Journal Articles

In vivo CAR T cell generation using retargeted and functionalized lentiviral vectors with reduced immunogenicity

Despite striking efficacy against hematologic malignancies, the cost and complexity of CAR T manufacturing present significant barriers to broader patient access. Beyond manufacturing challenges, ex vivo expansion of T cells may be detrimental to their function and persistence. Thus, delivery of CARs to reprogram host cells in vivo would represent a significant advance towards a readily available therapy, but has been limited by low efficiency, low specificity, and immunogenicity of viral vectors. Here, we describe the design of pseudotyped lentiviral vectors (LV) with superior functionality and high target specificity. We show that LV pseudotyped with chimeric envelope glycoproteins from dolphin morbillivirus (DMV) can be engineered to selectively infect human T cells and evade neutralizing antibody responses in measles-vaccinated human serum. We further demonstrate that camelid-derived nanobodies are a superior retargeting domain, overcoming limitations inherent to the use of single-chain variable fragment antibodies. Using a chimeric DMV-pseudotyped virus targeting the CD7 receptor, we demonstrate efficient and highly specific infection of T cells both in vitro and in vivo, generating functional CAR T cells and inducing therapeutic efficacy in a preclinical B cell lymphoma model.

Author Info: (1) Broad Institute of Massachusetts Institute of Technology and Harvard, Cambridge, MA, USA. Krantz Family Center for Cancer Research and Department of Medicine, Massachusetts Gen

Author Info: (1) Broad Institute of Massachusetts Institute of Technology and Harvard, Cambridge, MA, USA. Krantz Family Center for Cancer Research and Department of Medicine, Massachusetts General Hospital, Boston, MA, USA. (2) Broad Institute of Massachusetts Institute of Technology and Harvard, Cambridge, MA, USA. Krantz Family Center for Cancer Research and Department of Medicine, Massachusetts General Hospital, Boston, MA, USA. (3) Broad Institute of Massachusetts Institute of Technology and Harvard, Cambridge, MA, USA. Krantz Family Center for Cancer Research and Department of Medicine, Massachusetts General Hospital, Boston, MA, USA. (4) Broad Institute of Massachusetts Institute of Technology and Harvard, Cambridge, MA, USA. Krantz Family Center for Cancer Research and Department of Medicine, Massachusetts General Hospital, Boston, MA, USA. Dana-Farber Cancer Institute, Gastrointestinal Cancer Center, Boston, MA, USA. (5) Broad Institute of Massachusetts Institute of Technology and Harvard, Cambridge, MA, USA. Krantz Family Center for Cancer Research and Department of Medicine, Massachusetts General Hospital, Boston, MA, USA. (6) Broad Institute of Massachusetts Institute of Technology and Harvard, Cambridge, MA, USA. Krantz Family Center for Cancer Research and Department of Medicine, Massachusetts General Hospital, Boston, MA, USA. (7) Broad Institute of Massachusetts Institute of Technology and Harvard, Cambridge, MA, USA. Krantz Family Center for Cancer Research and Department of Medicine, Massachusetts General Hospital, Boston, MA, USA. (8) Broad Institute of Massachusetts Institute of Technology and Harvard, Cambridge, MA, USA. Krantz Family Center for Cancer Research and Department of Medicine, Massachusetts General Hospital, Boston, MA, USA. (9) Broad Institute of Massachusetts Institute of Technology and Harvard, Cambridge, MA, USA. Krantz Family Center for Cancer Research and Department of Medicine, Massachusetts General Hospital, Boston, MA, USA. (10) Broad Institute of Massachusetts Institute of Technology and Harvard, Cambridge, MA, USA. Krantz Family Center for Cancer Research and Department of Medicine, Massachusetts General Hospital, Boston, MA, USA. (11) Broad Institute of Massachusetts Institute of Technology and Harvard, Cambridge, MA, USA. yates@broadinstitute.org. Krantz Family Center for Cancer Research and Department of Medicine, Massachusetts General Hospital, Boston, MA, USA. yates@broadinstitute.org. (12) Broad Institute of Massachusetts Institute of Technology and Harvard, Cambridge, MA, USA. rmanguso@broadinstitute.org. Krantz Family Center for Cancer Research and Department of Medicine, Massachusetts General Hospital, Boston, MA, USA. rmanguso@broadinstitute.org.

