Targeting Immunoglobulin Superfamily Member 9 (IGSF9) to Overcome Acute Myeloid Leukemia Resistance to CAR-T Therapy

Targeting Immunoglobulin Superfamily Member 9 (IGSF9) to Overcome Acute Myeloid Leukemia Resistance to CAR-T Therapy | 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 Research Article Targeting Immunoglobulin Superfamily Member 9 (IGSF9) to Overcome Acute Myeloid Leukemia Resistance to CAR-T Therapy Xianhui Meng, Fangmin Li, Hong Yu, Chunling Li, Yuxiao Sun, Hongying Wang, and 7 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8606613/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 10 You are reading this latest preprint version Abstract Background: Chimeric antigen receptor (CAR)-T cell therapy faces substantial barriers in acute myeloid leukemia (AML), yet the mechanisms by which AML evades CAR-T cell cytotoxicity—through tumor-intrinsic factors and microenvironmental suppression—remain poorly defined. Immunoglobulin superfamily member 9 (IGSF9) has recently been identified as an immunosuppressive molecule selectively expressed on AML blasts, but its role in mediating resistance to CAR-T therapy is unknown. Methods : We examined IGSF9 expression in AML cells upon CAR-T challenge and conducted in vitro and in vivo assays to define its functional impact on CAR-T cell activity. To overcome IGSF9-mediated suppression, we evaluated two therapeutic strategies: antibody-mediated IGSF9 blockade and the generation of IGSF9-specific CAR-T cells (IG9BBz). Results: CAR-T–induced cytotoxic pressure upregulated IGSF9 on AML cells. IGSF9-positive AML cells exhibited resistance to CAR-T killing and impaired CAR-T persistence both in vitro and in vivo . Antibody blockade of IGSF9 restored CAR-T function in xenograft models. Moreover, IG9BBz CAR-T cells demonstrated potent and selective elimination of IGSF9-positive AML cells in both settings. Conclusions: Our findings identify a cytokine-driven IGSF9 resistance circuit that suppresses CAR-T cell function in AML. Therapeutic disruption of this pathway through IGSF9 blockade or IGSF9-specific CAR-T cells restores antitumor immunity and provides complementary strategies to overcome CAR-T resistance in AML. AML CAR-T IGSF9 Immune suppression Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Background Acute myeloid leukemia (AML) is an aggressive hematologic malignancy characterized by clonal expansion of myeloid progenitors. It accounts for over 80% of adult acute leukemias(1). Despite advances in therapy, the prognosis remains poor, with 5-year survival rates stagnating at 20-30%. Although FLT3 inhibitors and venetoclax-based regimens have improved outcomes for certain patient subgroups, elderly and high-risk cohorts experience relapse rates exceeding 60%. Allogeneic hematopoietic stem cell transplantation (allo-HSCT), though potentially curative, faces significant limitations including donor scarcity, graft-versus-host disease (GVHD) and relapse rates greater than 30%(2–4). These unmet clinical needs underscore the imperative for novel therapeutic strategies in AML. Chimeric antigen receptor (CAR) T-cell therapy, which enables major histocompatibility complex (MHC)-independent tumor targeting, has revolutionized the treatment of B-cell malignancies, achieving remission rates exceeding 90% in relapsed/refractory disease(5). However, its translation to AML has been largely ineffective, with clinical response rates consistently below 30% despite promising preclinical targets such as CD33, CD123, CLL1(6–8). This therapeutic failure stems not only from antigenic heterogeneity but also from the profoundly immunosuppressive AML microenvironment(9–12). The mechanisms by which AML evades CAR-T cell cytotoxicity-through both intrinsic tumor factors and microenvironmental suppression-remain poorly understood. Immunoglobulin superfamily member 9 (IGSF9) is a transmembrane protein composed of five immunoglobulin (Ig)-like domains, two fibronectin type III (FNIII) domains, a transmembrane region and an intracellular immunoreceptor tyrosine-based inhibitory motif (ITIM)(13). Originally implicated in neurodevelopment, IGSF9 is aberrantly overexpressed in multiple carcinomas, where it correlates with poor prognosis and drives metastasis through the induction of epithelial-to-mesenchymal transition(14–17). Critically, we recently identified IGSF9 as a pan-cancer immunosuppressive checkpoint that orchestrates immune-evasive microenvironments by directly impairing cytotoxic lymphocyte function. Blockade of IGSF9 reversed T cell dysfunction and suppressed tumor growth in various tumor models(18). Notably, we observed highly restricted IGSF9 expression on leukemic blasts from AML patients, with minimal or absent expression in normal hematopoietic stem cells and most other major tissues(19). This tumor-selective expression profiles IGSF9 as a compelling antigenic target for AML immunotherapy. IFNγ has been identified as a major inducer of IGSF9 expression in AML (19). Given that IFNγ is abundantly secreted by activated CAR-T cells, we hypothesized that cytokine-driven upregulation of IGSF9 contributes to AML resistance against CAR-T therapy. However, the mechanisms by which AML cells exploit IGSF9 to evade immune surveillance, particularly CAR-T–mediated cytotoxicity, remain undefined. Here, we delineate the role of IGSF9 in orchestrating AML immune escape and engineer next-generation IG9BBz CAR-T cells to neutralize this immunosuppressive pathway. Our findings establish a mechanistic and therapeutic framework for overcoming resistance in AML immunotherapy. Methods Cells and patient samples All cell lines were preserved by our lab and verified by STR(18,19). The AML cell lines (THP-1, MOLM13, U937, MV4-11) were cultured in RPMI1640 medium (10% FBS, 1% penicillin/streptomycin), The adherent cells (293T, LL/2, A549, H1299) were cultured in DMEM medium (10% FBS, 1% penicillin/streptomycin). Human primary T cells were cultured in RPMI1640 medium (10% FBS, 1% penicillin/streptomycin, 5ng/ml IL-7, 5ng/ml IL-15). Mouse primary T cells were cultured in RPMI1640 medium (10% FBS, 1% penicillin/streptomycin, 10ng/ml IL-2). Fresh bone marrow aspirates of AML patients were obtained from Yantai Yuhuangding Hospital with informed consent. Production of CAR-T cells Human peripheral blood mononuclear cells (PBMC) were isolated from healthy blood, which obtained from Yantai central blood station. Then the cells were stimulated by CD3/CD28 Dynabeads (ThermoFisher, Cat.No.11141D) with a ratio of 1:1 and cultured with RPMI1640 medium (10% FBS, 1% PS, 5ng/ml IL-7, 5ng/ml IL-15). To transduce CAR genes, the lentivirus was produced in 293T cells which has been transfected with CAR plasmid and packing plasmids (MD2.G and PsPAXP2). Transduction was performed 48 hours after PBMC isolation. The activated T cells were transduced with lentivirus supernatants on RetroNectin (Takara, Cat.No.T100A) coated plates by centrifuge at 2,000×g for 2 hours at 32℃. Transduction efficiency of CAR-T cells was determined after three days. To generate mouse CAR-T cells, the splenocytes were isolated from C57BL/6 wild type mice and activated by Concanavalin A (2μg/ml). To prepare retrovirus for transduction, the GP2-293 cells were transfected with CAR plasmid and pCAG-Eco envelop vector. 48 hours after transfection, the retrovirus supernatants were collected and concentrated by centrifuge at 20,000×g for 2 hours at 4℃. Transduction of CAR-T cells were performed 24 hours after splenocytes isolation on RetroNectin coated plats by spin centrifugation. Co-culture of CAR-T cells with AML cells MOLM13-CD19 or MOLM13-CD19-IGSF9 cells (5×10⁵) were co-cultured with 19BBz or 33BBz CAR-T cells (5×10⁵) in 24-well plates using complete RPMI 1640 medium. After 20 hours, cells were harvested for flow cytometry. PD-L1 and IGSF9 surface expression was evaluated on gated CD19⁺ MOLM13 cells. To assess IGSF9 on antigen-negative AML cells, MOLM13-CD19 and MOLM13-GFP cells were mixed 1:1. This mixture (5×10⁵ cells) was co-cultured with an equal number of CAR-T cells under identical conditions. For conditioned medium experiments, 33BBz CAR-T cells and MOLM13 cells were co-cultured (1:1 ratio). After 20 hours, medium was collected, filtered (0.45 µm), and mixed 1:1 with fresh complete RPMI 1640. MOLM13, U937, MV4-11 cells or patient-derived AML cells were then treated with this mixture for 24 hours, followed by IGSF9 surface expression analysis via flow cytometry. To assess degranulation (CD107a) and cytokines production, 1×10⁵ 33BBz CAR-T cells were co-cultured with equal numbers of MOLM13 or MOLM13-IGSF9 cells in complete RPMI1640 medium supplemented with 3μg/ml Brefeldin A (MCE, Cat.NO.HY-16592) and 1mM Monensin (MCE, Cat.NO.HY-N0150) in a 96-well plate, then incubated for 6 hours prior to flow cytometry analysis. Repeated stimulation of CAR-T cells For repeated stimulation, irradiated THP-1 (WT) or THP-1 (IGSF9 KO) cells (5×10⁵) were seeded in 24-well plates. CD33-specific CAR-T cells (5×10⁵) were added (E/T = 1/1). Fresh target cells were replenished at the same E/T ratio every 3 days. On day 12 following four stimulations, CAR-T cells were harvested and differentiation states were assessed by flow cytometry. C57BL/6 mouse model Four- to six-week-old female C57BL/6 mice were purchased from Charles River Laboratories. Individual mice were randomly assigned to treatment/control groups Mice (n=4/group) received subcutaneous injections of 1×10⁶ LL/2-hCD19 or LL/2-hCD19-mIGSF9 cells. When tumors reached ~50 mm³ (day 9), 2 × 10⁶ 19m28z CAR-T cells were administered intravenously. Mice were euthanized two weeks post-CAR-T infusion. Tumors were enzymatically dissociated, and exhaustion/differentiation states of tumor-infiltrating GFP⁺ CAR-T cells were assessed by flow cytometry. Xenograft mouse model Four- to six-week-old female NOD/SCID-IL2Rg-null (NSG) mice were purchased from Shanghai Model Organisms. Individual mice were randomly assigned to treatment/control groups. For xenograft model with IGSF9 blockade, NSG mice received intravenous injection of 0.5×10⁶ luciferase-expressing, GFP labeled MOLM13-IGSF9 cells. Three days post engraftment, mice were administered 0.5×10⁶ 33BBz CAR-T or untransduced T (UnT) cells via tail vein. Anti-IGSF9 blocking antibody or mouse IgG control (0.2 mg/mouse) was delivered intravenously starting 24 hours post CAR-T infusion, with repeat dosing every 4 days (total 3 doses). Tumor burden was quantified weekly by bioluminescence imaging (IVIS Spectrum, PerkinElmer). For flow cytometric analysis of CAR-T cell function, mice received two antibody doses every 3 days beginning 24 hours post CAR-T infusion. At the terminal endpoint (day 12 post CAR-T infusion), the peripheral blood, bone marrow and spleen tissue were harvested. The frequency of GFP⁺ tumor cells and mCherry⁺ CAR-T cells in vivo was determined by flow cytometry. CAR-T cell differentiation status was evaluated based on surface expression of CD45RA and CD62L. For in vivo experiments of IGSF9-specific CAR-T cells, NSG mice received intravenous injection of 0.2×10⁶ luciferase-expressing MOLM13-IGSF9 cells. Three days post engraftment, mice were administered 2×10⁶ IG9BBz CAR-T or UnT cells via tail vein. Tumor burden was quantified weekly by bioluminescence imaging. Mice survival was monitored until death or reaching humane endpoint (Severe weight loss or paralysis). The probability of survival was described by Kaplan-Meier curves using log-rank (Mantel-Cox) tests. To quantify tumor burden and adoptive T cell persistence, bone marrow was harvested from mice at 14 days post-T cell infusion. GFP⁺ tumor cells and infused T cells were subsequently analyzed by flow cytometry to determine their frequency and phenotype For IGSF9 endogenously expressed AML model, NSG mice were engrafted with THP-1 tumors via intravenous injection of 0.5×10⁶ THP-1-GFP-Luc cells. The next day, 5×10⁶ IG9BBz CAR-T cells or untransduced T cells were administered by tail vein injection. Tumor burden was monitored longitudinally through serial bioluminescence imaging. Bone marrow was harvested at day 25 post-T cell infusion, followed