Journal Articles

Targeted TNF Potentiates the Activity of Bispecific T-cell Engagers in Solid Tumors by Turning Cold Tumors Hot Spotlight 

As colorectal cancer immunotherapy has shown limited success, Thorhallsdottir et al. developed a dual-modality approach. L19-TNF, a TNF-based fusion protein directed to pan-tumor stromal extradomain B of fibronectin (to induce intratumoral inflammation) was combined with a CEA-targeted CD3-based T cell engager (CEAxCD3 TCE) to promote CD8+ T cell proliferation and antigen-specific cytotoxicity. In two immunocompetent models, L19-TNF plus CEAxCD3 resulted in >50% CRs, prolonged survival, and durable memory, with a tolerable safety profile. Mechanistically, the combination revealed enhanced TCE extravasation and TIME remodeling.

Contributed by Katherine Turner

As colorectal cancer immunotherapy has shown limited success, Thorhallsdottir et al. developed a dual-modality approach. L19-TNF, a TNF-based fusion protein directed to pan-tumor stromal extradomain B of fibronectin (to induce intratumoral inflammation) was combined with a CEA-targeted CD3-based T cell engager (CEAxCD3 TCE) to promote CD8+ T cell proliferation and antigen-specific cytotoxicity. In two immunocompetent models, L19-TNF plus CEAxCD3 resulted in >50% CRs, prolonged survival, and durable memory, with a tolerable safety profile. Mechanistically, the combination revealed enhanced TCE extravasation and TIME remodeling.

Contributed by Katherine Turner

ABSTRACT: Colorectal cancer remains a major global health burden and an area of urgent unmet medical need. Immunotherapy has shown limited success in colorectal cancer as most patients present with an immune-excluded, "cold" tumor microenvironment (TME). In this study, we report a dual-modality approach to treating colorectal cancer by combining the tumor necrosis factor (TNF)-based fusion protein directed to the extradomain B (EDB) of fibronectin, L19-TNF, which induces localized intratumoral inflammation and facilitates T-cell infiltration, with a CD3-based bispecific T-cell engager (TCE) targeting carcinoembryonic antigen (CEA), which mediates antigen-specific cytotoxicity. Together, these agents aim to remodel the TME, convert "cold" tumors into inflamed "hot" lesions, and broaden the therapeutic reach of immunotherapy in colorectal cancer. Immunohistochemistry confirmed coexpression of CEA and EDB across microsatellite-stable and -instable tumors. In vitro, L19-TNF in combination with a CEAxCD3 TCE significantly enhanced tumor cell killing and CD8+ T-cell proliferation. In vivo, the combination induced complete tumor regression in most animals, prolonged survival, and conferred durable protection against tumor rechallenge. Furthermore, mechanistic analyses revealed enhanced TCE extravasation, upregulated intercellular adhesion molecule 1 expression, and increased CD8+ T-cell infiltration, indicating vascular modulation and remodeling of the TME toward an inflamed "hot" phenotype. These findings confirm that targeted delivery of TNF to the TME can effectively enhance the activity of immunotherapeutic agents, such as T cell-redirecting therapies, in challenging tumor settings.

Author Info: (1) Philochem AG, Otelfingen, Switzerland. Swiss Federal Institute of Technology, ETH ZŸrich, Zurich, Switzerland. ROR: https://ror.org/05a28rw58 (2) Philochem AG, Otelfingen, Swit

Author Info: (1) Philochem AG, Otelfingen, Switzerland. Swiss Federal Institute of Technology, ETH ZŸrich, Zurich, Switzerland. ROR: https://ror.org/05a28rw58 (2) Philochem AG, Otelfingen, Switzerland. (3) University of Pisa , Pisa, Italy. ROR: https://ror.org/03ad39j10 (4) University of Pisa , Pisa, Italy. ROR: https://ror.org/03ad39j10 (5) Philochem AG, Otelfingen, Switzerland. (6) Philochem AG, Otelfingen, Switzerland. (7) Philochem AG, Otelfingen, Switzerland. Swiss Federal Institute of Technology, ETH ZŸrich, Zurich, Switzerland. ROR: https://ror.org/05a28rw58 (8) Philochem AG, Otelfingen, Switzerland. (9) Philochem AG, Otelfingen, Switzerland. Philogen SpA, Siena, Italy. (10) Philochem AG, Otelfingen, Switzerland. Swiss Federal Institute of Technology, ETH ZŸrich, Zurich, Switzerland. ROR: https://ror.org/05a28rw58 Philogen SpA, Siena, Italy. (11) Philochem AG, Otelfingen, Switzerland.

Activated T cell extracellular vesicle DNA transfer enhances antigen presentation and anti-tumor immunity Spotlight 

Hu and Liu et al. found that activated T cells secreted abundant extracellular vesicular DNA (AT-EVDNA) that was mainly from newly made genomic DNA and was rich in immune-related genes. Upon uptake of EVs by tumor cells or dendritic cells, granzyme B encapsulated in the EVs disrupted the nuclear envelope and facilitated entry of EVDNA into the nucleus, where transient expression of the EVDNA increased antigen processing and presentation machinery and cytokine production, enhancing immunogenicity. In mouse models, AT-EVs overcame immune evasion and boosted immune checkpoint blockade, supporting their potential use as an acellular immunotherapy.

Contributed by Lauren Hitchings

Hu and Liu et al. found that activated T cells secreted abundant extracellular vesicular DNA (AT-EVDNA) that was mainly from newly made genomic DNA and was rich in immune-related genes. Upon uptake of EVs by tumor cells or dendritic cells, granzyme B encapsulated in the EVs disrupted the nuclear envelope and facilitated entry of EVDNA into the nucleus, where transient expression of the EVDNA increased antigen processing and presentation machinery and cytokine production, enhancing immunogenicity. In mouse models, AT-EVs overcame immune evasion and boosted immune checkpoint blockade, supporting their potential use as an acellular immunotherapy.

Contributed by Lauren Hitchings

ABSTRACT: Antigen processing and presentation (APP) is essential for adaptive immunosurveillance. We uncover a mechanism whereby activated T cell-derived extracellular vesicles (AT(EVs)) drive a positive feedback loop that enhances antigen presentation and immune responses in normal physiology and cancer. AT(EV)-induced immunogenicity relies on extracellular vesicular double-stranded DNA (EV(DNA)), which is notably abundant and primarily composed of genomic DNA enriched in immune-related genes, including those encoding APP machinery. Mechanistically, granzyme B (Gzmb) packaged by AT(EVs) disrupts the nuclear envelope of recipient cells, facilitating intranuclear transfer and subsequent transient expression of EV(DNA) encoding APP genes. DNase treatment removes most AT-EV(DNA), abrogating APP upregulation and thus T cell activation and recruitment to tumors. Notably, AT(EVs) hold promise as an acellular immunotherapy, restoring APP and synergizing with checkpoint blockade in immunotherapy-refractory tumors. Collectively, our findings uncover a mechanism of transient, non-viral gene delivery by AT(EVs) that boosts APP and anti-tumor immunity while limiting autoimmunity.