Mitochondrial metabolism and signaling direct dendritic cell function in antitumor immunity

Antitumor immunity requires conventional type 1 dendritic cells (cDC1s). How cDC1s maintain functional fitness in the tumor microenvironment remains unclear. In this study, we established that intratumoral cDC1s exhibited discrete mitochondrial states and that OPA1-mediated mitochondrial energy and redox metabolism dictated cDC1 antitumor responses. Mechanistically, OPA1 orchestrated antigen presentation and the CD8(+) T cell priming function of cDC1s by promoting nuclear respiratory factor 1 (NRF1) expression and electron transport chain integrity, thereby supporting bioenergetics and NAD(+)/NADH balance. During tumor progression, mitochondrial membrane potential and volume, as well as OPA1-NRF1 signaling, declined in intratumoral cDC1s. Furthermore, intratumoral administration of cDC1s with polarized mitochondria showed immunotherapeutic benefits in mice, particularly in combination with immune checkpoint blockade. Collectively, our findings reveal mitochondrial metabolism and signaling as putative targets to reinvigorate cDC1 function for cancer immunotherapy.

Author Info: (1) Department of Immunology, St. Jude Children's Research Hospital, Memphis, TN, USA. (2) Department of Immunology, St. Jude Children's Research Hospital, Memphis, TN, USA. (3) De

Author Info: (1) Department of Immunology, St. Jude Children's Research Hospital, Memphis, TN, USA. (2) Department of Immunology, St. Jude Children's Research Hospital, Memphis, TN, USA. (3) Department of Immunology, St. Jude Children's Research Hospital, Memphis, TN, USA. (4) Department of Immunology, St. Jude Children's Research Hospital, Memphis, TN, USA. (5) Department of Immunology, St. Jude Children's Research Hospital, Memphis, TN, USA. (6) Department of Immunology, St. Jude Children's Research Hospital, Memphis, TN, USA. (7) Department of Immunology, St. Jude Children's Research Hospital, Memphis, TN, USA. (8) Department of Immunology, St. Jude Children's Research Hospital, Memphis, TN, USA. (9) Department of Immunology, St. Jude Children's Research Hospital, Memphis, TN, USA. (10) Department of Immunology, St. Jude Children's Research Hospital, Memphis, TN, USA. (11) Department of Immunology, St. Jude Children's Research Hospital, Memphis, TN, USA. (12) Department of Immunology, St. Jude Children's Research Hospital, Memphis, TN, USA. (13) Department of Immunology, St. Jude Children's Research Hospital, Memphis, TN, USA. (14) Department of Immunology, St. Jude Children's Research Hospital, Memphis, TN, USA. (15) Cell and Tissue Imaging Center, St. Jude Children's Research Hospital, Memphis, TN, USA. (16) Department of Immunology, St. Jude Children's Research Hospital, Memphis, TN, USA.

Agonistic anti-CD40 antibody treatment converts resident regulatory T cells into activated type 1 effectors within the tumor microenvironment

Maltez et al. reported that in combination with anti-PD-1 and anti-CTLA-4, treatment with agonist anti-CD40 induced spatial reorganization of Tregs within PDAC tumor microenvironments, and supported the conversion of conventional Tregs into “ExTregs”. These effects were dependent on cDC1s through Cxcl9/Cxcr3-mediated recruitment, IFNγ and IL-12 stimulation, and direct TCR–MHC-II interactions with Tregs in the tumor periphery. In Tregs, these interactions activated nuclear translocation of NFAT1, leading to Foxp3 loss and acquisition of Th1-like features, including Tbet and IFNγ expression. Observations in patient samples were consistent with this pattern, and loss of Tregs was associated with longer disease-free survival.

Maltez et al. reported that in combination with anti-PD-1 and anti-CTLA-4, treatment with agonist anti-CD40 induced spatial reorganization of Tregs within PDAC tumor microenvironments, and supported the conversion of conventional Tregs into “ExTregs”. These effects were dependent on cDC1s through Cxcl9/Cxcr3-mediated recruitment, IFNγ and IL-12 stimulation, and direct TCR–MHC-II interactions with Tregs in the tumor periphery. In Tregs, these interactions activated nuclear translocation of NFAT1, leading to Foxp3 loss and acquisition of Th1-like features, including Tbet and IFNγ expression. Observations in patient samples were consistent with this pattern, and loss of Tregs was associated with longer disease-free survival.