by flow cytometric analysis of GFP⁺ tumor cells and infused T cells. Flow cytometry The production of human IGSF9 antibody was described previously(18). To detect IGSF9 by flow cytometry, cells were incubated with IGSF9 antibody for 30 minutes at room temperature, and then stained with APC-conjugated anti-mouse IgG antibody (Biolegend, Cat.No.405308) for 20 minutes at 4℃ in the dark. Direct staining of surface antigens was performed by incubating cells with fluorochrome-conjugated antibodies for 20 minutes at 4℃ in the dark. For intracellular staining of IFNγ and TNFα, cells were fixed and permeabilized using a Fixation/Permeabilization buffer (eBioscience, Cat.No.00-5123-43). Antibodies targeting human PD-L1 (Cat.No.329734), CD19 (Cat.No.302208), CD3 (Cat.No.300317), CD4 (Cat.No.317432), CD8 (Cat.No.344750), CD107a (Cat.No.328609), TNFα (Cat.No.376203)，IFNγ (Cat.No.502510) PD-1 (Cat.No.367406), TIM-3 (Cat.No.345041), LAG-3 (Cat.No.369317), CD45RA (Cat.No.304142), CD62L (Cat.No.304810), CD69 (Cat.NO.310904) and mouse PD-1 (Cat.No.135221), TIM-3(Cat.No.134008), CD44 (Cat.No.103012), CD62L (Cat.No.104424), CD13 (Cat.No.301703), CD33 (Cat.No.303441), CD117 (Cat.No.313217) were purchased from Biolegend. Flow cytometry was performed on a BD Fortessa flow cytometer (BD Bioscience) or an Aurora Spectral Flow Cytometer (Cytek). Data were analyzed with FlowJo software (V10, TreeStar). Western blot A549 and A549-IGSF9-HA cells were lysed using RIPA buffer (Solarbio) containing protease inhibitors (Beyotime Biotechnology). The samples were first loaded onto an SDS-PAGE gel and then transferred to a PVDF membrane. The following primary antibodies were used for protein probing: mouse anti-human IGSF9(1:1000 dilution, home-made), mouse anti-HA tag (1:6000 dilution, Proteintech, Cat.No.51064-2-AP), Rabbit anti-human GAPDH (1:6000 dilution, Affinity, Cat.No.AF7021). After incubation with HRP-conjugated secondary antibodies (Proteintech) and ECL reagent (Biosharp), the protein bands were visualized by a chemiluminescent imaging system (Tanon). In vitro cytotoxicity assay CAR-T cell cytotoxicity was assessed using a luciferase-based assay. Target cells expressing luciferase (5×10⁵) were co-cultured with CAR-T cells at specified E:T ratios in 96-well black-bottom plates. After 18–20 hours, D-luciferin substrate was added, and bioluminescence (BL) was measured using a luminescence reader (LUX-P110, BLT). Lysis rates were calculated as [(1- BL of each sample)/BL of target cells alone]×100%. IGSF9-ECD-Fc binding assay Jurkat T cells expressing 19BBz or IG9BBz CAR constructs (1×10 6 ) were incubated with 2μg IGSF9-ECD-Fc protein (prepared as described(18)) or human IgG1 control at 4℃ for 30 minutes. Cells were washed with PBS, then stained with APC-conjugated anti-human IgG Fc antibody (Biolegend, 410712) at 4℃ for 30 minutes. Cytokines quantification by ELISA IG9BBz CAR-T cells were co-cultured with K562 or K562-IGSF9 cells at 1:1 ratio in complete RPMI1640 medium in a six-well plate. After 20 hours, culture supernatants were collected. Human IFNγ ELISA Kit (Abcam, ab46025), Human TNFα SimpleStep ELISA Kit (Abcam, ab181421) and Human IL-2 ELISA Kit (Proteintech, KE00017) were used to quantify IFNγ, TNFα and IL-2 secretion following manufacturer’s instruction. Statistical analysis GraphPad Prism 8 was used for statistical analysis. A two-tail Student’s t-test was used for comparing two groups. Data are presented as mean±SEM. *P<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Results CAR-T cell cytotoxicity induces IGSF9 upregulation in AML cells Given that IGSF9 is an IFNγ-inducible immune checkpoint molecule and CAR-T cells release cytotoxic cytokines (e.g., IFNγ) upon tumor engagement, we hypothesized that CAR-T cell attack triggers IGSF9 expression on AML cells. Co-culturing AML cells with second-generation 4-1BBz CAR-T cells targeting CD33 (33BBz; an endogenous AML antigen) or CD19 (19BBz; a clinically validated target) significantly upregulated both PD-L1 (confirming IFNγ pathway activation) and IGSF9 on target MOLM13-CD19⁺ cells after 20 hours (Figure 1A, Supplementary Figure S1A). Mechanistically, in mixed co-cultures containing both MOLM13-CD19⁺ and MOLM13-GFP⁺ cells, both populations exhibited comparable IGSF9 upregulation when exposed to either 19BBz or 33BBz CAR-T cells, confirming antigen-independence induction (Figure 1B, Supplementary Figure S1B). Furthermore, conditioned medium (CM) from 33BBz CAR-T/MOLM13 co-cultures, unlike that from untransduced T cell controls, robustly induced IGSF9 expression in fresh AML cells (Figure 1C, D, Supplementary Figure S1C). These findings demonstrate that soluble mediators, such as IFNγ, released during CAR-T cells/tumor interactions drive IGSF9 upregulation in AML cells, thereby establishing a feedforward inhibitory loop. IGSF9 expression on tumor cells impairs CAR-T cell anti-tumor functions To determine whether tumor-expressed IGSF9 suppresses CAR-T cell activity, we performed luciferase-based cytotoxicity assays. Various CAR-T target cells were generated to express CD19 antigen and different level of IGSF9 (Supplementary Figure S2A). Knockout of IGSF9 in THP-1-CD19⁺ cells significantly enhanced tumor lysis by both 19BBz and 1928z CAR-T cells (Figure 2A, B). Conversely, IGSF9 overexpression in A549-CD19⁺ cells markedly reduced killing by both 19BBz and 1928z CAR-T cells (Figure 2C, D), indicating suppression independent CAR structure. Murine-derived 19m28z CAR-T cells targeting LL/2 cells in which human CD19 (hCD19) was overexpressed exhibited significantly diminished cytotoxicity against LL/2-hCD19-mIGSF9⁺ cells compared to LL/2-hCD19 controls, confirming similar suppressive function of mouse-derived IGSF9 (Figure 2E). Similarly, MOLM13-IGSF9⁺ cells showed enhanced resistance to 33BBz and 3328z CAR-T cell-mediated killing compared to parental MOLM13 (Figure 2F, G). As a control, 1928z CAR-T cells targeting MOLM13 or MOLM13-IGSF9 + cells did not elicit remarkable tumor lysis effect (Supplementary Figure S2B). Mechanistically, co-cultured with MOLM13-IGSF9 elicited lower expression of CD107a of 33BBz CAR-T cells than MOLM13 groups (Figure 2H), indicating repressed degranulation of CAR-T cells by IGSF9. The cytokines release was also compared. CAR-T cells stimulated by MOLM13-IGSF9 produced comparable level of IFNγ but significantly reduced level of TNFα compared to MOLM13 groups (Figure 2I, J). Moreover, repeated stimulation of CD33-specific CAR-T cells with irradiated THP-1 (IGSF9 KO) cells ( vs . THP-1 WT) elevated the frequency of CD45RA - CD62L + central memory T cells (Supplementary Figure S3A-C). In vivo , tumors derived from LL/2-hCD19-mIGSF9⁺ cells in CAR-T-treated mice exhibited reduced CD44⁺CD62L⁺ memory T cell subsets and increased (though not significant, p=0.2655) PD-1⁺TIM-3⁺ population compared to LL/2-hCD19⁺ controls (Supplementary Figure S3D-G). Collectively, these data demonstrate that tumor-intrinsic IGSF9 impairs CAR-T cell functions by suppressing T cells degranulation, cytokines release and memory differentiation. Blockade of IGSF9 enhances CAR-T cell anti-tumor activity in vivo While IGSF9 blockade has been shown to improve T cell function in solid tumors(18), its potential to enhance CAR-T cell efficacy against AML in vivo remained unexplored. To address this, we employed a combination therapy approach in an AML xenograft model. NSG mice engrafted with GFP labeled MOLM13-IGSF9 cells were administered either 33BBz CAR-T cells or untransduced T cells (UnT). Starting one day after CAR-T cells infusion, mice were administered anti-IGSF9 or isotype control IgG every four days for a total of three doses (Figure 3A). Bioluminescence imaging revealed a significant reduction in tumor burden by day 7 in CAR-T cell-treated mice compared to UnT controls. Notably, anti-IGSF9 co-administration further suppressed tumor progression in the CAR-T group (Figure 3B-D). Furthermore, CAR-T cell treatment significantly prolonged survival, and this effect that was further enhanced by anti-IGSF9 treatment (Figure 3E). We next evaluated the impact of anti-IGSF9 treatment on the persistence of CAR-T cells in vivo . One day after CAR-T cell injection, tumor-engrafted mice received anti-IGSF9 or control IgG, administered in two doses every four days. Flow cytometry analysis at 12 days post-CAR-T injection revealed that 33BBz CAR-T cells significantly reduced the frequency of GFP⁺ tumor cells (Figure 4A, Supplementary Figure S4A). Notably, in anti-IGSF9 combination group, GFP + tumor cells were nearly undetectable (Figure 4A). Furthermore, anti-IGSF9 treatment markedly increased the proportion of human CD3⁺ T cells in peripheral blood (Figure 4A, B). We then quantified CAR-T cells (mCherry⁺) in the bone marrow and spleen. Compared to IgG controls, anti-IGSF9 treatment elevated the proportion of mCherry⁺ CAR-T cells in these organs, resulting in a significantly higher mCherry⁺/GFP⁺ cell ratio (Figure 4C, D). Finally, we evaluated the differentiation state of CAR-T cells in the spleen, and found that anti-IGSF9 treatment significantly increased the proportion of CAR-T cells exhibiting a central memory phenotype (CD45RA⁻CD62L⁺) (Figure 4E, F, Supplementary Figure S4B). Collectively, these results suggest that IGSF9 blockage enhances CAR-T cells persistence and promotes CAR-T cells anti-AML functions in vivo . Design and Functional Validation of IGSF9-Specific CAR-T Cells Given the restricted expression of IGSF9 to AML blasts and its role as a critical suppressor of T-cell function, we hypothesized that selective elimination of IGSF9⁺ AML cells would limit disease progression. To this end, we engineered novel IGSF9-specific CAR-T cells (IG9BBz). Western blot analysis confirmed the specificity of the anti-IGSF9 antibody for IGSF9⁺ human cells (Figure 5A). Structural modeling predicted that the Ig-like 2 and fibronectin type III-2 (FNIII-2) domains constitute the core epitope recognized by the scFv antibody (Figure 5B), which is consistent with our previous report(19). Flow cytometry demonstrated robust labeling of membrane-expressed IGSF9-mEGFP in HEK293T cells using the fluorophore-conjugated anti-IGSF9 antibody, confirming efficient binding to cell-surface IGSF9 (Figure 5C). The IG9BBz CAR construct incorporated an scFv derived from the anti-IGSF9 antibody as the antigen-binding domain, coupled with 4-1BB and CD3z intracellular signaling domains, and a P2A-linked mCherry reporter for tracking CAR expression (Figure 5D). Jurkat cells expressing IG9BBz bound recombinant IGSF9-ECD-Fc, whereas control CAR variants lacking the scFv showed no binding, confirming antigen specificity (Figure 5E). To evaluate antigen-dependent activation, primary IG9BBz CAR-T cells were generated and co-cultured with tumor targets for 20 hours (Supplementary Figure S5). Exposure to H1299-IGSF9 or K562-IGSF9 cells, but not parental K562 cells, markedly upregulated CD69 expression on CAR-T cells (Figure 5F). Moreover, co-culture with K562-IGSF9 cells, but not parental K562 cells, induced a pronounced shift of CAR-T cells from a naïve to an effector state (Figure 5G, H), indicating antigen-specific activation of IG9BBz CAR-T cells. To further determine CAR-T cell functions, cytokine secretion was quantified by ELISA. Co-culture with K562-IGSF9 cells, but not parental K562 cells elicited abundant secretion of IFNγ, IL-2, and TNFα (Figure 5I). In cytotoxicity assays, IG9BBz CAR-T cells efficiently eliminated H1299-IGSF9 and MOLM13-IGSF9 cells within 18 hours, as measured by bioluminescence (Fig. 5J). Potent anti-AML activity of IG9BBz CAR-T cells across preclinical models The anti-tumor efficacy of IG9BBz CAR-T cells was first evaluated in NSG mice bearing MOLM13-IGSF9-Luc AML xenografts (Figure 6A). IG9BBz CAR-T cell administration significantly reduced tumor burden, as quantified by bioluminescence imaging (Figures 6B, C), and extended survival compared to UnT controls (Figure 6D). To confirm direct tumor elimination, bone marrow was analyzed at 14 days post-infusion. IG9BBz CAR-T treatment markedly decreased GFP⁺ tumor burden while increasing the