC cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCD8\u003csup\u003e+\u003c/sup\u003e T cells play an important role in cytotoxicity, cytokine production, and immune surveillance (28). We hypothesized that blocking the PD-1/PD-L1 interaction with TER would enhance CD8\u003csup\u003e+\u003c/sup\u003e T-cell-mediated cytotoxicity against CRC cells. The direct effects of TER on hPD-L1 MC38 cells were first determined. Cells were treated with TER for 72 h, and cell viability was measured, which showed a dose-dependent reduction (Figure 5A). To further examine T-cell-mediated cytotoxicity, tumor formation was induced in hPD-1 knock-in mice using hPD-L1 MC38 cells, and CD8\u003csup\u003e+\u003c/sup\u003e T cells were isolated from the tumors. These tumor-infiltrating CD8\u003csup\u003e+\u003c/sup\u003e T cells were co-cultured with hPD-L1 MC38 cells as the target cells. The results indicated that TER treatment resulted in a dose-dependent increase in cancer cell cytotoxicity, confirming its role in augmentation of T-cell activity (Figure 5B). Western blot analysis was used to determine whether TER modulates PD-L1 expression in a T-cell co-culture setting. The results indicated a dose-dependent reduction in PD-L1 expression (Figure 5C). Furthermore, analysis of key immune-related factors in the co-culture media, including granzyme B (GrB), interleukin-2 (IL-2), and interferon-\u0026gamma; (IFN-\u0026gamma;), which play important roles in T-cell-mediated cytotoxicity and immune activation, revealed that their levels were significantly increased at higher TER doses (Figure 5D). Overall, these findings indicate that TER enhances CD8\u003csup\u003e+\u003c/sup\u003e T-cell cytotoxicity by inhibiting the PD-1/PD-L1 interaction, resulting in increased immune activation and tumor cell elimination.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTER promotes CD8\u003csup\u003e+\u003c/sup\u003e T cell infiltration and inhibits tumor growth \u003cem\u003ein\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003e\u0026nbsp;vivo\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo determine the effects of TER on tumor suppression, \u003cem\u003ein vivo\u003c/em\u003e allograft experiments were carried out using hPD-1 knock-in mice bearing hPD-L1 MC38 tumors. TER was administered at various doses [10 and 30 mpk], and tumor growth was assessed over time. Body and spleen weight remained similar across all groups, indicating that TER does not induce systemic toxicity or remarkable adverse effects (Fig. 6A, B). In contrast, TER treatment significantly reduced tumor volume and weight compared with the vehicle control group (Figure 6C, D).\u003c/p\u003e\n\u003cp\u003eTo determine the effects of TER on CD8\u003csup\u003e+\u003c/sup\u003e T-cell infiltration, we analyzed the proportion of CD8\u003csup\u003e+\u003c/sup\u003e T cells within tumors. Flow cytometry revealed a dose-dependent increase in CD8\u003csup\u003e+\u003c/sup\u003e T-cell infiltration in the TER-treated groups (Figure 6E). We then measured PD-L1 expression levels in tumor tissues. Western blot analysis revealed a dose-dependent decrease in PD-L1 expression following TER treatment, indicating that TER downregulates PD-L1 \u003cem\u003ein vivo\u003c/em\u003e (Figure 6F). Moreover, immunohistochemistry (IHC) revealed a dose-dependent increase in CD8\u003csup\u003e+\u003c/sup\u003e T-cell infiltration and GrB expression in tumor tissues following TER treatment (Figure 6G).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCD8\u003csup\u003e+\u003c/sup\u003e T cell depletion abrogates the antitumor effects of TER \u003cem\u003ein\u003c/em\u003e\u003cem\u003e\u0026nbsp;vivo\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo determine whether the \u003cem\u003ein vivo\u003c/em\u003e antitumor effects of TER are CD8\u003csup\u003e+\u003c/sup\u003e T-cell-dependent, we conducted additional allograft experiments using CD8 depletion antibodies. The mice were divided into four experimental groups: vehicle\u0026ndash;isotype control, vehicle\u0026ndash;CD8 depletion antibody, TER\u0026ndash;isotype control, and TER\u0026ndash;CD8 depletion antibody. TER was administered at a dose of 30 mpk. To elucidate the role of CD8\u003csup\u003e+\u003c/sup\u003e T cells in TER-mediated tumor suppression, the mice in the CD8 depletion groups were treated with a CD8-depleting antibody. No significant changes in body or spleen weight were observed in the groups, suggesting that TER treatment does not cause significant toxicity (Fig. 7A, B). In contrast, CD8 depletion abolished the antitumor effects of TER, as evidenced by the loss of tumor growth suppression in the TER\u0026ndash;CD8 depletion group compared with the TER\u0026ndash;isotype control group (Figure 7C, D). These results indicate that TER\u0026apos;s efficacy is dependent on CD8\u003csup\u003e+\u003c/sup\u003e T cells.