Author Info: (1) Children's Cancer and Blood Foundation Laboratories, Departments of Pediatrics, and Cell and Developmental Biology, Drukier Institute for Children's Health, Meyer Cancer Center

Author Info: (1) Children's Cancer and Blood Foundation Laboratories, Departments of Pediatrics, and Cell and Developmental Biology, Drukier Institute for Children's Health, Meyer Cancer Center, Weill Cornell Medicine, New York, NY, USA; Division of Pharmaceutics and Pharmacology, College of Pharmacy, The Ohio State University, Columbus, OH, USA. (2) Children's Cancer and Blood Foundation Laboratories, Departments of Pediatrics, and Cell and Developmental Biology, Drukier Institute for Children's Health, Meyer Cancer Center, Weill Cornell Medicine, New York, NY, USA. (3) Children's Cancer and Blood Foundation Laboratories, Departments of Pediatrics, and Cell and Developmental Biology, Drukier Institute for Children's Health, Meyer Cancer Center, Weill Cornell Medicine, New York, NY, USA. (4) Department of Physiology, Biophysics, and Systems Biology, Weill Cornell Medicine, New York, NY, USA. (5) Department of Physiology, Biophysics, and Systems Biology, Weill Cornell Medicine, New York, NY, USA. (6) Department of Physiology, Biophysics, and Systems Biology, Weill Cornell Medicine, New York, NY, USA. (7) Department of Systems Biology, Columbia University, New York, NY, USA. (8) Children's Cancer and Blood Foundation Laboratories, Departments of Pediatrics, and Cell and Developmental Biology, Drukier Institute for Children's Health, Meyer Cancer Center, Weill Cornell Medicine, New York, NY, USA. (9) Children's Cancer and Blood Foundation Laboratories, Departments of Pediatrics, and Cell and Developmental Biology, Drukier Institute for Children's Health, Meyer Cancer Center, Weill Cornell Medicine, New York, NY, USA; Thoracic Surgery Service, Department of Surgery, Memorial Sloan Kettering Cancer Center, New York, NY, USA; Department of Respiratory Medicine, Juntendo University, Tokyo, Japan. (10) Children's Cancer and Blood Foundation Laboratories, Departments of Pediatrics, and Cell and Developmental Biology, Drukier Institute for Children's Health, Meyer Cancer Center, Weill Cornell Medicine, New York, NY, USA; Department of Biomedical and Clinical Sciences, Linkšping University, Linkšping, Sweden. (11) Children's Cancer and Blood Foundation Laboratories, Departments of Pediatrics, and Cell and Developmental Biology, Drukier Institute for Children's Health, Meyer Cancer Center, Weill Cornell Medicine, New York, NY, USA. (12) Children's Cancer and Blood Foundation Laboratories, Departments of Pediatrics, and Cell and Developmental Biology, Drukier Institute for Children's Health, Meyer Cancer Center, Weill Cornell Medicine, New York, NY, USA. (13) Drukier Institute for Children's Health and Department of Pediatrics, Weill Cornell Medicine, New York, NY, USA. (14) Drukier Institute for Children's Health and Department of Pediatrics, Weill Cornell Medicine, New York, NY, USA. (15) Children's Cancer and Blood Foundation Laboratories, Departments of Pediatrics, and Cell and Developmental Biology, Drukier Institute for Children's Health, Meyer Cancer Center, Weill Cornell Medicine, New York, NY, USA. (16) Children's Cancer and Blood Foundation Laboratories, Departments of Pediatrics, and Cell and Developmental Biology, Drukier Institute for Children's Health, Meyer Cancer Center, Weill Cornell Medicine, New York, NY, USA. (17) Children's Cancer and Blood Foundation Laboratories, Departments of Pediatrics, and Cell and Developmental Biology, Drukier Institute for Children's Health, Meyer Cancer Center, Weill Cornell Medicine, New York, NY, USA. (18) Drukier Institute for Children's Health and Department of Pediatrics, Weill Cornell Medicine, New York, NY, USA. (19) Drukier Institute for Children's Health and Department of Pediatrics, Weill Cornell Medicine, New York, NY, USA. (20) Children's Cancer and Blood Foundation Laboratories, Departments of Pediatrics, and Cell and Developmental Biology, Drukier Institute for Children's Health, Meyer Cancer Center, Weill Cornell Medicine, New York, NY, USA. (21) Children's Cancer and Blood Foundation Laboratories, Departments of Pediatrics, and Cell and Developmental Biology, Drukier Institute for Children's Health, Meyer Cancer Center, Weill Cornell Medicine, New York, NY, USA. (22) Children's Cancer and Blood Foundation Laboratories, Departments of Pediatrics, and Cell and Developmental Biology, Drukier Institute for Children's Health, Meyer Cancer Center, Weill Cornell Medicine, New York, NY, USA. (23) Department of Molecular Cell Biology, The Weizmann Institute of Science, Rehovot, Israel. (24) Proteomics Resource Center, The Rockefeller University, New York, NY 10065, USA. (25) Hepatopancreatobiliary Service, Department of Surgery, Memorial Sloan Kettering Cancer Center, New York, NY, USA. (26) Department of Molecular Cell Biology, The Weizmann Institute of Science, Rehovot, Israel. (27) Department of Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA. (28) Department of Pathology, University of California, San Diego, La Jolla, CA, USA. (29) Laboratory of Tumor Microenvironment and Therapeutic Resistance, KU Leuven, Leuven, Belgium. (30) Laboratory of Biology of Tumor and Development, UniversitŽ de Lige, Lige, Belgium. (31) Eppley Institute for Research in Cancer and Allied Diseases, Fred & Pamela Buffett Cancer Center, University of Nebraska Medical Center, Omaha, NE, USA. (32) Eppley Institute for Research in Cancer and Allied Diseases, Fred & Pamela Buffett Cancer Center, University of Nebraska Medical Center, Omaha, NE, USA. (33) Cancer Center, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, NY 11724, USA. (34) Children's Cancer and Blood Foundation Laboratories, Departments of Pediatrics, and Cell and Developmental Biology, Drukier Institute for Children's Health, Meyer Cancer Center, Weill Cornell Medicine, New York, NY, USA. (35) Department of Medicine, Memorial Sloan Kettering Cancer Center, New York, NY, USA. (36) Gastrointestinal Oncology Service, Memorial Sloan Kettering Cancer Center, New York, NY, USA. (37) Thoracic Surgery Service, Department of Surgery, Memorial Sloan Kettering Cancer Center, New York, NY, USA. (38) Department of Radiation Oncology, Weill Cornell School of Medicine, New York, NY, USA. (39) Drukier Institute for Children's Health and Department of Pediatrics, Weill Cornell Medicine, New York, NY, USA. (40) Drukier Institute for Children's Health and Department of Pediatrics, Weill Cornell Medicine, New York, NY, USA. (41) Children's Cancer and Blood Foundation Laboratories, Departments of Pediatrics, and Cell and Developmental Biology, Drukier Institute for Children's Health, Meyer Cancer Center, Weill Cornell Medicine, New York, NY, USA; Yonsei Cancer Center, Division of Medical Oncology, Department of Internal Medicine, Yonsei University College of Medicine, Seoul, South Korea. (42) Department of Physiology, Biophysics, and Systems Biology, Weill Cornell Medicine, New York, NY, USA. (43) Children's Cancer and Blood Foundation Laboratories, Departments of Pediatrics, and Cell and Developmental Biology, Drukier Institute for Children's Health, Meyer Cancer Center, Weill Cornell Medicine, New York, NY, USA. Electronic address: haz2005@med.cornell.edu. (44) Children's Cancer and Blood Foundation Laboratories, Departments of Pediatrics, and Cell and Developmental Biology, Drukier Institute for Children's Health, Meyer Cancer Center, Weill Cornell Medicine, New York, NY, USA. Electronic address: irm2224@med.cornell.edu. (45) Children's Cancer and Blood Foundation Laboratories, Departments of Pediatrics, and Cell and Developmental Biology, Drukier Institute for Children's Health, Meyer Cancer Center, Weill Cornell Medicine, New York, NY, USA. Electronic address: dcl2001@med.cornell.edu.

Intratumoral Treg cell ablation elicits NK cell-mediated control of CD8 T cell-resistant tumors

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Zhang et al. found that intratumoral depletion of Tregs elicited potent antitumor NK cell responses that controlled MHC-I-deficient and even MHC-I-proficient cancers expressing sufficient NKG2D ligands. This effect was dependent on cDC2-mediated activation of CD4+ T cells and their subsequent production of IL-2, which directly enhanced NK cell activation and cytotoxic potential. Antibody-mediated depletion of intratumoral Tregs or administration of exogenous IL-2 had similar effects.