CD3⁺/GFP⁺ ratio (Figures 6E, G). Residual tumor cells maintained IGSF9 expression in both groups (Supplementary Figure S6A). Notably, the IGSF9 percentage in residual GFP⁺ cells were significantly lower in IG9BBz-treated mice vs. UnT controls, indicating preferential targeting of IGSF9-high tumor cells (Supplementary Figure S6A). Phenotypic characterization revealed distinct T-cell differentiation patterns: UnT cells predominantly exhibited an effector phenotype (CD45RA⁺CD62L⁻), whereas IG9BBz CAR-T cells (CD3⁺mCherry⁺) primarily comprised effector memory (CD45RA⁻CD62L⁻) and central memory (CD45RA⁻CD62L⁺) subsets (Figures 6E, F). Critically, IG9BBz CAR-T cells demonstrated significantly lower expression of inhibitory receptors (PD-1, TIM-3, LAG-3) versus UnT controls (Figures 6E, H). These findings indicate tumor antigen-driven activation and favorable differentiation of IG9BBz CAR-T cells. The emergence of memory subpopulations coupled with reduced exhaustion suggests enhanced persistence potential of this CAR construct. To evaluate IG9BBz CAR-T cell activity against AML with endogenous IGSF9 expression, NSG mice bearing THP-1-GFP-Luc xenografts received IG9BBz CAR-T cells or UnT controls (Figure 7A). Longitudinal bioluminescence imaging revealed progressive tumor expansion in UnT-treated mice, whereas 3 of 4 IG9BBz CAR-T-treated mice exhibited minimal disease progression (Figures 7B). Bone marrow analysis at day 25 post-infusion confirmed significant tumor reduction by IG9BBz CAR-T treatment, demonstrating decreased GFP⁺ tumor burden and increased CD3⁺/GFP⁺ ratios (Figure 7C, D). The GFP + tumor cells were further verified to be IGSF9 positive (Figure 7C). Notably, one IG9BBz-treated mouse showing elevated bioluminescent signal exhibited low marrow tumor infiltration, suggesting predominant extramedullary tumor engraftment in this individual. To assess clinical relevance, bone marrow aspirates from two AML patients were analyzed. Treatment with conditioned medium from 33BBz CAR-T/MOLM13 co-cultures significantly upregulated IGSF9 expression in the CD3⁻ compartment of AML (Figure 7E). When co-cultured with AML blasts, IG9BBz CAR-T cells exhibited elevated CD69 expression, confirming antigen-specific activation (Figure 7F). Notably, IGSF9⁺ cells predominantly resided within the primitive CD13⁺CD33⁺CD117⁺ population (Supplementary Figure S6B). IG9BBz CAR-T co-culture selectively reduced this target-enriched subset in AML samples (Figure 7G). Collectively, these data establish that IG9BBz CAR-T cells exert potent anti-leukemic activity against IGSF9-expressing AML in vivo , and demonstrate translational potential through CAR-T-induced IGSF9 upregulation that enhances blast elimination. Discussion Immune escape remains a major barrier to durable remission after CAR-T therapy for AML. Here, we identify IGSF9 as an immune suppressor that drives resistance to CAR-T cells in AML. Cytotoxic cytokines released by activated CAR-T cells induced robust IGSF9 expression on AML blasts. The release of inflammatory cytokines, including IFNγ, IL-2, and TNFα, represents a primary mechanism by which CAR-T cells exert cytotoxic effects(20). Our previous work identified IFNγ as a key driver of IGSF9 upregulation via the JAK1-STAT1 signaling pathway(19). Whether additional cytokines contribute to this induction remains to be determined. Nevertheless, we show that the cytokine milieu generated during CAR-T cytotoxicity is sufficient to induce substantial IGSF9 expression in AML cells. This antigen-independent induction by soluble mediators establishes a feed-forward immunosuppressive loop: CAR-T cells activation triggers cytokine release, which in turn upregulates IGSF9 on AML blasts, thereby amplifying its inhibitory effect and progressively impairing T-cell function. This mechanism represents a distinct form of immune evasion that is independent of antigen loss(21). Disrupting this immune-evasive niche is paramount for advancing AML CAR-T therapy. In vitro , AML cells with high IGSF9 expression confer treatment resistance against CAR-T cytotoxicity, potentially through impairing effector functions—specifically by suppressing degranulation and TNFα secretion. Repeated antigen stimulation further compromised CAR-T memory differentiation, thereby undermining durable antitumor immunity. Although the structural basis of IGSF9 signaling remains undefined, two non-mutually exclusive mechanisms are plausible: (1) direct activation of inhibitory pathways, dampening CAR-T cell activation, and (2) signaling through the IGSF9 intracellular ITIM motif in AML cells, potentially enhancing their survival upon T cell contact. The identity of the cognate IGSF9 receptor remains challenging. Our preliminary data nominate TMUB1 (Transmembrane and Ubiquitin-Like Domain-Containing 1) as a candidate mediator of IGSF9-dependent immunosuppression. Definitive molecular validation of this interaction warrants further investigation. Using xenogeneic IGSF9 positive AML models, we demonstrate that monoclonal antibody–mediated IGSF9 blockade attenuates tumor progression and prolongs survival. Beyond direct blockade of IGSF9’s inhibitory signaling, the modest tumor control observed with anti-IGSF9 plus untransduced T cells points to an additional contribution from antibody-dependent cellular phagocytosis (ADCP). Nevertheless, In vivo , IGSF9 inhibition enhanced CAR-T cell persistence, restored the CD62L⁺CD44⁺ central-memory T cell pool, and reduced the frequency of exhausted T cell subsets. These findings establish IGSF9 as a tractable therapeutic target capable of reprogramming the AML microenvironment from a tolerogenic to an immunogenic state. We further developed IGSF9-specific CAR-T cells designed to integrate tumor antigen recognition with simultaneous ablation of an inhibitory ligand. IGSF9 expression is restricted to leukemic blasts and absent on normal CD34⁺ hematopoietic stem cells(19), minimizing the risk of on-target off-tumor toxicity. In vitro and in vivo , IG9BBz CAR-T cells demonstrated potent cytotoxicity against IGSF9 positive AML cells. This strategy leverages the dual role of IGSF9-its tumor-restricted expression and immunosuppressive function-enabling simultaneous clearance of malignant cells and disruption of the inhibitory niche. The potential emergence of IGSF9-negative escape variants could be mitigated by employing tandem CAR constructs that co-target IGSF9 and CD33 or CD123 under OR-gate logic, thereby ensuring robust activity against both antigen-low subclones and microenvironmentally suppressive cells(22). Limitations of this study include the use of immunodeficient AML models for CAR-T therapy, which cannot fully recapitulate endogenous T cell-AML interactions or allow assessment of graft-versus-host disease. Future studies will employ humanized models and primary AML xenografts with autologous CAR-T manufacturing to better evaluate patient-specific heterogeneity and immune dynamics. Moreover, this study does not assess potential on-target/off-tumor toxicity of IGSF8-directed therapies, which requires urgent investigation given its trace expression in neuronal tissues (Human Protein Atlas, proteinatlas.org)(23). Conclusions In summary, we delineate a cytokine-driven IGSF9-mediated resistance circuit that undermines CAR-T cell activity in AML and validate two complementary therapeutic strategies-IGSF9 blockade and IGSF9-directed CAR-T cells-to disrupt this immunosuppressive axis. These findings provide a mechanistic framework for next-generation immunotherapies that integrate tumor antigen targeting with reprogramming of the immune microenvironment, offering a promising path toward sustained remission in high-risk AML. Declarations Ethics approval All studies were approved by the Medical Ethics Committee of Binzhou Medical University (No: 2025-003), and the Animal Ethics Committee of Binzhou Medical University (No: 2025-015). All animal experiments were performed under the national standards of Institutional Animal Care and Use Committee. Consent for publication Not applicable. Availability of data and material All data generated or analysed during this study are included in this published article [and its supplementary information files]. Competing interests The authors declare that they have no competing interests. Funding This study was funded by the National Natural Science Foundation of China (82150101, 81872332, 82070225) to Zunling Li, Shandong Natural Science Foundation (ZR2024LZL010) to Zunling Li, and (ZR2023QC190) to Xianhui Meng. Authors’ contributions X.M., Y.W. and Z.L. designed the study and wrote the manuscript. X.M., FM.L. and H.Y. performed the experiments and analyzed the data. C.L., Y.S., H.W., G.L., J.Z., L.H., F.L. and S.W. contributed to animal studies and data analysis. All authors reviewed and approved the final manuscript. The corresponding author (Z.L.) is the guarantor for the publication, taking responsibility for the integrity of the entire work. Acknowledgements This work was supported by the Special Funding for the "Case-by-Case Introduction of Top Talent (Teams)" Program in Yantai. References Toma MM, Skorski T. Star wars against leukemia: attacking the clones. Leukemia. 2024 Nov;38(11):2293–302. Kantarjian HM, DiNardo CD, Kadia TM, Daver NG, Altman JK, Stein EM, et al. Acute myeloid leukemia management and research in 2025. CA Cancer J Clin. 2025 Jan;75(1):46–67. Thol F, Döhner H, Ganser A. How I treat refractory and relapsed acute myeloid leukemia. Blood. 2024 Jan 4;143(1):11–20. Mathioudaki A, Wang X, Sedloev D, Huth R, Kamal A, Hundemer M, et al. The remission status of AML patients after allo-HCT is associated with a distinct single-cell bone marrow T-cell signature. Blood. 2024 Mar 28;143(13):1269–81. Cappell KM, Kochenderfer JN. Long-term outcomes following CAR T cell therapy: what we know so far. Nat Rev Clin Oncol. 2023 June;20(6):359–71. Haubner S, Subklewe M, Sadelain M. Honing CAR T cells to tackle acute myeloid leukemia. Blood. 2025 Mar 13;145(11):1113–25. Tettamanti S, Pievani A, Biondi A, Dotti G, Serafini M. Catch me if you can: how AML and its niche escape immunotherapy. Leukemia. 2022 Jan;36(1):13–22. Foldvari Z, Brennan MS, Titov A, Jacobsen SEW, Olweus J. Targeting the roots of myeloid malignancies with T cell receptors. Nat Rev Cancer. 2025 Aug 21;1–21. Ciantra Z, Paraskevopoulou V, Aifantis I. The rewired immune microenvironment in leukemia. Nat Immunol. 2025 Mar;26(3):351–65. Vadakekolathu J, Rutella S. Escape from T-cell–targeting immunotherapies in acute myeloid leukemia. Blood. 2024 June 27;143(26):2689–700. Zuo S, Li C, Sun X, Deng B, Zhang Y, Han Y, et al. C-JUN overexpressing CAR-T cells in acute myeloid leukemia: preclinical characterization and phase I trial. Nat Commun. 2024 July 22;15(1):6155. Lasry A, Nadorp B, Fornerod M, Nicolet D, Wu H, Walker CJ, et al. An inflammatory state remodels the immune microenvironment and improves risk stratification in acute myeloid leukemia. Nat Cancer. 2023 Jan;4(1):27–42. Hansen M, Walmod PS. IGSF9 family proteins. Neurochem Res. 2013 June;38(6):1236–51. Shi SH, Cheng T, Jan LY, Jan YN. The immunoglobulin family member dendrite arborization and synapse maturation 1 (Dasm1) controls excitatory synapse maturation. Proc Natl Acad Sci U S A. 2004 Sept 7;101(36):13346–51. Shi SH, Cox DN, Wang D, Jan LY, Jan YN. Control of dendrite arborization by an ig family member, dendrite arborization and synapse maturation 1 (Dasm1). Proc Natl Acad Sci U S A. 2004 Sept 7;101(36):13341–5. Li Y, Deng Y, Zhao Y, Zhang W, Zhang S, Zhang L, et al. Immunoglobulin superfamily 9 (IGSF9) is trans-activated by p53, inhibits breast cancer metastasis via FAK. Oncogene. 2022 Oct;41(41):4658–72. Luan H, Wang T, Li F, Sun S, Wang Z, Zhao X, et al. IGSF9 promotes tumor invasion and metastasis through GSK-3β/β-catenin mediated EMT in lung cancer. Neoplasia N Y N. 2024 Dec;58:101067. Liu Y, Wang H, Zhao X, Zhang J, Zhao Z, Lian X, et al. Targeting the immunoglobulin IGSF9 enhances antitumor T-cell activity and sensitivity to anti-PD-1 immunotherapy. Cancer Res. 2023 Oct 13;83(20):3385–99. Hui L, Xiao J, Zhao Z, Zhang J, Luan H, Zhang J, et al. IGSF9-targeted therapy inhibits the progression of acute myeloid leukemia. Blood Adv. 2025 June 17;bloodadvances.2025016432. Young RM, Engel NW, Uslu U, Wellhausen N, June CH. Next-generation CAR T-cell therapies. Cancer Discov. 