\u003c/p\u003e\n\u003cp\u003eFlow cytometry revealed that TER treatment resulted in an increase in CD8\u003csup\u003e+\u003c/sup\u003e T-cell infiltration within the tumor. However, this effect was abolished by CD8 depletion, confirming the dependency of its antitumor effects on CD8\u003csup\u003e+\u003c/sup\u003e T cells (Figure 7E). In addition, IHC analysis revealed an increase in CD8\u003csup\u003e+\u003c/sup\u003e T-cell infiltration and GrB expression in tumor tissues following TER treatment; however, these effects were reversed by CD8\u003csup\u003e+\u003c/sup\u003e T-cell depletion, thus confirming the essential role of CD8\u003csup\u003e+\u003c/sup\u003e T cells in TER-mediated tumor suppression (Figure 7F).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eWe demonstrated that TER, an FDA-approved drug for multiple sclerosis, acts as a dual inhibitor of both PD-1 and PD-L1, promoting CD8\u003csup\u003e+\u003c/sup\u003e T-cell-mediated antitumor immunity in CRC models. TER directly binds to PD-1, preventing its interaction with PD-L1, and also downregulates PD-L1 expression in tumor cells. These results highlight the potential of TER as a novel immune checkpoint modulator with dual functionality targeting both PD-1 and PD-L1 to enhance the antitumor immune response in CRC.\u003c/p\u003e\u003cp\u003eCRC is a leading cause of cancer-related mortality worldwide, and ICIs exhibit significant efficacy in a subset of patients (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e). PD-1/PD-L1 blockade has shown clinical benefit primarily in microsatellite instability-high (MSI-H) or mismatch repair-deficient (dMMR) CRC, which exhibits a high tumor mutational burden and increased immune cell infiltration (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e). However, the majority of CRC cases are MSS, which are generally unresponsive to PD-1/PD-L1 inhibitors because of the immunosuppressive tumor microenvironment (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e). This emphasizes the need for alternative or complementary strategies to enhance antitumor immunity in CRC, particularly in MSS subtypes (\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e). TER may enhance antitumor immunity by modulating the immune response, including enhancing CD8\u003csup\u003e+\u003c/sup\u003e T-cell activation and modifying the tumor microenvironment. This may overcome some of the resistance mechanisms observed with traditional immune checkpoint therapies. Because of its dual ability to block PD-1 and reduce PD-L1 expression, TER represents a promising candidate for addressing these limitations.\u003c/p\u003e\u003cp\u003ePrevious studies have primarily focused on PD-1 or PD-L1 inhibitors, with several monoclonal antibodies, such as pembrolizumab and nivolumab, showing efficacy in various cancers (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e). However, many patients exhibit limited response because of immune resistance mechanisms and insufficient T-cell activation (\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e). In contrast, TER uniquely targets both PD-1 and PD-L1 simultaneously, offering a more comprehensive strategy for enhancing the immune response. Our study builds on previous work showing the importance of PD-1/PD-L1 blockade in overcoming tumor-induced immune suppression and also introduces TER as a dual-action immune modulator.\u003c/p\u003e\u003cp\u003eOur results suggest that TER enhances CD8\u003csup\u003e+\u003c/sup\u003e T-cell infiltration and activation within tumors, likely through its direct inhibition of PD-1 and a reduction of PD-L1 expression. Moreover, TER may induce apoptosis in tumor cells, which further contributes to its antitumor effects (\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e). Flow cytometry and IHC analyses confirmed that TER significantly increases CD8\u003csup\u003e+\u003c/sup\u003e T-cell infiltration and GrB expression, indicating enhanced cytotoxic activity against cancer cells. CD8 depletion experiments further confirmed the essential role of CD8\u003csup\u003e+\u003c/sup\u003e T cells in mediating the antitumor effects of TER. These results are consistent with those of other studies showing that CD8\u003csup\u003e+\u003c/sup\u003e T-cell activation is important for the positive effects of ICIs (\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eIn the present study, we examined the potential of TER as a dual inhibitor targeting both PD-1 and PD-L1. Although TER demonstrated a strong binding affinity for PD-1, as confirmed by SPR analysis, no binding was