Zhang et al. found that intratumoral depletion of Tregs elicited potent antitumor NK cell responses that controlled MHC-I-deficient and even MHC-I-proficient cancers expressing sufficient NKG2D ligands. This effect was dependent on cDC2-mediated activation of CD4+ T cells and their subsequent production of IL-2, which directly enhanced NK cell activation and cytotoxic potential. Antibody-mediated depletion of intratumoral Tregs or administration of exogenous IL-2 had similar effects.

ABSTRACT: Cancer cells frequently lose major histocompatibility complex class I (MHC I) to evade CD8 T cell recognition. Natural killer (NK) cells are poised to target MHC I-deficient cancer cells, but MHC I loss alone is often insufficient to unleash fully effective NK cell responses. Here, we show that selective intratumoral (IT) ablation of regulatory T cells (T(reg) cells) elicited potent antitumor NK cell responses that controlled MHC I-deficient and even MHC I(+) cancers that expressed NKG2D ligands. T(reg) cells controlled the activation, maturation, and antitumor cytotoxic activity of NK cells within the tumor microenvironment. Mechanistically, depletion of IT-T(reg) cells relieved the inhibition of cDC2-dependent induction of IL-2 production by conventional CD4 T cells that was necessary for NK cell activation. Systemically administered antibodies that selectively depleted IT-T(reg) cells similarly empowered NK cell-dependent tumor control. These findings expand the breadth of T(reg) cell-mediated cancer immunosuppression to encompass antitumor NK cells and suggest that therapeutic targeting of T(reg) cells in tumors can control CD8 T cell-resistant cancers.

Author Info: (1) Division of Immunology and Molecular Medicine, Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720, USA. (2) Department of Electric

Author Info: (1) Division of Immunology and Molecular Medicine, Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720, USA. (2) Department of Electrical Engineering and Computer Sciences, University of California, Berkeley, Berkeley, CA 94720, USA. Center for Computational Biology, University of California, Berkeley, Berkeley, CA 94720, USA. (3) Division of Immunology and Molecular Medicine, Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720, USA. (4) Division of Immunology and Molecular Medicine, Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720, USA. (5) Division of Immunology and Molecular Medicine, Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720, USA. (6) Division of Immunology and Molecular Medicine, Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720, USA. (7) Division of Immunology and Molecular Medicine, Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720, USA. (8) Division of Immunology and Molecular Medicine, Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720, USA. (9) Division of Immunology and Molecular Medicine, Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720, USA. (10) Division of Immunology and Molecular Medicine, Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720, USA. (11) Division of Immunology and Molecular Medicine, Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720, USA. (12) Department of Molecular and Cellular Physiology and Howard Hughes Medical Institute, Stanford University School of Medicine, Palo Alto, CA 94305, USA. (13) Division of Immunology and Molecular Medicine, Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720, USA. Department of Electrical Engineering and Computer Sciences, University of California, Berkeley, Berkeley, CA 94720, USA. Center for Computational Biology, University of California, Berkeley, Berkeley, CA 94720, USA. (14) Division of Immunology and Molecular Medicine, Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720, USA. (15) Division of Immunology and Molecular Medicine, Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720, USA.

Spleen-targeted neoantigen mRNA vaccine induces ISG15+ CD8+ T cell-mediated tertiary lymphoid structure formation in hepatocellular carcinoma

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Lin et al. engineered a spleen-targeted neoantigen mRNA vaccine (STNvac) using a two-component LNP formulation that selectively delivered mRNA to splenic DCs and prompted robust neoantigen-specific CD8+ T cell response in an orthotopic Hepa1-6 HCC model. STNvac induced a distinct ISG15+ CD8+ T cell subset with enhanced cytotoxicity that mediated antigen-specific tumor clearance. Single-cell and spatial analyses showed interaction between ISG15+ CD8+ T cells and intratumoral APCs via a GZMA–F2R axis, which drove ISG15+ CD8+ T cell activation, proliferation, and organization into TLSs in human and mouse HCC specimens.

Contributed by Shishir Pant

Lin et al. engineered a spleen-targeted neoantigen mRNA vaccine (STNvac) using a two-component LNP formulation that selectively delivered mRNA to splenic DCs and prompted robust neoantigen-specific CD8+ T cell response in an orthotopic Hepa1-6 HCC model. STNvac induced a distinct ISG15+ CD8+ T cell subset with enhanced cytotoxicity that mediated antigen-specific tumor clearance. Single-cell and spatial analyses showed interaction between ISG15+ CD8+ T cells and intratumoral APCs via a GZMA–F2R axis, which drove ISG15+ CD8+ T cell activation, proliferation, and organization into TLSs in human and mouse HCC specimens.

Contributed by Shishir Pant

ABSTRACT: The efficacy of neoantigen vaccine for advanced hepatocellular carcinoma (HCC) is limited largely due to insufficient T cell mobilization and activation. Herein, we develop a spleen-targeted neoantigen mRNA vaccine (STNvac) with highly efficient spleen-selective mRNA transfection. Using a three-dose vaccination regimen, STNvac demonstrates remarkable therapeutic efficacy in orthotopic HCC model with a high likelihood of complete tumor regression and significantly improved survival rates (p < 0.0001). Notably, we identify a distinct ISG15(+) CD8(+) T cell population as crucial mediators of STNvac-induced immunity with potent antigen-processing and cytotoxic capacities. Intriguingly, STNvac promotes the formation of tertiary lymphoid structures (TLSs) through GZMA-F2R-mediated interactions between ISG15(+) CD8(+) T cells and antigen-presenting cells (APCs), which is also confirmed in HCC patients. Taken together, our findings demonstrate the potent antitumor efficacy of spleen-targeted mRNA vaccine and reveal its underlying immune cell interactive mechanisms, presenting high potential for clinical translation.

Author Info: (1) The United Innovation of Mengchao Hepatobiliary Technology Key Laboratory of Fujian Province, Mengchao Hepatobiliary Hospital of Fujian Medical University, Fuzhou, Fujian 35000