2022 July 6;12(7):1625–33. Bhagwat AS, Torres L, Shestova O, Shestov M, Mellors PW, Fisher HR, et al. Cytokine-mediated CAR T therapy resistance in AML. Nat Med. 2024 Dec;30(12):3697–708. Haubner S, Mansilla-Soto J, Nataraj S, Kogel F, Chang Q, De Stanchina E, et al. Cooperative CAR targeting to selectively eliminate AML and minimize escape. Cancer Cell. 2023 Nov;41(11):1871-1891.e6. Mishra A, Traut MH, Becker L, Klopstock T, Stein V, Klein R. Genetic evidence for the adhesion protein IgSF9/Dasm1 to regulate inhibitory synapse development independent of its intracellular domain. J Neurosci Off J Soc Neurosci. 2014 Mar 19;34(12):4187–99. Additional Declarations No competing interests reported. Supplementary Files SupplementaryFiguresMeng.docx Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 02 Mar, 2026 Reviews received at journal 02 Mar, 2026 Reviews received at journal 23 Feb, 2026 Reviewers agreed at journal 27 Jan, 2026 Reviewers agreed at journal 26 Jan, 2026 Reviewers agreed at journal 25 Jan, 2026 Reviewers invited by journal 19 Jan, 2026 Editor assigned by journal 19 Jan, 2026 Submission checks completed at journal 19 Jan, 2026 First submitted to journal 14 Jan, 2026 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. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8606613","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":574867978,"identity":"ed4a26dc-46cc-4878-8f4e-aac231ea15b2","order_by":0,"name":"Xianhui Meng","email":"","orcid":"","institution":"Binzhou Medical University","correspondingAuthor":false,"prefix":"","firstName":"Xianhui","middleName":"","lastName":"Meng","suffix":""},{"id":574867979,"identity":"2e2a3288-587d-4bab-8623-b793028702b4","order_by":1,"name":"Fangmin Li","email":"","orcid":"","institution":"Binzhou Medical 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Li","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA5ElEQVRIiWNgGAWjYBACPmYQWSDBwMDeAxU6QEALG1iLAVALzxlitYBJAyCWyCFWCzv7M+kCA4s8+ci3hz/dqGGQ47uRwPi5AL/D0qRnGEgUG97OSzDOOcZgLHkjgVl6Bn4tx6R5DCQSN87OMUjObWBI3HAjgY2ZB68WxjaIlplnDA4DtdQToYWZDaxlvgSPYTNQS4IBYS1szNYgLRt48pKZc45JGM4887BZGp8Wfv7jD2/zVNQlzm8/e/hzTo2NPN/x5IOf8WmBA4MDYAoYpwyMDcRoYGCQJ1LdKBgFo2AUjEAAANWOP251x+DTAAAAAElFTkSuQmCC","orcid":"","institution":"Binzhou Medical University","correspondingAuthor":true,"prefix":"","firstName":"Zunling","middleName":"","lastName":"Li","suffix":""}],"badges":[],"createdAt":"2026-01-15 03:53:14","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8606613/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8606613/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":104374885,"identity":"f9ffeebd-438a-47ad-9028-fa23da7eb92e","added_by":"auto","created_at":"2026-03-11 06:11:17","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":332648,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCAR-T Cell Co-culture Induces Contact-Independent IGSF9 Upregulation in AML Cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003eMOLM13-CD19 cells were co-cultured with 19BBz or 33BBz CAR-T cells for 20 hours. PD-L1 and IGSF9 expression on CD19⁺ tumor cells were analyzed by flow cytometry versus untreated controls.\u003cbr\u003e\n \u003cstrong\u003e(B)\u003c/strong\u003e MOLM13-CD19 and MOLM13-GFP cells (1:1 ratio) were co-cultured with 19BBz or 33BBz CAR-T cells. IGSF9 expression was assessed on CD19⁺GFP⁻ (antigen-expressing) and CD19⁻GFP⁺ (bystander) populations.\u003cbr\u003e\n \u003cstrong\u003e(C)\u003c/strong\u003e Conditioned medium (CM) from MOLM13/33BBz CAR-T co-cultures was applied to U937, MV4-11, and MOLM13 cells. IGSF9 levels were measured by flow cytometry versus fresh medium controls.\u003cbr\u003e\n \u003cstrong\u003e(D)\u003c/strong\u003e Quantification of IGSF9 expression proportion from panels C (mean ± SEM; n=3 biological replicates), ****P\u0026lt;0.0001.\u003c/p\u003e","description":"","filename":"Picture1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8606613/v1/2ede31717fb5e91def14c53c.jpg"},{"id":104374840,"identity":"0c475173-8a17-489b-bab4-8a54c8196760","added_by":"auto","created_at":"2026-03-11 06:11:07","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":316503,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIGSF9 Expression on Tumor Cells Impairs CAR-T Cell Cytotoxic Function\u003c/strong\u003e\u003cbr\u003e\n\u003cstrong\u003e(A–G)\u003c/strong\u003e CAR-T cell cytotoxicity against luciferase-expressing tumor cells was assessed by luminescence after 18-20 hours of co-culture.\u003cbr\u003e\n\u003cstrong\u003e(A)\u003c/strong\u003e Cytotoxicity of 19BBz CAR-T cells against THP-1-CD19 versus IGSF9-knockout (KO) THP-1-CD19 cells.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(B)\u003c/strong\u003e Cytotoxicity of 1928z CAR-T cells against THP-1-CD19 versus IGSF9-knockout (KO) THP-1-CD19 cells.\u003cbr\u003e\n\u003cstrong\u003e(C)\u003c/strong\u003e Cytotoxicity of 19BBz CAR-T cells against A549-CD19 versus A549-CD19 overexpressing IGSF9 (A549-CD19-IGSF9).\u003cbr\u003e\n\u003cstrong\u003e(D)\u003c/strong\u003e Cytotoxicity of 1928z CAR-T cells against A549-CD19 versus A549-CD19-IGSF9.\u003cbr\u003e\n\u003cstrong\u003e(E)\u003c/strong\u003e Cytotoxicity of mouse 19m28z CAR-T cells against LL2-CD19 versus LL2-CD19 overexpressing mouse IGSF9 (LL2-CD19-mIGSF9).\u003cbr\u003e\n\u003cstrong\u003e(F)\u003c/strong\u003e Cytotoxicity of 33BBz CAR-T cells against MOLM13 versus MOLM13 overexpressing IGSF9 (MOLM13-IGSF9).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(G)\u003c/strong\u003e Cytotoxicity of 3328z CAR-T cells against MOLM13 versus MOLM13 overexpressing IGSF9 (MOLM13-IGSF9).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(H-J) \u003c/strong\u003e33BBz CAR-T cells were co-cultured with MOLM13 or MOLM13-IGSF9 cells in a 1:1 ratio. Six hours later, effector response of the mCherry\u003csup\u003e+\u003c/sup\u003e CAR-T cells were quantified by the surface expression of CD107a (H), and production of IFNγ (I) and TNFα (J) by flow cytometry.\u003c/p\u003e","description":"","filename":"Picture2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8606613/v1/25210ed799784959c73bc451.jpg"},{"id":104374887,"identity":"eadfc14f-3e9c-46c9-bd65-783acd154176","added_by":"auto","created_at":"2026-03-11 06:11:18","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":317027,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCombined IGSF9 Blockade Synergizes with CAR-T Cell Therapy to Control Tumor Growth\u003c/strong\u003e\u003cbr\u003e\n \u003cstrong\u003e(A)\u003c/strong\u003e Treatment schema: Mice engrafted with MOLM13-IGSF9-GFP cells received 0.5×10⁶ 33BBz CAR-T cells or untransduced T cells (UnT), followed by three doses of anti-IGSF9 or IgG control antibody (0.2 mg/dose) (n=4, 4, 5, 5).\u003cbr\u003e\n \u003cstrong\u003e(B)\u003c/strong\u003e Representative bioluminescence images showing tumor burden dynamics.\u003cbr\u003e\n \u003cstrong\u003e(C-D)\u003c/strong\u003e Quantification of total flux (photons/second) across experiment and statistical assay of the total flux at day 7 (mean ± SEM).\u003cbr\u003e\n \u003cstrong\u003e(E)\u003c/strong\u003e Kaplan-Meier survival curves.\u003c/p\u003e","description":"","filename":"Picture3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8606613/v1/376768c64c603dded32c581d.jpg"},{"id":104374882,"identity":"535a6b42-1ffa-4366-9378-79ae9e286518","added_by":"auto","created_at":"2026-03-11 06:11:16","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":289581,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIGSF9 Blockade Enhances CAR-T Cell Persistence and Memory Differentiation in Tissues\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003e Tumor burden (GFP\u003csup\u003e+\u003c/sup\u003e cells) and infused T cell (human CD3\u003csup\u003e+\u003c/sup\u003e) frequency in peripheral blood 12 days post-infusion (after two antibody doses). Representative plots shown.\u003cbr\u003e\n\u003cstrong\u003e(B)\u003c/strong\u003e Quantification of tumor and CAR-T cell frequencies in peripheral blood (mean ± SEM; n=4 mice/group).\u003cbr\u003e\n\u003cstrong\u003e(C)\u003c/strong\u003e Tumor burden and CAR-T cell (mCherry\u003csup\u003e+\u003c/sup\u003e) frequency in bone marrow and spleen.\u003cbr\u003e\n\u003cstrong\u003e(D)\u003c/strong\u003e Quantification of tumor and CAR-T cell frequencies in bone marrow/spleen (mean ± SEM; n=4 mice/group).\u003cbr\u003e\n\u003cstrong\u003e(E)\u003c/strong\u003e Central memory differentiation (CD45RA\u003csup\u003e-\u003c/sup\u003eCD62L\u003csup\u003e+\u003c/sup\u003e) of splenic CD8\u003csup\u003e+\u003c/sup\u003eCAR-T cells (mCherry\u003csup\u003e+\u003c/sup\u003e).\u003cbr\u003e\n\u003cstrong\u003e(F)\u003c/strong\u003e Quantification of central memory CD8\u003csup\u003e+\u003c/sup\u003eCAR-T cells frequencies in spleen (mean ± SEM; n=4 mice/group).\u003c/p\u003e","description":"","filename":"Picture4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8606613/v1/5e18bbb6a6f944ec7e2dd436.jpg"},{"id":104374844,"identity":"7ee3f288-8789-44df-a972-fa36a1180ba6","added_by":"auto","created_at":"2026-03-11 06:11:08","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":461902,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAntigen specificity of IGSF9-Specific CAR Construct\u003c/strong\u003e\u003cbr\u003e\n\u003cstrong\u003e(A)\u003c/strong\u003e anti-IGSF9 antibody recognized IGSF9-HA overexpression in engineered A549-IGSF9-HA cells versus parental A549 controls by western blot.\u003cbr\u003e\n\u003cstrong\u003e(B)\u003c/strong\u003e Epitope binding of anti-IGSF9 scFv to human IGSF9 extracellular domain (ECD) as predicted by Alphafold 3.\u003cbr\u003e\n\u003cstrong\u003e(C)\u003c/strong\u003e anti-IGSF9 antibody recognized IGSF9-mEGFP surface expression in transiently transfected 293T cells by flow cytometry.\u003cbr\u003e\n\u003cstrong\u003e(D)\u003c/strong\u003e IG9BBz CAR architecture: Anti-IGSF9 scFv-41BB-CD3z signaling domains with mCherry reporter linked via P2A self-cleaving peptide.\u003cbr\u003e\n\u003cstrong\u003e(E)\u003c/strong\u003e Specific binding of IGSF9-ECD-Fc fusion protein to IG9BBz CAR\u003csup\u003e+\u003c/sup\u003e Jurkat cells versus control (19BBz CAR).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(F)\u003c/strong\u003e Representative histograms of CD69 expression on IG9BBz CAR-T cells after 20-hour co-culture with IGSF9\u003csup\u003e+\u003c/sup\u003e tumor lines (H1299-IGSF9, K562-IGSF9) versus IGSF9- controls (K562) at 1:1 E:T ratio.\u003cbr\u003e\n\u003cstrong\u003e(G)\u003c/strong\u003e Memory phenotype of CAR-T cells using CD45RA and CD62L post-co-culture with K562-IGSF9.\u003cbr\u003e\n\u003cstrong\u003e(H)\u003c/strong\u003e Memory subset distribution in CD4\u003csup\u003e+\u003c/sup\u003e and CD8\u003csup\u003e+\u003c/sup\u003e CAR-T cell compartments (mean ± SEM; n=3; subsets defined: Tcm [CD45RA-CD62L+], Tem [CD45RA\u003csup\u003e-\u003c/sup\u003eCD62L\u003csup\u003e-\u003c/sup\u003e], Tnaive [CD45RA\u003csup\u003e+\u003c/sup\u003eCD62L\u003csup\u003e+\u003c/sup\u003e], Teff [CD45RA\u003csup\u003e+\u003c/sup\u003eCD62L\u003csup\u003e-\u003c/sup\u003e]).\u003cbr\u003e\n\u003cstrong\u003e(I)\u003c/strong\u003e ELISA showed antigen-dependent cytokine secretion (IFNγ, IL-2, TNFα) following 20-hour co-culture with K562-IGSF9 versus parental K562 (mean ± SEM; n=3).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(J)\u003c/strong\u003e Dose-dependent cytotoxicity against IGSF9+ solid (H1299-IGSF9) and liquid (MOLM13-IGSF9) tumor models at indicated E:T ratios (luminescence-based killing; mean ± SEM; n=3).