observed between TER and PD-L1 (data not shown). This suggests that TER exerts its immune checkpoint inhibition primarily through a direct interaction with PD-1, rather than directly affecting PD-L1. This finding aligns with that of previous studies indicating that many PD-1 inhibitors function by disrupting the PD-1/PD-L1 interaction without binding directly to PD-L1 (\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e). Interestingly, our data clearly demonstrate that TER effectively downregulates PD-L1 expression via the lysosomal degradation pathway. Nevertheless, a direct interaction between TER and PD-L1 was not detected in our experiments, suggesting that the upstream mechanism initiating this process remains unclear. These findings underscore a current limitation of our study and highlight the complexity of TER\u0026rsquo;s mode of action. We speculate that TER may function through an indirect mechanism, such as by interacting with an unknown intermediary protein or by modulating an upstream signaling pathway that targets PD-L1 for degradation. Therefore, future studies are warranted to precisely identify the upstream mediators of this effect. Overall, these results underscore the complex nature of TER\u0026rsquo;s immunomodulatory effects, in which direct PD-1 blockade enhances CD8\u0026thinsp;+\u0026thinsp;T-cell activation, while a distinct, indirect mechanism of PD-L1 downregulation contributes to a broader antitumor immune response.\u003c/p\u003e\u003cp\u003eTER is approved for the treatment of multiple sclerosis, in which it acts as an immunomodulator by inhibiting dihydroorotate dehydrogenase (DHODH) and limiting the proliferation of activated lymphocytes (\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e). This immunoregulatory function of TER suggests that its effects on the PD-1/PD-L1 axis may extend beyond direct checkpoint inhibition. The ability of TER to influence T-cell responses and immune regulation in an inflammatory disease setting may translate into enhanced antitumor immunity, which warrants further examination into its broader immunomodulatory effects.\u003c/p\u003e\u003cp\u003eAn important aspect of our findings is the apparent paradox that TER, a known immunosuppressant, induces potent antitumor immunity within the tumor microenvironment. Although TER suppresses the immune system during multiple sclerosis by inhibiting the proliferation of activated lymphocytes (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e), we demonstrated its function as a CD8\u003csup\u003e+\u003c/sup\u003e T-cell activator through a PD-1/PD-L1 axis blockade. This dual functionality may be explained by the unique cellular dynamics of the TME. For example, the primary mechanism of TER, which is DHODH inhibition, targets highly proliferative cells. This may affect not only effector T cells, but also proliferative immunosuppressive populations, such as regulatory T cells (Tregs), which are abundant in the TME (\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e). Consequently, the suppression of the Treg proliferation within the TME may synergize with the direct blockade of PD-1 by TER. This results in a net positive effect that enhances CD8\u003csup\u003e+\u003c/sup\u003e T-cell-mediated antitumor immunity, and suggests that TER is not merely an immunosuppressant, but a sophisticated immunomodulator whose function is contingent on a specific immunological context.\u003c/p\u003e\u003cp\u003eAlthough the use of murine models presents inherent limitations in terms of translational relevance to human cancers, we used humanized models involving human PD-1 knock-in mice and hPD-L1 MC38 cells. These models provide a more accurate representation of human tumor\u0026ndash;immune interactions compared with traditional murine models (\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e). Although this approach is not fully representative of human physiology, the use of these humanized models enables the evaluation of TER\u0026rsquo;s effects on the PD-1/PD-L1 axis and CD8\u003csup\u003e+\u003c/sup\u003e T-cell-mediated immunity, thus enhancing the reliability of our findings.\u003c/p\u003e\u003cp\u003eIn addition to its potential efficacy as a monotherapy, TER may also exhibit synergy when combined with existing PD-1/PD-L1 inhibitors. Monoclonal antibodies targeting PD-1 or PD-L1, such as pembrolizumab and nivolumab, have demonstrated remarkable clinical success; however, a significant proportion of patients do not respond or develop resistance to these agents (\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e). The ability of TER to block PD-1 while simultaneously downregulating PD-L1 suggests its potential to enhance the efficacy of these antibody-based therapies by providing an additional checkpoint inhibition mechanism. Moreover, as a small-molecule inhibitor, TER offers advantages such as enhanced tissue penetration and suitability for oral delivery, which may improve patient compliance and expand therapeutic accessibility. Future studies should examine the efficacy of TER combined with PD-1/PD-L1 antibodies for enhancing the immune response and overcoming resistance in non-responding patients.