Author Info: (1) The United Innovation of Mengchao Hepatobiliary Technology Key Laboratory of Fujian Province, Mengchao Hepatobiliary Hospital of Fujian Medical University, Fuzhou, Fujian 350007, P.R. China; Mengchao Med-X Center, Fuzhou University, Fuzhou, Fujian 350116, P.R. China; The Liver Center of Fujian Province, Fujian Medical University, Fuzhou, Fujian 350007, P.R. China. (2) The United Innovation of Mengchao Hepatobiliary Technology Key Laboratory of Fujian Province, Mengchao Hepatobiliary Hospital of Fujian Medical University, Fuzhou, Fujian 350007, P.R. China; Mengchao Med-X Center, Fuzhou University, Fuzhou, Fujian 350116, P.R. China; The Liver Center of Fujian Province, Fujian Medical University, Fuzhou, Fujian 350007, P.R. China. (3) The United Innovation of Mengchao Hepatobiliary Technology Key Laboratory of Fujian Province, Mengchao Hepatobiliary Hospital of Fujian Medical University, Fuzhou, Fujian 350007, P.R. China; Mengchao Med-X Center, Fuzhou University, Fuzhou, Fujian 350116, P.R. China; The Liver Center of Fujian Province, Fujian Medical University, Fuzhou, Fujian 350007, P.R. China. (4) The United Innovation of Mengchao Hepatobiliary Technology Key Laboratory of Fujian Province, Mengchao Hepatobiliary Hospital of Fujian Medical University, Fuzhou, Fujian 350007, P.R. China; Mengchao Med-X Center, Fuzhou University, Fuzhou, Fujian 350116, P.R. China; The Liver Center of Fujian Province, Fujian Medical University, Fuzhou, Fujian 350007, P.R. China. (5) The United Innovation of Mengchao Hepatobiliary Technology Key Laboratory of Fujian Province, Mengchao Hepatobiliary Hospital of Fujian Medical University, Fuzhou, Fujian 350007, P.R. China; Mengchao Med-X Center, Fuzhou University, Fuzhou, Fujian 350116, P.R. China; The Liver Center of Fujian Province, Fujian Medical University, Fuzhou, Fujian 350007, P.R. China. (6) The United Innovation of Mengchao Hepatobiliary Technology Key Laboratory of Fujian Province, Mengchao Hepatobiliary Hospital of Fujian Medical University, Fuzhou, Fujian 350007, P.R. China; Mengchao Med-X Center, Fuzhou University, Fuzhou, Fujian 350116, P.R. China; The Liver Center of Fujian Province, Fujian Medical University, Fuzhou, Fujian 350007, P.R. China. (7) The United Innovation of Mengchao Hepatobiliary Technology Key Laboratory of Fujian Province, Mengchao Hepatobiliary Hospital of Fujian Medical University, Fuzhou, Fujian 350007, P.R. China; Mengchao Med-X Center, Fuzhou University, Fuzhou, Fujian 350116, P.R. China; The Liver Center of Fujian Province, Fujian Medical University, Fuzhou, Fujian 350007, P.R. China. (8) The United Innovation of Mengchao Hepatobiliary Technology Key Laboratory of Fujian Province, Mengchao Hepatobiliary Hospital of Fujian Medical University, Fuzhou, Fujian 350007, P.R. China; Mengchao Med-X Center, Fuzhou University, Fuzhou, Fujian 350116, P.R. China; The Liver Center of Fujian Province, Fujian Medical University, Fuzhou, Fujian 350007, P.R. China. (9) The United Innovation of Mengchao Hepatobiliary Technology Key Laboratory of Fujian Province, Mengchao Hepatobiliary Hospital of Fujian Medical University, Fuzhou, Fujian 350007, P.R. China; Mengchao Med-X Center, Fuzhou University, Fuzhou, Fujian 350116, P.R. China; The Liver Center of Fujian Province, Fujian Medical University, Fuzhou, Fujian 350007, P.R. China. (10) The United Innovation of Mengchao Hepatobiliary Technology Key Laboratory of Fujian Province, Mengchao Hepatobiliary Hospital of Fujian Medical University, Fuzhou, Fujian 350007, P.R. China; Mengchao Med-X Center, Fuzhou University, Fuzhou, Fujian 350116, P.R. China; The Liver Center of Fujian Province, Fujian Medical University, Fuzhou, Fujian 350007, P.R. China. (11) The United Innovation of Mengchao Hepatobiliary Technology Key Laboratory of Fujian Province, Mengchao Hepatobiliary Hospital of Fujian Medical University, Fuzhou, Fujian 350007, P.R. China; Mengchao Med-X Center, Fuzhou University, Fuzhou, Fujian 350116, P.R. China; The Liver Center of Fujian Province, Fujian Medical University, Fuzhou, Fujian 350007, P.R. China. (12) State Key Laboratory of Drug Research & Center of Pharmaceutics, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China. (13) The United Innovation of Mengchao Hepatobiliary Technology Key Laboratory of Fujian Province, Mengchao Hepatobiliary Hospital of Fujian Medical University, Fuzhou, Fujian 350007, P.R. China; Mengchao Med-X Center, Fuzhou University, Fuzhou, Fujian 350116, P.R. China; The Liver Center of Fujian Province, Fujian Medical University, Fuzhou, Fujian 350007, P.R. China. Electronic address: caizhixiong1985@163.com. (14) The United Innovation of Mengchao Hepatobiliary Technology Key Laboratory of Fujian Province, Mengchao Hepatobiliary Hospital of Fujian Medical University, Fuzhou, Fujian 350007, P.R. China; Mengchao Med-X Center, Fuzhou University, Fuzhou, Fujian 350116, P.R. China; The Liver Center of Fujian Province, Fujian Medical University, Fuzhou, Fujian 350007, P.R. China. Electronic address: xiaoloong.liu@gmail.com.

PD-1 antibody-bound progenitor-exhausted CD8+ T cells in lymph nodes boost PD-1-blockade anti-tumor immunity in gastrointestinal cancer

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Utilizing scRNA/TCRseq, CITEseq, and a novel assay for cell-bound anti-PD-1 to study the dynamics of T cells targeted by anti-PD-1, Nose and Yasumizu et al. first found that abundance of progenitor-exhausted CD8+ T cells (Tpex) in metastasis-free lymph nodes (LNs), but not tumors or metastatic LNs, correlated with better prognosis in patients with anti-PD-1-naive gastric cancer. Anti-PD-1 promoted the proliferation of anti-PD-1high-bound Tpex in LNs, and clonotypes overlapped with intratumoral anti-PD-1-bound exhausted T cells (Tex), suggesting that anti-PD-1high-bound Tpex migrate to the tumor, where they differentiate into Tex.

Contributed by Ute Burkhardt

Utilizing scRNA/TCRseq, CITEseq, and a novel assay for cell-bound anti-PD-1 to study the dynamics of T cells targeted by anti-PD-1, Nose and Yasumizu et al. first found that abundance of progenitor-exhausted CD8+ T cells (Tpex) in metastasis-free lymph nodes (LNs), but not tumors or metastatic LNs, correlated with better prognosis in patients with anti-PD-1-naive gastric cancer. Anti-PD-1 promoted the proliferation of anti-PD-1high-bound Tpex in LNs, and clonotypes overlapped with intratumoral anti-PD-1-bound exhausted T cells (Tex), suggesting that anti-PD-1high-bound Tpex migrate to the tumor, where they differentiate into Tex.

Contributed by Ute Burkhardt

ABSTRACT: While progenitor-exhausted T cells (Tpex) expressing TCF1 and PD-1 are crucial for the therapeutic effect of immune checkpoint inhibitors (ICIs) with therapeutic anti-PD-1 antibodies (aPD-1), the dynamics of ICI-bound Tpex are not fully understood. In this study, we investigate ICI-bound T cells in detail using combined sequencing analysis at the single-cell level. By analyzing samples from gastrointestinal cancer patients with or without ICI treatment, we find that Tpex are enriched in proximal lymph nodes (LNs) and proliferate at a high rate after ICI treatment. Importantly, aPD-1 high-bound Tpex in LNs share T-cell receptor clonotypes with intratumoral exhausted CD8(+) T cells (Tex), suggesting their migration to tumor sites after ICI treatment. This study thus provides new insights into how ICIs enhance anti-tumor immunity by acting on Tpex in LNs, deepening our understanding of the cellular mechanisms underlying ICI therapy.

Author Info: (1) Department of Gastroenterological Surgery, Graduate School of Medicine, The University of Osaka, Suita, Japan. Department of Clinical Research in Tumor Immunology, Graduate Sch