ABSTRACT: In pancreatic ductal adenocarcinoma (PDAC), agonistic anti-CD40 (αCD40) reduces frequencies of intratumoral regulatory T (Treg) cells despite a lack of CD40 expression on Treg cells. Here, we leveraged spatiotemporal imaging and lineage tracing approaches to examine intratumoral Treg cell fate in a mouse model of PDAC, where immune checkpoint blockade (ICB) (αPD-1 + αCTLA-4) combined with αCD40 controls tumor growth. Intratumoral Foxp3+ Treg cell numbers collapsed upon treatment, dependent on CD40-activated dendritic cells (DCs) and induction of interleukin (IL)-12 and interferon (IFN)-γ. This reduction corresponded with cellular alterations; Treg cells acquired an "ExTreg" phenotype characterized by loss of Foxp3 expression and acquisition of T helper 1 (Th1)-like features (Tbet+IFN-γ+). αCD40 promoted a spatially reorganized tumor microenvironment (TME), with Cxcr3⁺ Treg and ExTreg cells localized to the tumor periphery with Cxcl9-expressing DCs. Through in situ analyses of T cell receptor (TCR) signaling, we found that ExTreg cells had the highest antigen-driven activation among tumor-infiltrating T cells. Reprogramming of intratumoral Treg cells into Th1-like effectors reveals plasticity and an anti-tumor capacity of these cells.

Author Info: (1) Postdoctoral Research Associate Training (PRAT) Program Fellow, NIGMS, NIH, Bethesda, MD, USA; Lymphocyte Biology Section, Laboratory of Immune System Biology, NIAID, NIH, Beth

Author Info: (1) Postdoctoral Research Associate Training (PRAT) Program Fellow, NIGMS, NIH, Bethesda, MD, USA; Lymphocyte Biology Section, Laboratory of Immune System Biology, NIAID, NIH, Bethesda, MD, USA. (2) Abramson Cancer Center, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA. (3) Department of Cell, Developmental and Cancer Biology, Oregon Health and Science University, Portland, OR, USA. (4) Department of Cell, Developmental and Cancer Biology, Oregon Health and Science University, Portland, OR, USA. (5) Department of Cell, Developmental and Cancer Biology, Oregon Health and Science University, Portland, OR, USA; Graduate Program in Biomedical Sciences, Oregon Health and Science University, Portland, OR, USA. (6) Abramson Cancer Center, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA. (7) Lymphocyte Biology Section, Laboratory of Immune System Biology, NIAID, NIH, Bethesda, MD, USA. (8) Department of Biomedical Engineering, Oregon Health and Science University, Portland, OR, USA; The Knight Cancer Institute, Oregon Health and Science University, Portland, OR, USA. (9) Department of Cell, Developmental and Cancer Biology, Oregon Health and Science University, Portland, OR, USA; The Knight Cancer Institute, Oregon Health and Science University, Portland, OR, USA. (10) The Knight Cancer Institute, Oregon Health and Science University, Portland, OR, USA; Department of Molecular and Medical Genetics, Oregon Health and Science University, Portland, OR, USA; Brenden-Colson Center for Pancreatic Care, Oregon Health and Science University, Portland, OR, USA. (11) Department of Cell, Developmental and Cancer Biology, Oregon Health and Science University, Portland, OR, USA; The Knight Cancer Institute, Oregon Health and Science University, Portland, OR, USA. (12) Abramson Cancer Center, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA; Parker Institute for Cancer Immunotherapy, University of Pennsylvania, Philadelphia, PA, USA. (13) Lymphocyte Biology Section, Laboratory of Immune System Biology, NIAID, NIH, Bethesda, MD, USA; Center for Advanced Tissue Imaging (CAT-I), NIAID and NCI, NIH, Bethesda, MD, USA. Electronic address: rgermain@niaid.nih.gov. (14) Department of Cell, Developmental and Cancer Biology, Oregon Health and Science University, Portland, OR, USA; The Knight Cancer Institute, Oregon Health and Science University, Portland, OR, USA; Brenden-Colson Center for Pancreatic Care, Oregon Health and Science University, Portland, OR, USA; Parker Institute for Cancer Immunotherapy, University of Pennsylvania, Philadelphia, PA, USA. Electronic address: byrneka@ohsu.edu.

Developing a multimodal therapy for glioblastoma using oncolytic virus delivering CD19 and EGFRvIII antigens and bi-specific CARs

ABSTRACT: Glioblastoma is the most aggressive primary brain tumor with no cure, largely because of tumor heterogeneity and immunosuppressive tumor microenvironment. Chimeric antigen receptor (CAR)-T cell therapy is highly effective in blood cancers but exhibits limited efficacy in glioblastoma due to heterogeneous tumor antigen expression, antigen loss and poor persistence of tumor-targeting immune cells in glioblastoma. Here we show a multimodal immunotherapy strategy that integrates engineered immune cells with oncolytic viruses to overcome these barriers. We have developed bispecific CAR-T and CAR-NK cells in combination with oncolytic virus that delivers two tumor antigens to glioblastoma cells for effective CAR targeting. Moreover, oncolytic virus armed with membrane-bound interleukin-15 and interleukin-21 enhances immune cell expansion/persistence and cytotoxic activity. This combined approach improves anti-tumor efficacy in vitro and in vivo by limiting immune escape and enhancing anti-tumor immunity. Together, these findings establish a promising platform for multimodal immunotherapy targeting glioblastoma and other solid tumors.