\u003c/p\u003e\u003cp\u003eAs TER is already an FDA-approved drug, the barrier for clinical translation is lower compared with novel drug candidates (\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e). Most small-molecule inhibitors or monoclonal antibodies targeting the PD-1/PD-L1 axis require extensive safety and pharmacokinetic evaluation before reaching clinical trials (\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e). Because TER has already been approved for treating multiple sclerosis, its established safety profile, pharmacodynamics, and clinical tolerability provide a strong foundation for its potential repurposing in cancer immunotherapy (\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e). Repositioning an existing drug for oncological applications offers several advantages, including reduced time and cost associated with drug development, a faster regulatory approval process, and improved accessibility for patients. Given its demonstrated efficacy in enhancing CD8\u003csup\u003e+\u003c/sup\u003e T-cell-mediated antitumor responses, further clinical studies are needed to evaluate the efficacy of TER as an immune checkpoint modulator for cancer treatment.\u003c/p\u003e\u003cp\u003eIn conclusion, TER shows potential as a dual PD-1/PD-L1 inhibitor, enhancing CD8\u003csup\u003e+\u003c/sup\u003e T-cell-mediated antitumor immunity in CRC. Because of its established safety profile as an FDA-approved drug, TER represents a promising therapeutic drug for enhancing cancer immunotherapy, particularly in patients who are unresponsive to traditional PD-1/PD-L1 inhibitors.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003eReagents\u003c/h2\u003e\u003cp\u003eTER was purchased from Targetmol (Boston, MA, USA). The PD-1/PD-L1 Blockade Bioassay kit was obtained from Promega (Madison, WI, USA). The cell counting kit-8 was purchased from Dojindo Molecular Technologies (Rockville, MD, USA). For cell dissociation and purification, a tumor dissociation kit, MACSmix tube rotator, gentleMACS C tubes, and gentleMACS dissociator were obtained from Miltenyi Biotec (Auburn, CA, USA). The anti-PD-L1 antibodies were purchased from BPS Bioscience (San Diego, CA, USA). Anti-PD-1 antibodies were purchased from Selleck Chemicals (Houston, TX, USA). A magnet for column-free cell separation, a Mouse CD8\u003csup\u003e+\u003c/sup\u003e T Cell Isolation Kit, and polystyrene round-bottom tubes were purchased from STEMCELL Technologies (Vancouver, BC, Canada). Mouse IL-2 and IFN-gamma ELISAs were purchased from BD Biosciences (San Diego, CA, USA), and a Mouse GrB ELISA was purchased from Thermo Fisher Scientific (Waltham, MA, USA). Anti-CD8 antibodies were obtained from Cell Signaling Technology (Danvers, MA, USA).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eCell culture\u003c/h2\u003e\u003cp\u003eHumanized PD-L1-expressing MC38 cells, derived from C57BL/6 mouse CRC, were obtained from the Shanghai Model Organisms Center (Shanghai, China). The cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM) containing 10% heat-inactivated fetal bovine serum (FBS) and antibiotics (100 units/ml penicillin).\u003c/p\u003e\u003cp\u003eRecombinant Jurkat-T cells expressing human PD-1 and an NFAT reporter gene (hPD-1/NFAT Jurkat-T cells, #60535) and recombinant CHO-K1 cells expressing human PD-L1 and a T-cell receptor (TCR) activator (hPD-L1/TCR CHO-K1 cells, #60536) were obtained from BPS Bioscience. Jurkat and CHO-K1 cells were infected with human PD-1- and PD-L1-expressing lentiviruses (GeneChem, Shanghai). The cells were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium containing 10% heat-inactivated FBS and antibiotics (100 units/ml penicillin).\u003c/p\u003e\u003cp\u003eHuman CRC cells (DLD-1, HCT116, and RKO) were obtained from the Korean Cell Line Bank (Seoul, Korea) and cultured in DMEM containing 10% heat-inactivated FBS and antibiotics (100 units/ml penicillin). All cell culture solutions were obtained from Hyclone Laboratories Inc. (Chicago, IL, USA). The cells were cultured at 37\u0026deg;C in a humidified 5% CO\u003csub\u003e2\u003c/sub\u003e incubator.