Author Info: (1) Department of Gastroenterological Surgery, Graduate School of Medicine, The University of Osaka, Suita, Japan. Department of Clinical Research in Tumor Immunology, Graduate School of Medicine, The University of Osaka, Suita, Japan. (2) Experimental Immunology, WPI Immunology Frontier Research Center, The University of Osaka, Suita, Japan. Integrated Frontier Research for Medical Science Division, Institute for Open and Transdisciplinary Research Initiatives (OTRI), The University of Osaka, Suita, Japan. (3) Department of Gastroenterological Surgery, Graduate School of Medicine, The University of Osaka, Suita, Japan. tsaito@gesurg.med.osaka-u.ac.jp. Department of Clinical Research in Tumor Immunology, Graduate School of Medicine, The University of Osaka, Suita, Japan. tsaito@gesurg.med.osaka-u.ac.jp. (4) Experimental Immunology, WPI Immunology Frontier Research Center, The University of Osaka, Suita, Japan. (5) Department of Gastroenterological Surgery, Graduate School of Medicine, The University of Osaka, Suita, Japan. Department of Clinical Research in Tumor Immunology, Graduate School of Medicine, The University of Osaka, Suita, Japan. (6) Department of Gastroenterological Surgery, Graduate School of Medicine, The University of Osaka, Suita, Japan. Department of Clinical Research in Tumor Immunology, Graduate School of Medicine, The University of Osaka, Suita, Japan. (7) Department of Gastroenterological Surgery, Graduate School of Medicine, The University of Osaka, Suita, Japan. (8) Department of Gastroenterological Surgery, Graduate School of Medicine, The University of Osaka, Suita, Japan. (9) Department of Gastroenterological Surgery, Graduate School of Medicine, The University of Osaka, Suita, Japan. (10) Department of Gastroenterological Surgery, Graduate School of Medicine, The University of Osaka, Suita, Japan. (11) Department of Gastroenterological Surgery, Graduate School of Medicine, The University of Osaka, Suita, Japan. (12) Department of Gastroenterological Surgery, Graduate School of Medicine, The University of Osaka, Suita, Japan. (13) Department of Clinical Research in Tumor Immunology, Graduate School of Medicine, The University of Osaka, Suita, Japan. Pharmaceutical Research Division, Shionogi & Co., Ltd., Toyonaka, Japan. (14) Department of Gastroenterological Surgery, Graduate School of Medicine, The University of Osaka, Suita, Japan. (15) Department of Pathology, Institute of Medical Science (Medical Research Center), Tokyo Medical University, Tokyo, Japan. (16) Experimental Immunology, WPI Immunology Frontier Research Center, The University of Osaka, Suita, Japan. Department of Basic Research in Tumor Immunology, Graduate School of Medicine, The University of Osaka, Osaka, Japan. (17) Experimental Immunology, WPI Immunology Frontier Research Center, The University of Osaka, Suita, Japan. (18) Department of Gastroenterological Surgery, Graduate School of Medicine, The University of Osaka, Suita, Japan. Department of Clinical Research in Tumor Immunology, Graduate School of Medicine, The University of Osaka, Suita, Japan. (19) Department of Gastroenterological Surgery, Graduate School of Medicine, The University of Osaka, Suita, Japan. (20) Department of Gastroenterological Surgery, Graduate School of Medicine, The University of Osaka, Suita, Japan.

The CD4+ T cell population partners with Tpex CD8+ T cells to mediate antitumor immunity in the tumor microenvironment

Spotlight 

Takei et al. identified IL-7Rhi CCR6+ Th1-like CD4+ T cells (Th7R) that were distinct from Th1 and Th17 states. Th7R cells expressed CXCL13 and lymphotoxin-β, localized to TLSs, and associated with high endothelial venules. Th7R abundance correlated with GZMK+GZMB- progenitor exhausted CD8+ T cells (Tpex) across tumors and lymph nodes. Adoptive transfer of Th7R cells into mice bearing MCA205 skin tumors expanded Tpex and Tex populations, supported Tpex maintenance and differentiation, and enhanced tumor control. Intratumoral and circulating Th7R correlated with response to PD-1 blockade, and improved clinical outcomes in patients with lung cancer.

Contributed by Shishir Pant

Takei et al. identified IL-7Rhi CCR6+ Th1-like CD4+ T cells (Th7R) that were distinct from Th1 and Th17 states. Th7R cells expressed CXCL13 and lymphotoxin-β, localized to TLSs, and associated with high endothelial venules. Th7R abundance correlated with GZMK+GZMB- progenitor exhausted CD8+ T cells (Tpex) across tumors and lymph nodes. Adoptive transfer of Th7R cells into mice bearing MCA205 skin tumors expanded Tpex and Tex populations, supported Tpex maintenance and differentiation, and enhanced tumor control. Intratumoral and circulating Th7R correlated with response to PD-1 blockade, and improved clinical outcomes in patients with lung cancer.

Contributed by Shishir Pant

ABSTRACT: CD4⁺ T cells support the priming, expansion, and function of CD8⁺ T cells through dendritic cells. Precursor exhausted T cells (Tpex) maintain self-renewal and supply cytotoxic CD8⁺ T cells in the tumor microenvironment (TME), but the identity of their CD4⁺ T-cell partners remains unclear. Here, we perform scRNA-seq, scTCR-seq, and mass cytometry analysis on peripheral blood, tumor, and lymph nodes primarily from lung cancer patients and, in part, renal cell carcinoma. We identify an IL-7Rhigh CCR6⁺ Th1-like CD4⁺ T cell-population, named Th7R, that is numerically and spatially partnered with Tpex. Th7R cells express lymphotoxin-β and CXCL13, correlate with high endothelial venules, and co-localize with Tpex in tertiary lymphoid structures. Th7R cell abundance correlates with Tpex numbers in the TME and lymph nodes, and adoptive transfer of Th7R increases Tpex in a preclinical mouse model. Intratumoral Th7R and Tpex associate with improved response to neoadjuvant PD-1 blockade therapy. These results suggest that Th7R cells act as partners of Tpex to sustain antitumor T-cell immunity.

Author Info: (1) Department of Respiratory Medicine, Saitama Medical University International Medical Center, 1397-1 Yamane, Hidaka, Saitama, Japan. Department of Respiratory Medicine, Kyoto Pr

Author Info: (1) Department of Respiratory Medicine, Saitama Medical University International Medical Center, 1397-1 Yamane, Hidaka, Saitama, Japan. Department of Respiratory Medicine, Kyoto Prefectural University of Medicine, 465 Kajii-cho, Kawaramachi-Hirokoji, Kamigyo-ku, Kyoto, Japan. (2) Department of Clinical Cancer Genomics, Saitama Medical University International Medical Center, 1397-1 Yamane, Hidaka, Saitama, Japan. (3) Department of Respiratory Medicine, Saitama Medical University International Medical Center, 1397-1 Yamane, Hidaka, Saitama, Japan. (4) Department of Respiratory Medicine, Saitama Medical University International Medical Center, 1397-1 Yamane, Hidaka, Saitama, Japan. (5) Department of Respiratory Medicine, Saitama Medical University International Medical Center, 1397-1 Yamane, Hidaka, Saitama, Japan. (6) Department of Respiratory Medicine, Saitama Medical University International Medical Center, 1397-1 Yamane, Hidaka, Saitama, Japan. (7) Department of Respiratory Medicine, Saitama Medical University International Medical Center, 1397-1 Yamane, Hidaka, Saitama, Japan. (8) Department of Respiratory Medicine, Saitama Medical University International Medical Center, 1397-1 Yamane, Hidaka, Saitama, Japan. (9) Department of Respiratory Medicine, Saitama Medical University International Medical Center, 1397-1 Yamane, Hidaka, Saitama, Japan. (10) Department of General Thoracic Surgery, Saitama Medical University International Medical Center, 1397-1 Yamane, Hidaka, Saitama, Japan. (11) Department of General Thoracic Surgery, Saitama Medical University International Medical Center, 1397-1 Yamane, Hidaka, Saitama, Japan. (12) Department of General Thoracic Surgery, Saitama Medical University International Medical Center, 1397-1 Yamane, Hidaka, Saitama, Japan. (13) Department of General Thoracic Surgery, Saitama Medical University International Medical Center, 1397-1 Yamane, Hidaka, Saitama, Japan. (14) Department of Respiratory Medicine, Saitama Medical University International Medical Center, 1397-1 Yamane, Hidaka, Saitama, Japan. (15) Department of Respiratory Medicine, Saitama Medical University International Medical Center, 1397-1 Yamane, Hidaka, Saitama, Japan. kagamu19@saitama-med.ac.jp.

Exposed phosphatidylserine is an inhibitory molecule in T cell exhaustion

Spotlight 

In a model of chronic LCMV infection, surface phosphatidylserine (PS) expression increased in virus-specific PD-1+CD8+ T cells over time, relative to naive CD8+ T cells and the setting of acute infection. PS expression increased with T cell differentiation state (stem-like to terminally differentiated). An anti-PS mAb enhanced DC costimulation, splenic PD-1+ stem-like CD8+ T cell proliferation and effector differentiation, and virus-specific CD8+ T cell counts across tissues. Anti-PS synergized with anti-PD-L1 to reduce LCMV burden. PD-1+CD8+ TILs from human renal cancer and NSCLC also expressed surface PS, which increased with T cell differentiation.