\u003c/p\u003e","description":"","filename":"Picture5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8606613/v1/c2704af1e2f8e86260c9c8c6.jpg"},{"id":104409635,"identity":"3ec6ee23-f4cb-42d2-ac4b-6f46c233d5a0","added_by":"auto","created_at":"2026-03-11 12:46:19","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":389844,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIG9BBz CAR-T Cells Eliminate IGSF9\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e MOLM13 Xenografts and Confer Survival Advantage\u003c/strong\u003e\u003cbr\u003e\n\u003cstrong\u003e(A)\u003c/strong\u003e Treatment schema: NSG mice (n=5 per group) received intravenous injection of 0.2×10⁶ MOLM13-IGSF9-GFP cells, followed by 2×10⁶ IG9BBz CAR-T cells or untransduced T cells (UnT) three days later.\u003cbr\u003e\n\u003cstrong\u003e(B)\u003c/strong\u003e Representative bioluminescence images showing tumor burden reduction in CAR-T-treated mice at serial timepoints.\u003cbr\u003e\n\u003cstrong\u003e(C)\u003c/strong\u003e Quantitative tumor burden (total flux, p/s; mean ± SEM).\u003cbr\u003e\n\u003cstrong\u003e(D)\u003c/strong\u003e Significant survival extension in CAR-T-treated cohort (Kaplan-Meier curves by log-rank test).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(E)\u003c/strong\u003e Representative data of the tumor/T cell frequencies, T cells differentiation and exhaustion in bone marrow.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(F)\u003c/strong\u003eMemory subset distribution in UnT and mCherry\u003csup\u003e+\u003c/sup\u003e CAR-T cell compartments (mean ± SEM; n=5)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(G)\u003c/strong\u003e Quantification of GFP\u003csup\u003e+\u003c/sup\u003e tumor frequencies and CD3\u003csup\u003e+\u003c/sup\u003e/GFP\u003csup\u003e+\u003c/sup\u003e ratio (mean ± SEM; n=5).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(H)\u003c/strong\u003e Quantification of T cell inhibitory receptors (PD-1, TIM-3 and LAG-3) (mean ± SEM; n=5).\u003c/p\u003e","description":"","filename":"Picture6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8606613/v1/df61d03eb160649a6f05950a.jpg"},{"id":104374924,"identity":"2c4b246e-80ec-4a2d-b9ae-029fbc72d9cf","added_by":"auto","created_at":"2026-03-11 06:11:19","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":349398,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAnti-tumor effect of IG9BBz CAR-T Cells on AML cells with endogenous IGSF9 expression\u003c/strong\u003e\u003cbr\u003e\n\u003cstrong\u003e(A)\u003c/strong\u003e Treatment schema: NSG mice (n=4 per group) received intravenous 0.5×10⁶ THP-1-GFP-Luc cells followed by 5×10⁶ IG9BBz CAR-T cells or untransduced T cells (UnT) at day +1.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(B)\u003c/strong\u003e Bioluminescence quantitative tumor burden of mice at serial timepoints. (total flux, p/s; mean ± SEM).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(C)\u003c/strong\u003e Representative bone marrow analysis: GFP\u003csup\u003e+\u003c/sup\u003e tumor infiltration vs. adoptive T cell engraftment.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(D)\u003c/strong\u003e Quantified CD3\u003csup\u003e+\u003c/sup\u003e cells, GFP\u003csup\u003e+\u003c/sup\u003e tumor cells, and CD3\u003csup\u003e+\u003c/sup\u003e/GFP\u003csup\u003e+\u003c/sup\u003e ratios in bone marrow (mean ± SEM; n=5).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(E)\u003c/strong\u003e IGSF9 expression of patient-derived AML cells (PD-AML) treated with conditioned media from MOLM13/33BBz CAR-T co-cultures (mean ± SEM; n=3).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(F)\u003c/strong\u003e CD69 activation on IG9BBz CAR-T cells after 24h co-culture with PD-AML (mean ± SEM; n=3).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(G)\u003c/strong\u003e Flow cytometry analysis and quantification of CD3\u003csup\u003e-\u003c/sup\u003e CD13\u003csup\u003e+\u003c/sup\u003eCD33\u003csup\u003e+\u003c/sup\u003eCD117\u003csup\u003e+\u003c/sup\u003e AML blasts before and after IG9BBz CAR-T cells treatment (mean ± SEM; n=3).\u003c/p\u003e","description":"","filename":"Picture7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8606613/v1/83b8f331cf6957e916566b39.jpg"},{"id":104413132,"identity":"7eec8bf5-e009-4fe6-b9c5-fd6c12961252","added_by":"auto","created_at":"2026-03-11 13:03:20","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3533657,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8606613/v1/359f4518-4d8e-4217-95c9-d9e0d57a9c2e.pdf"},{"id":104374849,"identity":"9dca6d14-1fe1-4290-bc89-e23fe519d39a","added_by":"auto","created_at":"2026-03-11 06:11:10","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":2471650,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFiguresMeng.docx","url":"https://assets-eu.researchsquare.com/files/rs-8606613/v1/53b99380d06c6ffaece2c81f.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Targeting Immunoglobulin Superfamily Member 9 (IGSF9) to Overcome Acute Myeloid Leukemia Resistance to CAR-T Therapy","fulltext":[{"header":"Background","content":"\u003cp\u003eAcute myeloid leukemia (AML) is an aggressive hematologic malignancy characterized by clonal expansion of myeloid progenitors. It accounts for over 80% of adult acute leukemias(1). Despite advances in therapy, the prognosis remains poor, with 5-year survival rates stagnating at 20-30%. Although FLT3 inhibitors and venetoclax-based regimens have improved outcomes for certain patient subgroups, elderly and high-risk cohorts experience relapse rates exceeding 60%. Allogeneic hematopoietic stem cell transplantation (allo-HSCT), though potentially curative, faces significant limitations including donor scarcity, graft-versus-host disease (GVHD) and relapse rates greater than 30%(2–4). These unmet clinical needs underscore the imperative for novel therapeutic strategies in AML.\u003c/p\u003e\n\u003cp\u003eChimeric antigen receptor (CAR) T-cell therapy, which enables major histocompatibility complex (MHC)-independent tumor targeting, has revolutionized the treatment of B-cell malignancies, achieving remission rates exceeding 90% in relapsed/refractory disease(5). However, its translation to AML has been largely ineffective, with clinical response rates consistently below 30% despite promising preclinical targets such as CD33, CD123, CLL1(6–8). This therapeutic failure stems not only from antigenic heterogeneity but also from the profoundly immunosuppressive AML microenvironment(9–12). The mechanisms by which AML evades CAR-T cell cytotoxicity-through both intrinsic tumor factors and microenvironmental suppression-remain poorly understood.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eImmunoglobulin superfamily member 9 (IGSF9) is a transmembrane protein composed of five immunoglobulin (Ig)-like domains, two fibronectin type III (FNIII) domains, a transmembrane region and an intracellular immunoreceptor tyrosine-based inhibitory motif (ITIM)(13). Originally implicated in neurodevelopment, IGSF9 is aberrantly overexpressed in multiple carcinomas, where it correlates with poor prognosis and drives metastasis through the induction of epithelial-to-mesenchymal transition(14–17). Critically, we recently identified IGSF9 as a pan-cancer immunosuppressive checkpoint that orchestrates immune-evasive microenvironments by directly impairing cytotoxic lymphocyte function. Blockade of IGSF9 reversed T cell dysfunction and suppressed tumor growth in various \u0026nbsp;tumor models(18). Notably, we observed highly restricted IGSF9 expression on leukemic blasts from AML patients, with minimal or absent expression in normal hematopoietic stem cells and most other major tissues(19). This tumor-selective expression profiles IGSF9 as a compelling antigenic target for AML immunotherapy.\u003c/p\u003e\n\u003cp\u003eIFNγ has been identified as a major inducer of IGSF9 expression in AML (19). Given that IFNγ is abundantly secreted by activated CAR-T cells, we hypothesized that cytokine-driven upregulation of IGSF9 contributes to AML resistance against CAR-T therapy. However, the mechanisms by which AML cells exploit IGSF9 to evade immune surveillance, particularly CAR-T–mediated cytotoxicity, remain undefined. Here, we delineate the role of IGSF9 in orchestrating AML immune escape and engineer next-generation IG9BBz CAR-T cells to neutralize this immunosuppressive pathway. Our findings establish a mechanistic and therapeutic framework for overcoming resistance in AML immunotherapy.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cstrong\u003eCells and patient samples\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll cell lines were preserved by our lab and verified by STR(18,19). The AML cell lines (THP-1, MOLM13, U937, MV4-11) were cultured in RPMI1640 medium (10% FBS, 1% penicillin/streptomycin), The adherent cells (293T, LL/2, A549, H1299) were cultured in DMEM medium (10% FBS, 1% penicillin/streptomycin). Human primary T cells were cultured in RPMI1640 medium (10% FBS, 1% penicillin/streptomycin, 5ng/ml IL-7, 5ng/ml IL-15). Mouse primary T cells were cultured in RPMI1640 medium (10% FBS, 1% penicillin/streptomycin, 10ng/ml IL-2). Fresh bone marrow aspirates of AML patients were obtained from Yantai Yuhuangding Hospital with informed consent.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eProduction of CAR-T cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHuman peripheral blood mononuclear cells (PBMC) were isolated from healthy blood, which obtained from Yantai central blood station. Then the cells were stimulated by CD3/CD28 Dynabeads (ThermoFisher, Cat.No.11141D) with a ratio of 1:1 and cultured with RPMI1640 medium (10% FBS, 1% PS, 5ng/ml IL-7, 5ng/ml IL-15). To transduce CAR genes, the lentivirus was produced in 293T cells which has been transfected with CAR plasmid and packing plasmids (MD2.G and PsPAXP2). Transduction was performed 48 hours after PBMC isolation. The activated T cells were transduced with lentivirus supernatants on RetroNectin (Takara, Cat.No.T100A) coated plates by centrifuge at 2,000\u0026times;g for 2 hours at 32℃. Transduction efficiency of CAR-T cells was determined after three days.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo generate mouse CAR-T cells, the splenocytes were isolated from C57BL/6 wild type mice and activated by Concanavalin A (2\u0026mu;g/ml). To prepare retrovirus for transduction, the GP2-293 cells were transfected with CAR plasmid and pCAG-Eco envelop vector. 48 hours after transfection, the retrovirus supernatants were collected and concentrated by centrifuge at 20,000\u0026times;g for 2 hours at 4℃. Transduction of CAR-T cells were performed 24 hours after splenocytes isolation on RetroNectin coated plats by spin centrifugation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCo-culture of CAR-T cells with AML cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMOLM13-CD19 or MOLM13-CD19-IGSF9 cells (5\u0026times;10⁵) were co-cultured with 19BBz or 33BBz CAR-T cells (5\u0026times;10⁵) in 24-well plates using complete RPMI 1640 medium. After 20 hours, cells were harvested for flow cytometry. PD-L1 and IGSF9 surface expression was evaluated on gated CD19⁺ MOLM13 cells. To assess IGSF9 on antigen-negative AML cells, MOLM13-CD19 and MOLM13-GFP cells were mixed 1:1. This mixture (5\u0026times;10⁵ cells) was co-cultured with an equal number of CAR-T cells under identical conditions. For conditioned medium experiments, 33BBz CAR-T cells and MOLM13 cells were co-cultured (1:1 ratio). After 20 hours, medium was collected, filtered (0.45 \u0026micro;m), and mixed 1:1 with fresh complete RPMI 1640. MOLM13, U937, MV4-11 cells or patient-derived AML cells were then treated with this mixture for 24 hours, followed by IGSF9 surface expression analysis via flow cytometry. To assess degranulation (CD107a) and cytokines production, 1\u0026times;10⁵ 33BBz CAR-T cells were co-cultured with equal numbers of MOLM13 or MOLM13-IGSF9 cells in complete RPMI1640 medium supplemented with 3\u0026mu;g/ml Brefeldin A (MCE, Cat.NO.HY-16592) and 1mM Monensin (MCE, Cat.NO.HY-N0150) in a 96-well plate, then incubated for 6 hours prior to flow cytometry analysis.