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eCell viability assay\u003c/h2\u003e\u003cp\u003eCell viability was measured using the CCK assay (#CK04, Dojindo Molecular Technologies, Inc., Rockville, MD, USA) based on the manufacturer's instructions. Briefly, cells were seeded into 96-well plates at a density of 1 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e cells per well and incubated overnight, followed by treatment with TER. A range of TER concentrations was added to the wells. After the designated incubation period, 10 \u0026micro;L of CCK solution was added to each well, and the plates were incubated for 2 h at 37\u0026deg;C. The absorbance at 450 nm was measured using a microplate reader (Molecular Devices i3, San Jose, CA, USA), and cell viability was calculated.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003eWestern blotting analysis\u003c/h2\u003e\u003cp\u003eThe cells were washed with 1\u0026times; PBS, and total protein lysates were prepared using RIPA buffer containing 1% NP-40 lysis buffer and protease inhibitor cocktail tablets (Roche, Basel, Switzerland). Protein concentration was measured using the Bio-Rad protein assay. Equivalent amounts of protein were separated by 8\u0026ndash;15% SDS-PAGE and transferred to nitrocellulose membranes (GE Healthcare, Munich, Germany). The membranes were blocked with 5% nonfat dry milk at room temperature (20\u0026ndash;25\u0026deg;C) for 1 h and incubated overnight at 4\u0026deg;C with primary antibodies. The membranes were washed three times with 1\u0026times; Tris-buffered saline for 10 min per wash, followed by incubation with the corresponding secondary antibody. After washing, protein detection was performed using the ChemiDoc Touch Imaging System (Bio-Rad, Hercules, USA). GAPDH was used as a loading control for protein normalization. The original uncropped images for all western blots are provided in the Supplementary Information (Supplementary Figures \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u0026ndash;S6).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003eQuantitative reverse transcription\u0026ndash;polymerase chain reaction (qRT\u0026ndash;PCR)\u003c/h2\u003e\u003cp\u003eTotal RNA was isolated using the RiboEx Total RNA Extraction Kit (GeneAll Biotechnology, Seoul, Korea). Complementary DNA (cDNA) was synthesized using the AccuPower CycleScript RT premix (dT20) (Bioneer, Daejeon, Korea). For the reverse transcription reaction, 1 \u0026micro;g of total RNA was used in a final cDNA reaction volume of 20 \u0026micro;L. Quantitative PCR was performed using a QuantStudio 3D Digital PCR System (Thermo Fisher Scientific, Waltham, MA, USA) along with the AccuPower 2X Greenstar qPCR master mix (Bioneer). The protocol consisted of 40 cycles of denaturation at 95\u0026deg;C for 15 sec, followed by annealing and extension at 60\u0026deg;C for 30 sec per cycle. All reagents were used according to the manufacturer\u0026rsquo;s instructions. The relative expression of target mRNA were normalized to GAPDH expression as an endogenous control.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003eCompetitive ELISA\u003c/h2\u003e\u003cp\u003eA competitive ELISA was performed using the PD-1/PD-L1 inhibitor ELISA screening kit (BPS Bioscience, #72005, San Diego, CA, USA), following the manufacturer's instructions. The αPD-L1 antibody (#71213) served as the positive control. Recombinant hPD-L1 (#71104, BPS Bioscience) was coated onto 96-well plates (Corning Inc., New York, NY, USA) at a concentration of 1 mg/mL in PBS and incubated overnight. The plates were then washed with PBS containing 0.1% Tween (PBS-T), blocked with 2% BSA in PBS for 1 h at room temperature, and re-washed. Next, 5 \u0026micro;L of 0.5 mg/mL biotinylated hPD-1 (#71109, BPS Bioscience) was added, and the plates were incubated for 2 h at room temperature. After three washes in PBS-T, 50 \u0026micro;L of 0.2 mg/mL HRP-conjugated streptavidin was added and the plates were incubated for 1 h. Finally, the plates were washed three times with PBS-T, and chemiluminescence was measured using a SpectraMax L Luminometer (Molecular Devices, San Jose, CA, USA).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003ePD-1/PD-L1 blockade bioassay\u003c/h2\u003e\u003cp\u003eTo determine the effectiveness of TER in blocking the PD-1/PD-L1 interaction, a cell-based PD-1/PD-L1 blockade bioassay was performed using the PD-1/PD-L1 blockade bioassay kit (#J4011, Promega, Madison, WI, USA), with slight modifications to the manufacturer\u0026rsquo;s instructions. In this assay, 5 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e PD-L1 aAPC/CHO-K1 cells were seeded into 96-well plates containing DMEM supplemented with 10% FBS. On the experiment day, the medium was removed, and PD-L1-blocking antibody or TER (as indicated) was added along with 1 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e PD-1 effector cells. Luminescence was measured using the GloMax\u0026reg; Explorer Multimode Microplate Reader (Promega) after adding Bio-Glo\u0026trade; Reagent (Promega, #G7940). The results are expressed as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error of the mean from four independent experiments.