Contributed by Alex Najibi

In a model of chronic LCMV infection, surface phosphatidylserine (PS) expression increased in virus-specific PD-1+CD8+ T cells over time, relative to naive CD8+ T cells and the setting of acute infection. PS expression increased with T cell differentiation state (stem-like to terminally differentiated). An anti-PS mAb enhanced DC costimulation, splenic PD-1+ stem-like CD8+ T cell proliferation and effector differentiation, and virus-specific CD8+ T cell counts across tissues. Anti-PS synergized with anti-PD-L1 to reduce LCMV burden. PD-1+CD8+ TILs from human renal cancer and NSCLC also expressed surface PS, which increased with T cell differentiation.

Contributed by Alex Najibi

ABSTRACT: In cancer and chronic infection, CD8 T cell exhaustion is hallmarked by expression of inhibitory receptors such as PD1, TIM3, LAG3 and others(1-3). Thus, inhibitory molecule focus has been limited to cell-surface proteins. Here we evaluate the surface lipid metabolite phosphatidylserine (PS) as a regulator of exhaustion. PS primarily localizes to the inner plasma membrane of live cells but is well known to be externalized to the outer membrane during cell death. The role of exposed PS on live immune cells is less clear. We show that viable, antigen-specific CD8 T cells externalize PS during lymphocytic choriomeningitis virus (LCMV) infection. T cell activation induced initial PS exposure, and chronic antigen stimulation sustained externalization. Transcriptomic and lipidomic analyses also identified PS accumulation in exhausted CD8 T cells. To evaluate a role for exposed PS in exhaustion, we treated LCMV chronically infected mice with a PS-targeting antibody (mch1N11)(4) and found that it expanded LCMV-specific CD8 responses. PD1(+)TCF1(+) stem-like CD8 T cells downregulated quiescence-associated gene modules and increased proliferation after antibody treatment, highlighting an inhibitory role for PS. Mechanistically, exposed PS on T cells functioned extrinsically to suppress dendritic cell immunostimulatory phenotypes, in turn limiting CD8 T cell responses. PS-targeting antibody with anti-PDL1 synergized to increase CD8 responses and improve viral control. Finally, we show that PD1(+) CD8 T cells from human tumours can also expose PS. In summary, we detail CD8 T cell PS biology and provide insight into a mechanism by which exposed PS functions as a 'non-classical' extrinsic inhibitory molecule in exhaustion.

Author Info: (1) Emory Vaccine Center, Emory University School of Medicine, Atlanta, GA, USA. Department of Microbiology and Immunology, Emory University School of Medicine, Atlanta, GA, USA. (

Author Info: (1) Emory Vaccine Center, Emory University School of Medicine, Atlanta, GA, USA. Department of Microbiology and Immunology, Emory University School of Medicine, Atlanta, GA, USA. (2) Department of Urology, Emory University School of Medicine, Atlanta, GA, USA. Winship Cancer Institute of Emory University, Atlanta, GA, USA. (3) The Jackson Laboratory for Genomic Medicine, Farmington, CT, USA. (4) Emory Vaccine Center, Emory University School of Medicine, Atlanta, GA, USA. Department of Microbiology and Immunology, Emory University School of Medicine, Atlanta, GA, USA. (5) The Jackson Laboratory for Genomic Medicine, Farmington, CT, USA. (6) Emory Vaccine Center, Emory University School of Medicine, Atlanta, GA, USA. (7) Emory Vaccine Center, Emory University School of Medicine, Atlanta, GA, USA. (8) Department of Surgery, University of Texas Southwestern Medical Center, Dallas, TX, USA. Hamon Center for Therapeutic Oncology Research, University of Texas Southwestern Medical Center, Dallas, TX, USA. (9) The Jackson Laboratory for Genomic Medicine, Farmington, CT, USA. (10) The Jackson Laboratory for Genomic Medicine, Farmington, CT, USA. (11) Winship Cancer Institute of Emory University, Atlanta, GA, USA. (12) Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA, USA. Department of Medicine, Harvard Medical School, Boston, MA, USA. (13) Department of Urology, Emory University School of Medicine, Atlanta, GA, USA. Winship Cancer Institute of Emory University, Atlanta, GA, USA. (14) Winship Cancer Institute of Emory University, Atlanta, GA, USA. (15) Emory Vaccine Center, Emory University School of Medicine, Atlanta, GA, USA. Department of Microbiology and Immunology, Emory University School of Medicine, Atlanta, GA, USA. Department of Urology, Emory University School of Medicine, Atlanta, GA, USA. Winship Cancer Institute of Emory University, Atlanta, GA, USA. (16) The Jackson Laboratory for Genomic Medicine, Farmington, CT, USA. Department of Immunology, University of Connecticut, Farmington, CT, USA. (17) Department of Surgery, University of Texas Southwestern Medical Center, Dallas, TX, USA. Hamon Center for Therapeutic Oncology Research, University of Texas Southwestern Medical Center, Dallas, TX, USA. (18) Emory Vaccine Center, Emory University School of Medicine, Atlanta, GA, USA. rahmed@emory.edu. Department of Microbiology and Immunology, Emory University School of Medicine, Atlanta, GA, USA. rahmed@emory.edu.

Chemokine-defined macrophage niches establish spatial organization of tumor immunity Spotlight 

Ghosh et al. demonstrated that tissue-resident interstitial macrophages (IMs) and recruited macrophages (recMacs) showed distinct gene expression profiles in B16F10 lung metastases and KPAR1.3 lung tumor models. CD206hi IM subsets (Cxcl13+, Cxcl9+, Cxcl10+) were localized in bronchovascular regions and promoted TLS formation and lymphocyte recruitment, whereas CD206lo Ccl2+ IMs recruited Ly6c2+Fn1+Vcan+ recMacs with tumor-promoting transcriptional programs. In tdLNs, Ly6C+ monocyte-derived dendritic cells acted as immunosuppressive APCs during neoantigen vaccination, and CCR5 blockade limited their migration, enhancing antitumor immunity.

Contributed by Shishir Pant

Ghosh et al. demonstrated that tissue-resident interstitial macrophages (IMs) and recruited macrophages (recMacs) showed distinct gene expression profiles in B16F10 lung metastases and KPAR1.3 lung tumor models. CD206hi IM subsets (Cxcl13+, Cxcl9+, Cxcl10+) were localized in bronchovascular regions and promoted TLS formation and lymphocyte recruitment, whereas CD206lo Ccl2+ IMs recruited Ly6c2+Fn1+Vcan+ recMacs with tumor-promoting transcriptional programs. In tdLNs, Ly6C+ monocyte-derived dendritic cells acted as immunosuppressive APCs during neoantigen vaccination, and CCR5 blockade limited their migration, enhancing antitumor immunity.

Contributed by Shishir Pant

ABSTRACT: Macrophages are among the most abundant immune cells in solid tumors, yet how macrophage lineage and spatial organization shape antitumor immunity remains unclear. Here we uncovered a division of labor between tissue-resident CD206(hi) and CD206(lo) interstitial macrophage (IM) subsets and Ly6c2(+)Fn1(+)Vcan(+) recruited macrophages (recMacs) in lung cancer. Using single-cell and spatial transcriptomics, we identified chemokine-expressing IM subsets with opposing functions. Cxcl13(+)CD206(hi) IMs, Cxcl9(+)CD206(hi) IMs and Cxcl10(+)CD206(hi) IMs positioned along bronchovascular regions drove tertiary lymphoid structure formation, lymphocyte recruitment and tumor control, whereas Ccl2(+) IMs, localized within tumor regions, recruited protumorigenic Ly6c2(+)Fn1(+)Vcan(+) recMacs. In addition, Ly6C(+)CD11b(+) monocyte-derived dendritic cells (moDCs) functioned as immunosuppressive antigen-presenting cells in tumor-draining lymph nodes. During neoantigen vaccination, CCR5 blockade with maraviroc selectively inhibited antigen-bearing moDC migration, enhancing dendritic cell-mediated antitumor immunity. These findings showed how macrophage lineage and spatial compartmentalization govern tumor immunity and identified strategies to preserve protective IM functions, while disrupting macrophage-driven immunosuppression.