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRepeated stimulation of CAR-T cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor repeated stimulation, irradiated THP-1 (WT) or THP-1 (IGSF9 KO) cells (5\u0026times;10⁵) were seeded in 24-well plates. CD33-specific CAR-T cells (5\u0026times;10⁵) were added (E/T = 1/1). Fresh target cells were replenished at the same E/T ratio every 3 days. On day 12 following four stimulations, CAR-T cells were harvested and differentiation states were assessed by flow cytometry.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eC57BL/6 mouse model\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFour- to six-week-old female C57BL/6 mice were purchased from Charles River Laboratories. Individual mice were randomly assigned to treatment/control groups Mice (n=4/group) received subcutaneous injections of 1\u0026times;10⁶ LL/2-hCD19 or LL/2-hCD19-mIGSF9 cells. When tumors reached ~50 mm\u0026sup3; (day 9), 2 \u0026times; 10⁶ 19m28z CAR-T cells were administered intravenously. Mice were euthanized two weeks post-CAR-T infusion. Tumors were enzymatically dissociated, and exhaustion/differentiation states of tumor-infiltrating GFP⁺ CAR-T cells were assessed by flow cytometry.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eXenograft mouse model\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFour- to six-week-old female NOD/SCID-IL2Rg-null (NSG) mice were purchased from Shanghai Model Organisms. Individual mice were randomly assigned to treatment/control groups. For xenograft model with IGSF9 blockade, NSG mice received intravenous injection of 0.5\u0026times;10⁶ luciferase-expressing, GFP labeled MOLM13-IGSF9 cells. Three days post engraftment, mice were administered 0.5\u0026times;10⁶ 33BBz CAR-T or untransduced T (UnT) cells via tail vein. Anti-IGSF9 blocking antibody or mouse IgG control (0.2 mg/mouse) was delivered intravenously starting 24 hours post CAR-T infusion, with repeat dosing every 4 days (total 3 doses). Tumor burden was quantified weekly by bioluminescence imaging (IVIS Spectrum, PerkinElmer). For flow cytometric analysis of CAR-T cell function, mice received two antibody doses every 3 days beginning 24 hours post CAR-T infusion. At the terminal endpoint (day 12 post CAR-T infusion), the peripheral blood, bone marrow and spleen tissue were harvested. The frequency of GFP⁺ tumor cells and mCherry⁺ CAR-T cells in vivo was determined by flow cytometry. CAR-T cell differentiation status was evaluated based on surface expression of CD45RA and CD62L.\u003c/p\u003e\n\u003cp\u003eFor in vivo experiments of IGSF9-specific CAR-T cells, NSG mice received intravenous injection of 0.2\u0026times;10⁶ luciferase-expressing MOLM13-IGSF9 cells. Three days post engraftment, mice were administered 2\u0026times;10⁶ IG9BBz CAR-T or UnT cells via tail vein. Tumor burden was quantified weekly by bioluminescence imaging. Mice survival was monitored until death or reaching humane endpoint (Severe weight loss or paralysis). The probability of survival was described by Kaplan-Meier curves using log-rank (Mantel-Cox) tests. To quantify tumor burden and adoptive T cell persistence, bone marrow was harvested from mice at 14 days post-T cell infusion. GFP⁺ tumor cells and infused T cells were subsequently analyzed by flow cytometry to determine their frequency and phenotype\u003c/p\u003e\n\u003cp\u003eFor IGSF9 endogenously expressed AML model, NSG mice were engrafted with THP-1 tumors via intravenous injection of 0.5\u0026times;10⁶ THP-1-GFP-Luc cells. The next day, 5\u0026times;10⁶ IG9BBz CAR-T cells or untransduced T cells were administered by tail vein injection. Tumor burden was monitored longitudinally through serial bioluminescence imaging. Bone marrow was harvested at day 25 post-T cell infusion, followed by flow cytometric analysis of GFP⁺ tumor cells and infused T cells.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFlow cytometry\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe production of human IGSF9 antibody was described previously(18). To detect IGSF9 by flow cytometry, cells were incubated with IGSF9 antibody for 30 minutes at room temperature, and then stained with APC-conjugated anti-mouse IgG antibody (Biolegend, Cat.No.405308) for 20 minutes at 4℃ in the dark. Direct staining of surface antigens was performed by incubating cells with fluorochrome-conjugated antibodies for 20 minutes at 4℃ in the dark. For intracellular staining of IFN\u0026gamma; and TNF\u0026alpha;, cells were fixed and permeabilized using a Fixation/Permeabilization buffer (eBioscience, Cat.No.00-5123-43). Antibodies targeting human PD-L1 (Cat.No.329734), CD19 (Cat.No.302208), CD3 (Cat.No.300317), CD4 (Cat.No.317432), CD8 (Cat.No.344750), CD107a (Cat.No.328609), TNF\u0026alpha; (Cat.No.376203)，IFN\u0026gamma; (Cat.No.502510) PD-1 (Cat.No.367406), TIM-3 (Cat.No.345041), LAG-3 (Cat.No.369317), CD45RA (Cat.No.304142), CD62L (Cat.No.304810), CD69 (Cat.NO.310904) and mouse PD-1 (Cat.No.135221), TIM-3(Cat.No.134008), CD44 (Cat.No.103012), CD62L (Cat.No.104424), CD13 (Cat.No.301703), CD33 (Cat.No.303441), CD117 (Cat.No.313217) were purchased from Biolegend. Flow cytometry was performed on a BD Fortessa flow cytometer (BD Bioscience) or an Aurora Spectral Flow Cytometer (Cytek). Data were analyzed with FlowJo software (V10, TreeStar).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWestern blot\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA549 and A549-IGSF9-HA cells were lysed using RIPA buffer (Solarbio) containing protease inhibitors (Beyotime Biotechnology). The samples were first loaded onto an SDS-PAGE gel and then transferred to a PVDF membrane. The following primary antibodies were used for protein probing: mouse anti-human IGSF9(1:1000 dilution, home-made), mouse anti-HA tag (1:6000 dilution, Proteintech, Cat.No.51064-2-AP), Rabbit anti-human GAPDH (1:6000 dilution, Affinity, Cat.No.AF7021). After incubation with HRP-conjugated secondary antibodies (Proteintech) and ECL reagent (Biosharp), the protein bands were visualized by a chemiluminescent imaging system (Tanon).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIn vitro cytotoxicity assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCAR-T cell cytotoxicity was assessed using a luciferase-based assay. Target cells expressing luciferase (5\u0026times;10⁵) were co-cultured with CAR-T cells at specified E:T ratios in 96-well black-bottom plates. After 18\u0026ndash;20 hours, D-luciferin substrate was added, and bioluminescence (BL) was measured using a luminescence reader (LUX-P110, BLT). Lysis rates were calculated as [(1- BL of each sample)/BL of target cells alone]\u0026times;100%.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIGSF9-ECD-Fc binding assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eJurkat T cells expressing 19BBz or IG9BBz CAR constructs (1\u0026times;10\u003csup\u003e6\u003c/sup\u003e) were incubated with 2\u0026mu;g IGSF9-ECD-Fc protein (prepared as described(18)) or human IgG1 control at 4℃ for 30 minutes. Cells were washed with PBS, then stained with APC-conjugated anti-human IgG Fc antibody (Biolegend, 410712) at 4℃ for 30 minutes.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCytokines quantification by ELISA\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIG9BBz CAR-T cells were co-cultured with K562 or K562-IGSF9 cells at 1:1 ratio in complete RPMI1640 medium in a six-well plate. After 20 hours, culture supernatants were collected. Human IFN\u0026gamma; ELISA Kit (Abcam, ab46025), Human TNF\u0026alpha; SimpleStep ELISA Kit (Abcam, ab181421) and Human IL-2 ELISA Kit (Proteintech, KE00017) were used to quantify IFN\u0026gamma;, TNF\u0026alpha; and IL-2 secretion following manufacturer\u0026rsquo;s instruction.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGraphPad Prism 8 was used for statistical analysis. A two-tail Student\u0026rsquo;s t-test was used for comparing two groups. Data are presented as mean\u0026plusmn;SEM. *P\u0026lt;0.05, **p\u0026lt;0.01, ***p\u0026lt;0.001, ****p\u0026lt;0.0001.\u003c/p\u003e"},{"header":"Results","content":"\u003ch2\u003eCAR-T cell cytotoxicity induces IGSF9 upregulation in AML cells\u0026nbsp;\u003c/h2\u003e\n\u003cp\u003eGiven that IGSF9 is an IFN\u0026gamma;-inducible immune checkpoint molecule and CAR-T cells release cytotoxic cytokines (e.g., IFN\u0026gamma;) upon tumor engagement, we hypothesized that CAR-T cell attack triggers IGSF9 expression on AML cells. Co-culturing AML cells with second-generation 4-1BBz CAR-T cells targeting CD33 (33BBz; an endogenous AML antigen) or CD19 (19BBz; a clinically validated target) significantly upregulated both PD-L1 (confirming IFN\u0026gamma; pathway activation) and IGSF9 on target MOLM13-CD19⁺ cells after 20 hours (Figure 1A, Supplementary Figure S1A). Mechanistically, in mixed co-cultures containing both MOLM13-CD19⁺ and MOLM13-GFP⁺ cells, both populations exhibited comparable IGSF9 upregulation when exposed to either 19BBz or 33BBz CAR-T cells, confirming antigen-independence induction (Figure 1B, Supplementary Figure S1B). Furthermore, conditioned medium (CM) from 33BBz CAR-T/MOLM13 co-cultures, unlike that from untransduced T cell controls, robustly induced IGSF9 expression in fresh AML cells (Figure 1C, D, Supplementary Figure S1C). These findings demonstrate that soluble mediators, such as IFN\u0026gamma;, released during CAR-T cells/tumor interactions drive IGSF9 upregulation in AML cells, thereby establishing a feedforward inhibitory loop.\u003c/p\u003e\n\u003ch2\u003eIGSF9 expression on tumor cells impairs CAR-T cell anti-tumor functions\u003c/h2\u003e\n\u003cp\u003eTo determine whether tumor-expressed IGSF9 suppresses CAR-T cell activity, we performed luciferase-based cytotoxicity assays. Various CAR-T target cells were generated to express CD19 antigen and different level of IGSF9 (Supplementary Figure S2A). Knockout of IGSF9 in THP-1-CD19⁺ cells significantly enhanced tumor lysis by both 19BBz and 1928z CAR-T cells (Figure 2A, B). Conversely, IGSF9 overexpression in A549-CD19⁺ cells markedly reduced killing by both 19BBz and 1928z CAR-T cells (Figure 2C, D), indicating suppression independent CAR structure. Murine-derived 19m28z CAR-T cells targeting LL/2 cells in which human CD19 (hCD19) was overexpressed exhibited significantly diminished cytotoxicity against LL/2-hCD19-mIGSF9⁺ cells compared to LL/2-hCD19 controls, confirming similar suppressive function of mouse-derived IGSF9 (Figure 2E). Similarly, MOLM13-IGSF9⁺ cells showed enhanced resistance to 33BBz and 3328z CAR-T cell-mediated killing compared to parental MOLM13 (Figure 2F, G). As a control, 1928z CAR-T cells targeting MOLM13 or MOLM13-IGSF9\u003csup\u003e+\u003c/sup\u003e cells did not elicit remarkable tumor lysis effect (Supplementary Figure S2B). Mechanistically, co-cultured with MOLM13-IGSF9 elicited lower expression of CD107a of 33BBz CAR-T cells than MOLM13 groups (Figure 2H), indicating repressed degranulation of CAR-T cells by IGSF9. The cytokines release was also compared. CAR-T cells stimulated by MOLM13-IGSF9 produced comparable level of IFN\u0026gamma; but significantly reduced level of TNF\u0026alpha; compared to MOLM13 groups (Figure 2I, J). Moreover, repeated stimulation of CD33-specific CAR-T cells with irradiated THP-1 (IGSF9 KO) cells (\u003cem\u003evs\u003c/em\u003e. THP-1 WT) elevated the frequency of CD45RA\u003csup\u003e-\u003c/sup\u003eCD62L\u003csup\u003e+\u003c/sup\u003e central memory T cells (Supplementary Figure S3A-C).\u003cem\u003e\u0026nbsp;In vivo\u003c/em\u003e, tumors derived from LL/2-hCD19-mIGSF9⁺ cells in CAR-T-treated mice exhibited reduced CD44⁺CD62L⁺ memory T cell subsets and increased (though not significant, p=0.2655) PD-1⁺TIM-3⁺ population compared to LL/2-hCD19⁺ controls (Supplementary Figure S3D-G).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eCollectively, these data demonstrate that tumor-intrinsic IGSF9 impairs CAR-T cell functions by suppressing T cells degranulation, cytokines release and memory differentiation.