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003eTumor-infiltrating t cells (TILs) isolation\u003c/h2\u003e\u003cp\u003eA humanized PD-1 mouse model (C57BL/6J background, genetically engineered to express the full-length human PD-1 protein) was obtained from the Shanghai Model Organisms Center (Shanghai, China). The animal experiments were conducted in compliance with the guidelines established by the Institutional Animal Care and Use Committee (IACUC) of the Korea Institute of Oriental Medicine (KIOM) and approved by the IACUC (approval number: KIOM-D-23-099). To extract tumor-infiltrating lymphocytes (TILs, CD8\u003csup\u003e+\u003c/sup\u003e T cells), MC38 tumor tissues were collected and digested with collagenase IV (0.5 mg/mL) at 37\u0026deg;C for 1 h. The resulting cell suspension was strained twice using 100-\u0026micro;m and 40-\u0026micro;m cell strainers (SPL) to isolate single cells. TILs were isolated from the single-cell suspension using negative selection beads according to the manufacturer\u0026rsquo;s instructions (STEMCELL Technologies Inc., Vancouver, Canada).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003eCo-culture experiments\u003c/h2\u003e\u003cp\u003eTumor-infiltrating CD8\u003csup\u003e+\u003c/sup\u003e T cells (1 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e cells) were used as effector cells and activated with Dynabeads T Activator CD3/CD28 (Life Technologies, Carlsbad, CA, USA) for 72 h at 37\u0026deg;C. Murine hPD-L1 MC38 cells, which served as target cells, were labeled using the CellTrace\u0026trade; Far Red Cell Proliferation Kit (Thermo Fisher Scientific, Waltham, MA, USA) and treated with IFN-γ (10 ng/mL) for 24 h at 37\u0026deg;C to induce PD-L1 expression. hPD-L1 MC38 cells (5 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e cells) were co-cultured with the activated CD8\u003csup\u003e+\u003c/sup\u003e T cells (2.5 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells) at an effector-to-target cell ratio of 5:1 for 72 h at 37\u0026deg;C in the presence of different concentrations of TER. Then, the plates were washed with PBS, and the remaining viable cancer cells were stained with crystal violet solution and quantified using a SpectraMax i3 microplate reader at 540 nm.\u003c/p\u003e\u003cp\u003e\u003cb\u003eIn vivo\u003c/b\u003e \u003cb\u003eanalysis\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe experimental mice (male, n\u0026thinsp;=\u0026thinsp;6) were housed in a specific pathogen-free facility and anesthetized using a 2% isoflurane\u0026ndash;oxygen mixture administered through an Avesko inhalation system. To induce subcutaneous tumor formation, hPD-L1 MC38 cells were injected in the right flank of humanized PD-1 mice. Each mouse received 200 \u0026micro;L of cell suspension (3.0 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e cells/mL, equivalent to 1.5 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells/mouse). Tumor progression was assessed biweekly using a digital caliper (Hi-Tech Diamond, Westmont, IL, USA). Tumor volume was calculated using the formula: (W\u003csup\u003e2\u003c/sup\u003e \u0026times; L)/2, where W represents the short diameter and L represents the long diameter.\u003c/p\u003e\u003cp\u003eOnce the tumors reached a volume of 100 mm3, the mice were divided into several experimental groups. The control group received vehicle [PBS, 10 mL/kg, orally (q.d., i.g.)], whereas the other groups were treated with either low or high doses of TER [10 or 30 mg/kg (mpk), administered intraperitoneally (q.d., i.p.)], or a CD8 depletion antibody (#BP0061, Bio Cell, West Lebanon, NH, USA). TER was administered continuously for 16 consecutive days. In contrast, the CD8 depletion antibody was administered twice a week through intragastric injections beginning after tumor implantation. On day 16 following treatment, the animals were euthanized for further analysis.