Author Info: (1) Department of Microbiology and Immunology, Dartmouth Geisel School of Medicine, Hanover, NH, USA. (2) Department of Microbiology and Immunology, Dartmouth Geisel School of Medi

Author Info: (1) Department of Microbiology and Immunology, Dartmouth Geisel School of Medicine, Hanover, NH, USA. (2) Department of Microbiology and Immunology, Dartmouth Geisel School of Medicine, Hanover, NH, USA. (3) Division of Oncology, Department of Medicine, Washington University School of Medicine, St Louis, MO, USA. (4) Department of Microbiology and Immunology, Dartmouth Geisel School of Medicine, Hanover, NH, USA. (5) Department of Microbiology and Immunology, Dartmouth Geisel School of Medicine, Hanover, NH, USA. (6) Department of Microbiology and Immunology, Dartmouth Geisel School of Medicine, Hanover, NH, USA. (7) Dartmouth Cancer Center, Dartmouth Geisel School of Medicine, Hanover, NH, USA. (8) Dartmouth Cancer Center, Dartmouth Geisel School of Medicine, Hanover, NH, USA. (9) Department of Microbiology and Immunology, Dartmouth Geisel School of Medicine, Hanover, NH, USA. claudia.jakubzick@dartmouth.edu.

Immunogenic tumor cell death and T-cell-derived IFN-γ elicit tumoricidal macrophages to potentiate OX40 immunotherapy

Spotlight 

Using a bilateral, humanized OX40 MC38 tumor model, Liu and Zhao et al. demonstrated that OX40 agonist Ab (agOX40) therapy increased infiltration of NOS2+ pro-inflammatory macrophages and effector CD8+ T cells. T cell-derived IFNγ synergized with DAMP-induced TLR4 signaling to reprogram TAMs toward a pro-inflammatory and tumoricidal NOS2+ state. agOX40-mediated depletion of OX40+Foxp3+ Tregs further potentiated NOS2+ TAM polarization. A combination of MPLA, IFNγ, and agOX40 reprogrammed TAMs, promoted DC maturation, and induced durable tumor regression. ICD-inducing cyclophosphamide enhanced agOX40 therapy.

Contributed by Shishir Pant

Using a bilateral, humanized OX40 MC38 tumor model, Liu and Zhao et al. demonstrated that OX40 agonist Ab (agOX40) therapy increased infiltration of NOS2+ pro-inflammatory macrophages and effector CD8+ T cells. T cell-derived IFNγ synergized with DAMP-induced TLR4 signaling to reprogram TAMs toward a pro-inflammatory and tumoricidal NOS2+ state. agOX40-mediated depletion of OX40+Foxp3+ Tregs further potentiated NOS2+ TAM polarization. A combination of MPLA, IFNγ, and agOX40 reprogrammed TAMs, promoted DC maturation, and induced durable tumor regression. ICD-inducing cyclophosphamide enhanced agOX40 therapy.

Contributed by Shishir Pant

ABSTRACT: Understanding the mechanisms limiting OX40 agonist antibody efficacy is critical for developing more effective combination immunotherapies. Tumor microenvironment (TME) analysis revealed that OX40-antibody-responsive mice harbored tumor-associated macrophages (TAMs) with elevated NOS2 expression and heightened pattern recognition receptor (PRR) activation and interferon gamma (IFN-γ) signaling. In addition, patients with more favorable treatment responses to OX40 antibody therapy exhibited increased NOS2 expression. Mechanistically, tumor-infiltrating T-cell-derived IFN-γ synergizes with endogenous ligands of PRR released during immunogenic cell death to drive NOS2+ TAMs reprogramming. Translating these insights into therapeutic strategy, a Combo approach composing of MPLA, IFN-γ, and OX40 agonist antibody is designed to actively polarize TAMs to express NOS2, which mediate tumor clearance through an NOS2-dependent cytotoxicity. Moreover, OX40-antibody-mediated regulatory T cell (Treg) depletion potentiated NOS2+ macrophage induction. This multimodal strategy offers a promising solution to overcome the limitations of OX40 antibody monotherapy and enhance outcomes of the OX40-targeted immunotherapies.

Author Info: (1) State Key Laboratory of Medicinal Chemical Biology, College of Life Sciences and Frontiers Science Center for Cell Responses, Academy for Advanced Interdisciplinary Studies, Na

Author Info: (1) State Key Laboratory of Medicinal Chemical Biology, College of Life Sciences and Frontiers Science Center for Cell Responses, Academy for Advanced Interdisciplinary Studies, Nankai University, Tianjin 300071, China; Henan Provincial People's Hospital & the People's Hospital of Zhengzhou University, Zhengzhou 450003, China; Henan Academy of Sciences, Zhengzhou 450046, China. (2) State Key Laboratory of Medicinal Chemical Biology, College of Life Sciences and Frontiers Science Center for Cell Responses, Academy for Advanced Interdisciplinary Studies, Nankai University, Tianjin 300071, China; College of Materials Science and Engineering, Shenzhen University, Shenzhen 518071, China. (3) State Key Laboratory of Medicinal Chemical Biology, College of Life Sciences and Frontiers Science Center for Cell Responses, Academy for Advanced Interdisciplinary Studies, Nankai University, Tianjin 300071, China. (4) State Key Laboratory of Medicinal Chemical Biology, College of Life Sciences and Frontiers Science Center for Cell Responses, Academy for Advanced Interdisciplinary Studies, Nankai University, Tianjin 300071, China. (5) State Key Laboratory of Medicinal Chemical Biology, College of Life Sciences and Frontiers Science Center for Cell Responses, Academy for Advanced Interdisciplinary Studies, Nankai University, Tianjin 300071, China. (6) State Key Laboratory of Medicinal Chemical Biology, College of Life Sciences and Frontiers Science Center for Cell Responses, Academy for Advanced Interdisciplinary Studies, Nankai University, Tianjin 300071, China. (7) State Key Laboratory of Medicinal Chemical Biology, College of Life Sciences and Frontiers Science Center for Cell Responses, Academy for Advanced Interdisciplinary Studies, Nankai University, Tianjin 300071, China. (8) State Key Laboratory of Medicinal Chemical Biology, College of Life Sciences and Frontiers Science Center for Cell Responses, Academy for Advanced Interdisciplinary Studies, Nankai University, Tianjin 300071, China. (9) State Key Laboratory of Medicinal Chemical Biology, College of Life Sciences and Frontiers Science Center for Cell Responses, Academy for Advanced Interdisciplinary Studies, Nankai University, Tianjin 300071, China. (10) State Key Laboratory of Medicinal Chemical Biology, College of Life Sciences and Frontiers Science Center for Cell Responses, Academy for Advanced Interdisciplinary Studies, Nankai University, Tianjin 300071, China. (11) State Key Laboratory of Medicinal Chemical Biology, College of Life Sciences and Frontiers Science Center for Cell Responses, Academy for Advanced Interdisciplinary Studies, Nankai University, Tianjin 300071, China. (12) State Key Laboratory of Medicinal Chemical Biology, College of Life Sciences and Frontiers Science Center for Cell Responses, Academy for Advanced Interdisciplinary Studies, Nankai University, Tianjin 300071, China. (13) State Key Laboratory of Medicinal Chemical Biology, College of Life Sciences and Frontiers Science Center for Cell Responses, Academy for Advanced Interdisciplinary Studies, Nankai University, Tianjin 300071, China. (14) State Key Laboratory of Medicinal Chemical Biology, College of Life Sciences and Frontiers Science Center for Cell Responses, Academy for Advanced Interdisciplinary Studies, Nankai University, Tianjin 300071, China. (15) State Key Laboratory of Medicinal Chemical Biology, College of Life Sciences and Frontiers Science Center for Cell Responses, Academy for Advanced Interdisciplinary Studies, Nankai University, Tianjin 300071, China. (16) State Key Laboratory of Medicinal Chemical Biology, College of Life Sciences and Frontiers Science Center for Cell Responses, Academy for Advanced Interdisciplinary Studies, Nankai University, Tianjin 300071, China. (17) State Key Laboratory of Medicinal Chemical Biology, College of Life Sciences and Frontiers Science Center for Cell Responses, Academy for Advanced Interdisciplinary Studies, Nankai University, Tianjin 300071, China. (18) Shanghai Institute for Advanced Immunochemical Studies, ShanghaiTech University, Shanghai 201210, China. (19) HiFiBiO (Shanghai) Co. Ltd., Cambridge, MA 02139, USA. (20) HiFiBiO (Shanghai) Co. Ltd., Cambridge, MA 02139, USA. (21) HiFiBiO (Shanghai) Co. Ltd., Cambridge, MA 02139, USA. (22) NovelBio Bio-Pharm Technology Co., Ltd., Shanghai 201114, China. (23) Faculty of Life Science, University College London, London WC1E 6BT, UK. (24) State Key Laboratory of Medicinal Chemical Biology, College of Life Sciences and Frontiers Science Center for Cell Responses, Academy for Advanced Interdisciplinary Studies, Nankai University, Tianjin 300071, China. (25) State Key Laboratory of Medicinal Chemical Biology, College of Life Sciences and Frontiers Science Center for Cell Responses, Academy for Advanced Interdisciplinary Studies, Nankai University, Tianjin 300071, China. (26) Henan Provincial People's Hospital & the People's Hospital of Zhengzhou University, Zhengzhou 450003, China. (27) State Key Laboratory of Medicinal Chemical Biology, College of Life Sciences and Frontiers Science Center for Cell Responses, Academy for Advanced Interdisciplinary Studies, Nankai University, Tianjin 300071, China; Shanghai Institute for Advanced Immunochemical Studies, ShanghaiTech University, Shanghai 201210, China. Electronic address: hongkai@nankai.edu.cn.