\u003c/p\u003e\n\u003ch2\u003eBlockade of IGSF9 enhances CAR-T cell anti-tumor activity \u003cem\u003ein vivo\u003c/em\u003e\u003c/h2\u003e\n\u003cp\u003eWhile IGSF9 blockade has been shown to improve T cell function in solid tumors(18), its potential to enhance CAR-T cell efficacy against AML\u003cem\u003e\u0026nbsp;in vivo\u003c/em\u003e remained unexplored. To address this, we employed a combination therapy approach in an AML xenograft model. NSG mice engrafted with GFP\u003csup\u003e\u0026nbsp;\u003c/sup\u003elabeled MOLM13-IGSF9 cells were administered either 33BBz CAR-T cells or untransduced T cells (UnT). Starting one day after CAR-T cells infusion, mice were administered anti-IGSF9 or isotype control IgG every four days for a total of three doses (Figure 3A). Bioluminescence imaging revealed a significant reduction in tumor burden by day 7 in CAR-T cell-treated mice compared to UnT controls. Notably, anti-IGSF9 co-administration further suppressed tumor progression in the CAR-T group (Figure 3B-D). Furthermore, CAR-T cell treatment significantly prolonged survival, and this effect that was further enhanced by anti-IGSF9 treatment (Figure 3E).\u003c/p\u003e\n\u003cp\u003eWe next evaluated the impact of anti-IGSF9 treatment on the persistence of CAR-T cells \u003cem\u003ein vivo\u003c/em\u003e. One day after CAR-T cell injection, tumor-engrafted mice received anti-IGSF9 or control IgG, administered in two doses every four days. Flow cytometry analysis at 12 days post-CAR-T injection revealed that 33BBz CAR-T cells significantly reduced the frequency of GFP⁺ tumor cells (Figure 4A, Supplementary Figure S4A). Notably, in anti-IGSF9 combination group, GFP\u003csup\u003e+\u003c/sup\u003e tumor cells were nearly undetectable (Figure 4A). Furthermore, anti-IGSF9 treatment markedly increased the proportion of human CD3⁺ T cells in peripheral blood (Figure 4A, B). We then quantified CAR-T cells (mCherry⁺) in the bone marrow and spleen. Compared to IgG controls, anti-IGSF9 treatment elevated the proportion of mCherry⁺ CAR-T cells in these organs, resulting in a significantly higher mCherry⁺/GFP⁺ cell ratio (Figure 4C, D). Finally, we evaluated the differentiation state of CAR-T cells in the spleen, and found that anti-IGSF9 treatment significantly increased the proportion of CAR-T cells exhibiting a central memory phenotype (CD45RA⁻CD62L⁺) (Figure 4E, F, Supplementary Figure S4B).\u003c/p\u003e\n\u003cp\u003eCollectively, these results suggest that IGSF9 blockage enhances CAR-T cells persistence and promotes CAR-T cells anti-AML functions \u003cem\u003ein vivo\u003c/em\u003e.\u003c/p\u003e\n\u003ch2\u003eDesign and Functional Validation of IGSF9-Specific CAR-T Cells\u003c/h2\u003e\n\u003cp\u003eGiven the restricted expression of IGSF9 to AML blasts and its role as a critical suppressor of T-cell function, we hypothesized that selective elimination of IGSF9⁺ AML cells would limit disease progression. To this end, we engineered novel IGSF9-specific CAR-T cells (IG9BBz). Western blot analysis confirmed the specificity of the anti-IGSF9 antibody for IGSF9⁺ human cells (Figure 5A). Structural modeling predicted that the Ig-like 2 and fibronectin type III-2 (FNIII-2) domains constitute the core epitope recognized by the scFv antibody (Figure 5B), which is consistent with our previous report(19). Flow cytometry demonstrated robust labeling of membrane-expressed IGSF9-mEGFP in HEK293T cells using the fluorophore-conjugated anti-IGSF9 antibody, confirming efficient binding to cell-surface IGSF9 (Figure 5C). The IG9BBz CAR construct incorporated an scFv derived from the anti-IGSF9 antibody as the antigen-binding domain, coupled with 4-1BB and CD3z intracellular signaling domains, and a P2A-linked mCherry reporter for tracking CAR expression (Figure 5D). Jurkat cells expressing IG9BBz bound recombinant IGSF9-ECD-Fc, whereas control CAR variants lacking the scFv showed no binding, confirming antigen specificity (Figure 5E). To evaluate antigen-dependent activation, primary IG9BBz CAR-T cells were generated and co-cultured with tumor targets for 20 hours (Supplementary Figure S5). Exposure to H1299-IGSF9 or K562-IGSF9 cells, but not parental K562 cells, markedly upregulated CD69 expression on CAR-T cells (Figure 5F). Moreover, co-culture with K562-IGSF9 cells, but not parental K562 cells, induced a pronounced shift of CAR-T cells from a na\u0026iuml;ve to an effector state (Figure 5G, H), indicating antigen-specific activation of IG9BBz CAR-T cells. To further determine CAR-T cell functions, cytokine secretion was quantified by ELISA. Co-culture with K562-IGSF9 cells, but not parental K562 cells elicited abundant secretion of IFN\u0026gamma;, IL-2, and TNF\u0026alpha; (Figure 5I). In cytotoxicity assays, IG9BBz CAR-T cells efficiently eliminated H1299-IGSF9 and MOLM13-IGSF9 cells within 18 hours, as measured by bioluminescence (Fig. 5J).\u0026nbsp;\u003c/p\u003e\n\u003ch2\u003ePotent anti-AML activity of IG9BBz CAR-T cells across preclinical models\u003c/h2\u003e\n\u003cp\u003eThe anti-tumor efficacy of IG9BBz CAR-T cells was first evaluated in NSG mice bearing MOLM13-IGSF9-Luc AML xenografts (Figure 6A). IG9BBz CAR-T cell administration significantly reduced tumor burden, as quantified by bioluminescence imaging (Figures 6B, C), and extended survival compared to UnT controls (Figure 6D). To confirm direct tumor elimination, bone marrow was analyzed at 14 days post-infusion. IG9BBz CAR-T treatment markedly decreased GFP⁺ tumor burden while increasing the CD3⁺/GFP⁺ ratio (Figures 6E, G). Residual tumor cells maintained IGSF9 expression in both groups (Supplementary Figure S6A). Notably, the IGSF9 percentage in residual GFP⁺ cells were significantly lower in IG9BBz-treated mice vs. UnT controls, indicating preferential targeting of IGSF9-high tumor cells (Supplementary Figure S6A). Phenotypic characterization revealed distinct T-cell differentiation patterns: UnT cells predominantly exhibited an effector phenotype (CD45RA⁺CD62L⁻), whereas IG9BBz CAR-T cells (CD3⁺mCherry⁺) primarily comprised effector memory (CD45RA⁻CD62L⁻) and central memory (CD45RA⁻CD62L⁺) subsets (Figures 6E, F). Critically, IG9BBz CAR-T cells demonstrated significantly lower expression of inhibitory receptors (PD-1, TIM-3, LAG-3) versus UnT controls (Figures 6E, H). These findings indicate tumor antigen-driven activation and favorable differentiation of IG9BBz CAR-T cells. The emergence of memory subpopulations coupled with reduced exhaustion suggests enhanced persistence potential of this CAR construct.\u003c/p\u003e\n\u003cp\u003eTo evaluate IG9BBz CAR-T cell activity against AML with endogenous IGSF9 expression, NSG mice bearing THP-1-GFP-Luc xenografts received IG9BBz CAR-T cells or UnT controls (Figure 7A). Longitudinal bioluminescence imaging revealed progressive tumor expansion in UnT-treated mice, whereas 3 of 4 IG9BBz CAR-T-treated mice exhibited minimal disease progression (Figures 7B). Bone marrow analysis at day 25 post-infusion confirmed significant tumor reduction by IG9BBz CAR-T treatment, demonstrating decreased GFP⁺ tumor burden and increased CD3⁺/GFP⁺ ratios (Figure 7C, D). The GFP\u003csup\u003e+\u003c/sup\u003e tumor cells were further verified to be IGSF9 positive (Figure 7C). Notably, one IG9BBz-treated mouse showing elevated bioluminescent signal exhibited low marrow tumor infiltration, suggesting predominant extramedullary tumor engraftment in this individual.\u003c/p\u003e\n\u003cp\u003eTo assess clinical relevance, bone marrow aspirates from two AML patients were analyzed. Treatment with conditioned medium from 33BBz CAR-T/MOLM13 co-cultures significantly upregulated IGSF9 expression in the CD3⁻ compartment of AML (Figure 7E). When co-cultured with AML blasts, IG9BBz CAR-T cells exhibited elevated CD69 expression, confirming antigen-specific activation (Figure 7F). Notably, IGSF9⁺ cells predominantly resided within the primitive CD13⁺CD33⁺CD117⁺ population (Supplementary Figure S6B). IG9BBz CAR-T co-culture selectively reduced this target-enriched subset in AML samples (Figure 7G).\u003c/p\u003e\n\u003cp\u003eCollectively, these data establish that IG9BBz CAR-T cells exert potent anti-leukemic activity against IGSF9-expressing AML \u003cem\u003ein vivo\u003c/em\u003e, and demonstrate translational potential through CAR-T-induced IGSF9 upregulation that enhances blast elimination.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eImmune escape remains a major barrier to durable remission after CAR-T therapy for AML. Here, we identify IGSF9 as an immune suppressor that drives resistance to CAR-T cells in AML. Cytotoxic cytokines released by activated CAR-T cells induced robust IGSF9 expression on AML blasts. The release of inflammatory cytokines, including IFNγ, IL-2, and TNFα, represents a primary mechanism by which CAR-T cells exert cytotoxic effects(20). Our previous work identified IFNγ as a key driver of IGSF9 upregulation via the JAK1-STAT1 signaling pathway(19). Whether additional cytokines contribute to this induction remains to be determined. Nevertheless, we show that the cytokine milieu generated during CAR-T cytotoxicity is sufficient to induce substantial IGSF9 expression in AML cells. This antigen-independent induction by soluble mediators establishes a feed-forward immunosuppressive loop: CAR-T cells activation triggers cytokine release, which in turn upregulates IGSF9 on AML blasts, thereby amplifying its inhibitory effect and progressively impairing T-cell function. This mechanism represents a distinct form of immune evasion that is independent of antigen loss(21). Disrupting this immune-evasive niche is paramount for advancing AML CAR-T therapy.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eIn vitro\u003c/em\u003e, AML cells with high IGSF9 expression confer treatment resistance against CAR-T cytotoxicity, potentially through impairing effector functions—specifically by suppressing degranulation and TNFα secretion. Repeated antigen stimulation further compromised CAR-T memory differentiation, thereby undermining durable antitumor immunity. Although the structural basis of IGSF9 signaling remains undefined, two non-mutually exclusive mechanisms are plausible: (1) direct activation of inhibitory pathways, dampening CAR-T cell activation, and (2) signaling through the IGSF9 intracellular ITIM motif in AML cells, potentially enhancing their survival upon T cell contact. The identity of the cognate IGSF9 receptor remains challenging. Our preliminary data nominate TMUB1 (Transmembrane and Ubiquitin-Like Domain-Containing 1) as a candidate mediator of IGSF9-dependent immunosuppression. Definitive molecular validation of this interaction warrants further investigation.\u003c/p\u003e\n\u003cp\u003eUsing xenogeneic IGSF9 positive AML models, we demonstrate that monoclonal antibody–mediated IGSF9 blockade attenuates tumor progression and prolongs survival. Beyond direct blockade of IGSF9’s inhibitory signaling, the modest tumor control observed with anti-IGSF9 plus untransduced T cells points to an additional contribution from antibody-dependent cellular phagocytosis (ADCP). Nevertheless, \u003cem\u003eIn vivo\u003c/em\u003e, IGSF9 inhibition enhanced CAR-T cell persistence, restored the CD62L⁺CD44⁺ central-memory T cell pool, and reduced the frequency of exhausted T cell subsets. These findings establish IGSF9 as a tractable therapeutic target capable of reprogramming the AML microenvironment from a tolerogenic to an immunogenic state.\u003c/p\u003e\n\u003cp\u003eWe further developed IGSF9-specific CAR-T cells designed to integrate tumor antigen recognition with simultaneous ablation of an inhibitory ligand. IGSF9 expression is restricted to leukemic blasts and absent on normal CD34⁺ hematopoietic stem cells(19), minimizing the risk of on-target off-tumor toxicity. \u003cem\u003eIn vitro\u0026nbsp;\u003c/em\u003eand\u003cem\u003e\u0026nbsp;in vivo\u003c/em\u003e, IG9BBz CAR-T cells demonstrated potent cytotoxicity against IGSF9 positive AML cells. This strategy leverages the dual role of IGSF9-its tumor-restricted expression and immunosuppressive function-enabling simultaneous clearance of malignant cells and disruption of the inhibitory niche. The potential emergence of IGSF9-negative escape variants could be mitigated by employing tandem CAR constructs that co-target IGSF9 and CD33 or CD123 under OR-gate logic, thereby ensuring robust activity against both antigen-low subclones and microenvironmentally suppressive cells(22).