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003eTissue dissociation for cell isolation and flow cytometry\u003c/h2\u003e\u003cp\u003eTumor tissues were collected in RPMI Medium 1640 containing 10% heat-inactivated FBS, 1% penicillin and streptomycin (all from HyClone\u0026trade;), minced, and incubated for 30 min in DPBS (Well-gene) with 0.5 mg/mL Collagenase IV (Sigma) at 37\u0026deg;C. The tissues were squeezed through a 100 \u0026micro;m cell strainer (#93100, SPL), re-filtered through a 70 \u0026micro;m cell strainer (#93070, SPL), and incubated for 5 min in Red Blood Cell Lysis Buffer (#10-548E, Lonza). Flow cytometry was conducted to determine cell surface antigen expression by a 30-min incubation on ice with the following antibodies: mouse-specific monoclonal antibodies PerCP/Cyanine5.5 anti-mouse CD45 (#103131, BioLegend), APC anti-mouse CD8a (#100712, BioLegend), corresponding isotype control mAbs PerCP/Cy5.5 Rat IgG2b (#400632, BioLegend), and APC Rat IgG2a (#400512, BioLegend). The cells were washed three times with PBS containing 2% FBS, fixed in suspension with 4% paraformaldehyde, and stored at 4\u0026deg;C until subsequent analysis with a CytoFLEX flow cell counter (Beckman). The data were analyzed using Kaluza analysis software (Beckman Coulter).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\u003ch2\u003eSurface plasmon resonance analysis\u003c/h2\u003e\u003cp\u003eSPR was used to measure binding affinity between PD-L1 and PD-1 proteins using a Biacore T200 biosensor (GE HealthCare Technologies, Inc., Chicago, IL, USA) with a nitrilotriacetic acid (NTA) sensor chip (GE HealthCare). The NTA surfaces were washed with 350 mM EDTA and loaded with 0.5 mM NiCl2. The hPD-1 protein (#10377-H08H, Sino Biologicals) was bound to the NTA sensor chip using an affinity capture method. Various concentrations of analytes were prepared by diluting in HBS-P buffer (10 mM HEPES, pH 7.4, 150 mM NaCl, and 0.005% surfactant P20). The equilibrium dissociation constants (KD; kd/ka) were calculated using sensorgrams.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e\u003ch2\u003eMolecular docking analysis\u003c/h2\u003e\u003cp\u003eTo examine the interaction between human PD-1 and TER, molecular docking was conducted using the CB-Dock2 platform, an automated docking tool capable of cavity detection and AutoDock Vina-based docking (\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e). The target protein structure was obtained from the Protein Data Bank (PDB ID: 3RRQ) (\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e) and used in its native form without any mutations or modifications. TER was used as a ligand, and its 3D structure file (.sdf) was downloaded from the PubChem database (CID: 54684141) (\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e). CB-Dock2 automatically detected potential binding cavities within the PD-1 structure, and the ligand was docked using AutoDock Vina. Subsequently, the interaction between PD-1 and Teriflunomide was visualized with the built-in visualization tools of the CB-Dock2 platform.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec22\" class=\"Section2\"\u003e\u003ch2\u003eStatistical analysis\u003c/h2\u003e\u003cp\u003eThe data are presented as ratios relative to their respective control values. The results are shown as means\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error of the mean (SEM) based on three independent experimental replicates. Differences between the mean values within each group were evaluated using a Student's t-test, whereas comparisons across multiple groups were done using a one-way analysis of variance, followed by Tukey\u0026rsquo;s post hoc test. The statistical analyses were performed using GraphPad Prism version 8.0 (GraphPad Software, San Diego, CA, USA).\u003c/p\u003e\u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors would like to thank Enago (www.enago.co.kr) for English language editing. This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Ministry of Science and ICT (NRF-2022R1A2C2092834), and by the Korea Institute of Oriental Medicine (KIOM) grant (KSN2412013), also provided by the Ministry of Science and ICT, Korea.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eJung Ho Han\u003c/strong\u003e: Data curation, Investigation, Visualization, Writing \u0026ndash; original draft, Writing \u0026ndash; review \u0026amp; editing. \u003cstrong\u003eEun-Ji Lee\u003c/strong\u003e: Conceptualization, Data curation, Investigation, Writing \u0026ndash; original draft. \u003cstrong\u003eYoung-Hoon Park\u003c/strong\u003e: Investigation. \u003cstrong\u003eJung-Hye Ha\u003c/strong\u003e: Investigation. \u003cstrong\u003eKazi Rejvee Ahmed\u003c/strong\u003e: Investigation. \u003cstrong\u003eJang-Gi Choi\u003c/strong\u003e: Conceptualization, Funding acquisition, Investigation, Writing \u0026ndash; review \u0026amp; editing. \u003cstrong\u003eHwan-Suck Chung\u003c/strong\u003e: Funding acquisition, Writing \u0026ndash; review\u0026nbsp;\u0026amp;\u0026nbsp;editing\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eWoo S-R, Turnis ME, Goldberg MV, Bankoti J, Selby M, Nirschl CJ, et al. 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Experimental biology and medicine. 