Reactivating exhausted tumor-infiltrating T cells by a bispecific DC-T cell engager in mice Spotlight 

Zhang, Gao, and Hu et al. addressed ways to enhance DC–T cell crosstalk in the TIME. BiDT, a bispecific DC–T cell engager (anti-Tim3–IFNα fusion), simultaneously bound Tim3 on exhausted TILs and activated DCs via the IFNAR receptor. In mouse models, BiDT resulted in potent antitumor activity, robust tumor specific memory, and synergized with anti-PD-L1 in an immune-cold tumor model. Mechanistically, BiDT depended on DCs and intratumoral, not LN, T cells, reactivated exhausted TIM3+ CD8+ TILs via anti-apoptotic Bcl-2 upregulation, and enhanced DC function via increased IL-2 production and B7/CD28 interactions. To address IFNα toxicity, an MMP-cleavable prodrug variant was generated.

Contributed by Katherine Turner

Zhang, Gao, and Hu et al. addressed ways to enhance DC–T cell crosstalk in the TIME. BiDT, a bispecific DC–T cell engager (anti-Tim3–IFNα fusion), simultaneously bound Tim3 on exhausted TILs and activated DCs via the IFNAR receptor. In mouse models, BiDT resulted in potent antitumor activity, robust tumor specific memory, and synergized with anti-PD-L1 in an immune-cold tumor model. Mechanistically, BiDT depended on DCs and intratumoral, not LN, T cells, reactivated exhausted TIM3+ CD8+ TILs via anti-apoptotic Bcl-2 upregulation, and enhanced DC function via increased IL-2 production and B7/CD28 interactions. To address IFNα toxicity, an MMP-cleavable prodrug variant was generated.

Contributed by Katherine Turner

ABSTRACT: Tumor infiltrating T cells (TIL) are key players in the anti-tumor immune response. However, chronic exposure to tumor-derived antigens drives the differentiation into 'exhausted' TILs. Whether intratumoral dendritic cells (DC) can mitigate TILs exhaustion and maintain function is unclear. Here, we develop a bispecific DC-T cell engager (BiDT), consisting of an anti-TIM3-IFN fusion protein, and demonstrate that, in preclinical mouse tumor models, this engager simultaneously targets TIM3 on exhausted TILs and activates DCs via the IFNAR receptor. Mechanistically, BiDT reactivates exhausted TIM3(+)TILs by preventing apoptosis through increased Bcl-2 expression and enhances DC function to reactivate T cells via IL-2 signalling and co-stimulatory CD80/86-CD28 interactions within the tumor microenvironment. Finally, to mitigate IFN_-induced toxicity, we engineer a Pro-BiDT engager featuring a pro-IFN_ and report potent antitumor activity with reduced systemic toxicity. Thus, by bridging DC-T cells together, BiDT treatment enhances the critical communication pathways and cellular circuits necessary for effective anti-tumor immunity.

Author Info: (1) Institute for Data-Driven Tumor Immunology, Chongqing Medical University, Chongqing, China. xuhaozhang@cqmu.edu.cn. School of Basic Medical Sciences, Tsinghua University, Beiji

Author Info: (1) Institute for Data-Driven Tumor Immunology, Chongqing Medical University, Chongqing, China. xuhaozhang@cqmu.edu.cn. School of Basic Medical Sciences, Tsinghua University, Beijing, China. xuhaozhang@cqmu.edu.cn. Tsinghua-Peking Center for Life Sciences, Tsinghua University, Beijing, China. xuhaozhang@cqmu.edu.cn. State Key Laboratory of Molecular oncology, Tsinghua University, Beijing, China. xuhaozhang@cqmu.edu.cn. (2) School of Basic Medical Sciences, Tsinghua University, Beijing, China. Tsinghua-Peking Center for Life Sciences, Tsinghua University, Beijing, China. State Key Laboratory of Molecular oncology, Tsinghua University, Beijing, China. (3) School of Basic Medical Sciences, Tsinghua University, Beijing, China. State Key Laboratory of Molecular oncology, Tsinghua University, Beijing, China. (4) School of Basic Medical Sciences, Tsinghua University, Beijing, China. State Key Laboratory of Molecular oncology, Tsinghua University, Beijing, China. (5) School of Basic Medical Sciences, Tsinghua University, Beijing, China. Tsinghua-Peking Center for Life Sciences, Tsinghua University, Beijing, China. State Key Laboratory of Molecular oncology, Tsinghua University, Beijing, China. (6) School of Basic Medical Sciences, Tsinghua University, Beijing, China. China National Clinical Research Center for Neurological Diseases, Beijing Tiantan Hospital, Capital Medical University, Beijing, China. (7) National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China. University of Chinese Academy of Sciences, Beijing, China. (8) Changping Laboratory, Beijing, China. (9) National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China. University of Chinese Academy of Sciences, Beijing, China. (10) Changping Laboratory, Beijing, China. (11) CAS Key Laboratory of Pathogen Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China. (12) Guangzhou National Laboratory, Bio-Island, Guangzhou, China. State Key Laboratory of Respiratory Disease, National Clinical Research Center for Respiratory Disease, Guangzhou Institute of Respiratory Health, The First Affiliated Hospital of Guangzhou Medical University, Guangzhou, China. (13) School of Basic Medical Sciences, Tsinghua University, Beijing, China. wywang2022@tsinghua.edu.cn. State Key Laboratory of Molecular oncology, Tsinghua University, Beijing, China. wywang2022@tsinghua.edu.cn. (14) School of Basic Medical Sciences, Tsinghua University, Beijing, China. yangxinfu@tsinghua.edu.cn. State Key Laboratory of Molecular oncology, Tsinghua University, Beijing, China. yangxinfu@tsinghua.edu.cn. Changping Laboratory, Beijing, China. yangxinfu@tsinghua.edu.cn.

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