\u003c/p\u003e\n\u003cp\u003eLimitations of this study include the use of immunodeficient AML models for CAR-T therapy, which cannot fully recapitulate endogenous T cell-AML interactions or allow assessment of graft-versus-host disease. Future studies will employ humanized models and primary AML xenografts with autologous CAR-T manufacturing to better evaluate patient-specific heterogeneity and immune dynamics. Moreover, this study does not assess potential on-target/off-tumor toxicity of IGSF8-directed therapies, which requires urgent investigation given its trace expression in neuronal tissues (Human Protein Atlas, proteinatlas.org)(23).\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eIn summary, we delineate a cytokine-driven IGSF9-mediated resistance circuit that undermines CAR-T cell activity in AML and validate two complementary therapeutic strategies-IGSF9 blockade and IGSF9-directed CAR-T cells-to disrupt this immunosuppressive axis. These findings provide a mechanistic framework for next-generation immunotherapies that integrate tumor antigen targeting with reprogramming of the immune microenvironment, offering a promising path toward sustained remission in high-risk AML.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll studies were approved by the Medical Ethics Committee of Binzhou Medical University (No: 2025-003), and the Animal Ethics Committee of Binzhou Medical University (No: 2025-015). All animal experiments were performed under the national standards of Institutional Animal Care and Use Committee.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and material\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated or analysed during this study are included in this published article [and its supplementary information files].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was funded by the National Natural Science Foundation of China (82150101, 81872332, 82070225) to Zunling Li, Shandong Natural Science Foundation (ZR2024LZL010) to Zunling Li, and (ZR2023QC190) to Xianhui Meng.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eX.M., Y.W. and Z.L. designed the study and wrote the manuscript. X.M., FM.L. and H.Y. performed the experiments and analyzed the data. C.L., Y.S., H.W., G.L., J.Z., L.H., F.L. and S.W. contributed to animal studies and data analysis. All authors reviewed and approved the final manuscript. The corresponding author (Z.L.) is the guarantor for the publication, taking responsibility for the integrity of the entire work.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the Special Funding for the \u0026quot;Case-by-Case Introduction of Top Talent (Teams)\u0026quot; Program in Yantai.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eToma MM, Skorski T. Star wars against leukemia: attacking the clones. Leukemia. 2024 Nov;38(11):2293\u0026ndash;302. \u003c/li\u003e\n\u003cli\u003eKantarjian HM, DiNardo CD, Kadia TM, Daver NG, Altman JK, Stein EM, et al. Acute myeloid leukemia management and research in 2025. CA Cancer J Clin. 2025 Jan;75(1):46\u0026ndash;67. \u003c/li\u003e\n\u003cli\u003eThol F, D\u0026ouml;hner H, Ganser A. How I treat refractory and relapsed acute myeloid leukemia. Blood. 2024 Jan 4;143(1):11\u0026ndash;20. \u003c/li\u003e\n\u003cli\u003eMathioudaki A, Wang X, Sedloev D, Huth R, Kamal A, Hundemer M, et al. The remission status of AML patients after allo-HCT is associated with a distinct single-cell bone marrow T-cell signature. Blood. 2024 Mar 28;143(13):1269\u0026ndash;81. \u003c/li\u003e\n\u003cli\u003eCappell KM, Kochenderfer JN. Long-term outcomes following CAR T cell therapy: what we know so far. Nat Rev Clin Oncol. 2023 June;20(6):359\u0026ndash;71. \u003c/li\u003e\n\u003cli\u003eHaubner S, Subklewe M, Sadelain M. Honing CAR T cells to tackle acute myeloid leukemia. Blood. 2025 Mar 13;145(11):1113\u0026ndash;25. \u003c/li\u003e\n\u003cli\u003eTettamanti S, Pievani A, Biondi A, Dotti G, Serafini M. Catch me if you can: how AML and its niche escape immunotherapy. Leukemia. 2022 Jan;36(1):13\u0026ndash;22. \u003c/li\u003e\n\u003cli\u003eFoldvari Z, Brennan MS, Titov A, Jacobsen SEW, Olweus J. Targeting the roots of myeloid malignancies with T cell receptors. Nat Rev Cancer. 2025 Aug 21;1\u0026ndash;21. \u003c/li\u003e\n\u003cli\u003eCiantra Z, Paraskevopoulou V, Aifantis I. The rewired immune microenvironment in leukemia. Nat Immunol. 2025 Mar;26(3):351\u0026ndash;65. \u003c/li\u003e\n\u003cli\u003eVadakekolathu J, Rutella S. Escape from T-cell\u0026ndash;targeting immunotherapies in acute myeloid leukemia. Blood. 2024 June 27;143(26):2689\u0026ndash;700. \u003c/li\u003e\n\u003cli\u003eZuo S, Li C, Sun X, Deng B, Zhang Y, Han Y, et al. C-JUN overexpressing CAR-T cells in acute myeloid leukemia: preclinical characterization and phase I trial. Nat Commun. 2024 July 22;15(1):6155. \u003c/li\u003e\n\u003cli\u003eLasry A, Nadorp B, Fornerod M, Nicolet D, Wu H, Walker CJ, et al. An inflammatory state remodels the immune microenvironment and improves risk stratification in acute myeloid leukemia. Nat Cancer. 2023 Jan;4(1):27\u0026ndash;42. \u003c/li\u003e\n\u003cli\u003eHansen M, Walmod PS. IGSF9 family proteins. Neurochem Res. 2013 June;38(6):1236\u0026ndash;51. \u003c/li\u003e\n\u003cli\u003eShi SH, Cheng T, Jan LY, Jan YN. The immunoglobulin family member dendrite arborization and synapse maturation 1 (Dasm1) controls excitatory synapse maturation. Proc Natl Acad Sci U S A. 2004 Sept 7;101(36):13346\u0026ndash;51. \u003c/li\u003e\n\u003cli\u003eShi SH, Cox DN, Wang D, Jan LY, Jan YN. Control of dendrite arborization by an ig family member, dendrite arborization and synapse maturation 1 (Dasm1). Proc Natl Acad Sci U S A. 2004 Sept 7;101(36):13341\u0026ndash;5. \u003c/li\u003e\n\u003cli\u003eLi Y, Deng Y, Zhao Y, Zhang W, Zhang S, Zhang L, et al. Immunoglobulin superfamily 9 (IGSF9) is trans-activated by p53, inhibits breast cancer metastasis via FAK. Oncogene. 2022 Oct;41(41):4658\u0026ndash;72. \u003c/li\u003e\n\u003cli\u003eLuan H, Wang T, Li F, Sun S, Wang Z, Zhao X, et al. IGSF9 promotes tumor invasion and metastasis through GSK-3\u0026beta;/\u0026beta;-catenin mediated EMT in lung cancer. Neoplasia N Y N. 2024 Dec;58:101067. \u003c/li\u003e\n\u003cli\u003eLiu Y, Wang H, Zhao X, Zhang J, Zhao Z, Lian X, et al. Targeting the immunoglobulin IGSF9 enhances antitumor T-cell activity and sensitivity to anti-PD-1 immunotherapy. Cancer Res. 2023 Oct 13;83(20):3385\u0026ndash;99. \u003c/li\u003e\n\u003cli\u003eHui L, Xiao J, Zhao Z, Zhang J, Luan H, Zhang J, et al. IGSF9-targeted therapy inhibits the progression of acute myeloid leukemia. Blood Adv. 2025 June 17;bloodadvances.2025016432. \u003c/li\u003e\n\u003cli\u003eYoung RM, Engel NW, Uslu U, Wellhausen N, June CH. Next-generation CAR T-cell therapies. Cancer Discov. 2022 July 6;12(7):1625\u0026ndash;33. \u003c/li\u003e\n\u003cli\u003eBhagwat AS, Torres L, Shestova O, Shestov M, Mellors PW, Fisher HR, et al. Cytokine-mediated CAR T therapy resistance in AML. Nat Med. 2024 Dec;30(12):3697\u0026ndash;708. \u003c/li\u003e\n\u003cli\u003eHaubner S, Mansilla-Soto J, Nataraj S, Kogel F, Chang Q, De Stanchina E, et al. Cooperative CAR targeting to selectively eliminate AML and minimize escape. Cancer Cell. 2023 Nov;41(11):1871-1891.e6. \u003c/li\u003e\n\u003cli\u003eMishra A, Traut MH, Becker L, Klopstock T, Stein V, Klein R. Genetic evidence for the adhesion protein IgSF9/Dasm1 to regulate inhibitory synapse development independent of its intracellular domain. J Neurosci Off J Soc Neurosci. 2014 Mar 19;34(12):4187\u0026ndash;99. \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":"journal-of-experimental-and-clinical-cancer-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jecc","sideBox":"Learn more about [Journal of Experimental \u0026 Clinical Cancer Research](http://jeccr.biomedcentral.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/jecc/default.aspx","title":"Journal of Experimental \u0026 Clinical Cancer Research","twitterHandle":"@OncoBioMed","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"AML, CAR-T, IGSF9, Immune suppression","lastPublishedDoi":"10.21203/rs.3.rs-8606613/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8606613/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground: \u003c/strong\u003eChimeric antigen receptor (CAR)-T cell therapy faces substantial barriers in acute myeloid leukemia (AML), yet the mechanisms by which AML evades CAR-T cell cytotoxicity—through tumor-intrinsic factors and microenvironmental suppression—remain poorly defined. Immunoglobulin superfamily member 9 (IGSF9) has recently been identified as an immunosuppressive molecule selectively expressed on AML blasts, but its role in mediating resistance to CAR-T therapy is unknown.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods\u003c/strong\u003e: We examined IGSF9 expression in AML cells upon CAR-T challenge and conducted in vitro and in vivo assays to define its functional impact on CAR-T cell activity. To overcome IGSF9-mediated suppression, we evaluated two therapeutic strategies: antibody-mediated IGSF9 blockade and the generation of IGSF9-specific CAR-T cells (IG9BBz).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults: \u003c/strong\u003eCAR-T–induced cytotoxic pressure upregulated IGSF9 on AML cells. IGSF9-positive AML cells exhibited resistance to CAR-T killing and impaired CAR-T persistence both \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e. Antibody blockade of IGSF9 restored CAR-T function in xenograft models. Moreover, IG9BBz CAR-T cells demonstrated potent and selective elimination of IGSF9-positive AML cells in both settings.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions: \u003c/strong\u003eOur findings identify a cytokine-driven IGSF9 resistance circuit that suppresses CAR-T cell function in AML. Therapeutic disruption of this pathway through IGSF9 blockade or IGSF9-specific CAR-T cells restores antitumor immunity and provides complementary strategies to overcome CAR-T resistance in AML.\u003c/p\u003e","manuscriptTitle":"Targeting Immunoglobulin Superfamily Member 9 (IGSF9) to Overcome Acute Myeloid Leukemia Resistance to CAR-T Therapy","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-03-11 06:10:12","doi":"10.21203/rs.3.rs-8606613/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-03-02T07:21:01+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-03-02T06:27:34+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-02-24T00:35:53+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"309918220292791715729338930002186006821","date":"2026-01-27T17:27:48+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"9270160112922829765739084895556169416","date":"2026-01-26T20:45:32+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"213897093199590356413327728725001357188","date":"2026-01-25T12:38:32+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-01-19T16:09:36+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-01-19T12:41:22+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-01-19T12:40:20+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of Experimental \u0026 Clinical Cancer Research","date":"2026-01-15T03:43:21+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"journal-of-experimental-and-clinical-cancer-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jecc","sideBox":"Learn more about [Journal of Experimental \u0026 Clinical Cancer Research](http://jeccr.biomedcentral.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/jecc/default.aspx","title":"Journal of Experimental \u0026 Clinical Cancer Research","twitterHandle":"@OncoBioMed","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"3cc8819a-dd99-4776-9591-e7c2ad894b33","owner":[],"postedDate":"March 11th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-05-14T14:10:19+00:00","versionOfRecord":[],"versionCreatedAt":"2026-03-11 06:10:12","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8606613","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8606613","identity":"rs-8606613","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}