2015;240(4):426-37. \u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"oncogenesis","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"oncsis","sideBox":"Learn more about [Oncogenesis](http://www.nature.com/oncsis/)","snPcode":"41389","submissionUrl":"https://mts-oncsis.nature.com/cgi-bin/main.plex","title":"Oncogenesis","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Teriflunomide, Colorectal cancer, Immunotherapy, PD-1/PD-L1 axis, CD8+ T cell","lastPublishedDoi":"10.21203/rs.3.rs-7590655/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7590655/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eInhibitors that target the programmed cell death protein 1 (PD-1)/programmed death-ligand 1 (PD-L1) immune checkpoint pathway have revolutionized cancer immunotherapy; however, many patients exhibit a limited response. In this study, we examined the potential of teriflunomide (TER), an FDA-approved drug for multiple sclerosis, as a novel immune checkpoint modulator for treating colorectal cancer (CRC). We determined the effect of TER on PD-L1 expression in human CRC cell lines, its direct binding to PD-1, and its impact on CD8\u003csup\u003e+\u003c/sup\u003e T-cell function. Antitumor activity was determined \u003cem\u003ein vivo\u003c/em\u003e using a humanized mouse model of hPD-1 knock-in mice implanted with hPD-L1 expressing MC38 tumor cells. TER treatment reduced PD-L1 expression in CRC cells and disrupted the PD-1/PD-L1 interaction directly. \u003cem\u003eIn vivo\u003c/em\u003e, TER significantly suppressed tumor growth without systemic toxicity, and enhanced the infiltration and activation of CD8\u003csup\u003e+\u003c/sup\u003e T cells within tumors, as evidenced by increased granzyme B expression. Moreover, the antitumor efficacy of TER was abolished by the depletion of CD8\u003csup\u003e+\u003c/sup\u003e T cells, which indicated its dependency on this cell population. These findings highlight TER as a promising immune checkpoint modulator that targets the PD-1/PD-L1 axis to promote CD8\u003csup\u003e+\u003c/sup\u003e T-cell-mediated antitumor immunity. Because of its established safety profile, TER is a readily translatable therapeutic for enhancing cancer immunotherapy in CRC.\u003c/p\u003e","manuscriptTitle":"Teriflunomide Modulates the PD-1/PD-L1 Axis and Enhances Antitumor Immunity in Colorectal Cancer","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-25 12:08:02","doi":"10.21203/rs.3.rs-7590655/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"revise","date":"2025-10-09T15:08:22+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"This content is not available.","date":"2025-10-09T07:13:51+00:00","index":2,"fulltext":"This content is not available."},{"type":"editorInvitedReview","content":"This content is not available.","date":"2025-09-27T10:07:59+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2025-09-23T18:06:24+00:00","index":2,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2025-09-23T00:46:30+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewersInvited","content":"","date":"2025-09-16T13:53:08+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-09-15T15:32:10+00:00","index":"","fulltext":""},{"type":"submitted","content":"Oncogenesis","date":"2025-09-15T01:41:16+00:00","index":"","fulltext":""},{"type":"checksFailed","content":"","date":"2025-09-12T12:57:14+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-09-11T10:18:41+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"oncogenesis","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"oncsis","sideBox":"Learn more about [Oncogenesis](http://www.nature.com/oncsis/)","snPcode":"41389","submissionUrl":"https://mts-oncsis.nature.com/cgi-bin/main.plex","title":"Oncogenesis","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"90f0177c-7852-4fa9-bd62-81b4764727e5","owner":[],"postedDate":"September 25th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":54817534,"name":"Biological sciences/Cancer/Tumour immunology"},{"id":54817535,"name":"Biological sciences/Immunology/Immunotherapy"}],"tags":[],"updatedAt":"2026-03-20T07:41:24+00:00","versionOfRecord":{"articleIdentity":"rs-7590655","link":"https://doi.org/10.1038/s41389-026-00607-3","journal":{"identity":"oncogenesis","isVorOnly":false,"title":"Oncogenesis"},"publishedOn":"2026-03-18 04:00:00","publishedOnDateReadable":"March 18th, 2026"},"versionCreatedAt":"2025-09-25 12:08:02","video":"","vorDoi":"10.1038/s41389-026-00607-3","vorDoiUrl":"https://doi.org/10.1038/s41389-026-00607-3","workflowStages":[]},"version":"v1","identity":"rs-7590655","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7590655","identity":"rs-7590655","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}