Author Info: (1) Department of Neurodegenerative Diseases, Beckman Research Institute of City of Hope, 1500 E. Duarte Rd., Duarte, CA, USA. (2) Department of Surgery, City of Hope, 1500 E. Duar

Author Info: (1) Department of Neurodegenerative Diseases, Beckman Research Institute of City of Hope, 1500 E. Duarte Rd., Duarte, CA, USA. (2) Department of Surgery, City of Hope, 1500 E. Duarte Rd., Duarte, CA, USA. (3) Department of Neurodegenerative Diseases, Beckman Research Institute of City of Hope, 1500 E. Duarte Rd., Duarte, CA, USA. (4) Department of Neurodegenerative Diseases, Beckman Research Institute of City of Hope, 1500 E. Duarte Rd., Duarte, CA, USA. (5) Department of Neurodegenerative Diseases, Beckman Research Institute of City of Hope, 1500 E. Duarte Rd., Duarte, CA, USA. (6) Department of Neurodegenerative Diseases, Beckman Research Institute of City of Hope, 1500 E. Duarte Rd., Duarte, CA, USA. (7) Department of Neurodegenerative Diseases, Beckman Research Institute of City of Hope, 1500 E. Duarte Rd., Duarte, CA, USA. (8) Department of Hematology & Hematopoietic Cell Transplantation, City of Hope, 1500 E. Duarte Rd., Duarte, CA, USA. (9) Department of Surgery, City of Hope, 1500 E. Duarte Rd., Duarte, CA, USA. (10) Cellular Immunotherapy Program Cancer Center, Massachusetts General Hospital, Boston, MA, USA. Harvard Medical School, Boston, MA, USA. (11) Department of Neurodegenerative Diseases, Beckman Research Institute of City of Hope, 1500 E. Duarte Rd., Duarte, CA, USA. yshi@coh.org.

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

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.

Nonsense-mediated mRNA decay inhibition reshapes the cancer immunopeptidome Featured  

Vendramin, Fu, Fernandez Patel, Zhao, et al. investigated nonsense-mediated mRNA decay (NMD) in cancer, and detected high activity of this pathway in tumors, with lower scores associated with better ICB responses in clinical data. Inhibition of SMG1 reduced NMD activity and resulted in significant increases in immunogenic MHC-I-presented neoantigens. This resulted in improved antitumor immune responses and synergized with ICB in vivo.

Vendramin, Fu, Fernandez Patel, Zhao, et al. investigated nonsense-mediated mRNA decay (NMD) in cancer, and detected high activity of this pathway in tumors, with lower scores associated with better ICB responses in clinical data. Inhibition of SMG1 reduced NMD activity and resulted in significant increases in immunogenic MHC-I-presented neoantigens. This resulted in improved antitumor immune responses and synergized with ICB in vivo.

ABSTRACT: DNA mutations are a well-characterized source of neoepitopes in immunotherapy. Here, we examined the contribution of dysregulated RNA processing to neoantigen production. Leveraging multi-omics and checkpoint inhibitor (CPI) response data from >1,000 patients, we identified reduced activity of the nonsense-mediated mRNA decay (NMD) pathway kinase SMG1 as a predictor of improved CPI response. NMD inhibition through SMG1 targeting stabilized transcripts containing premature termination codons, most of which were of non-mutational origin. This reshaped the major histocompatibility complex class I (MHC class I)-bound immunopeptidome and increased neoantigen abundance to levels comparable to high mutation burden tumors. Functionally, NMD inhibition drove antigen-dependent T cell-mediated tumor cell killing in vitro, promoted activation of tissue-resident T cells in patient-derived models ex vivo, and improved CPI efficacy in vivo. Our findings establish NMD inhibition as a strategy to harness a previously inaccessible source of canonical and non-canonical neoantigens, with the potential to increase tumor immunogenicity across cancers.

Author Info: (1) The Tumor Immunogenomics and Immunosurveillance Lab, University College London Cancer Institute, London, UK; Cancer Evolution and Genome Instability Lab, The Francis Crick Inst

Author Info: (1) The Tumor Immunogenomics and Immunosurveillance Lab, University College London Cancer Institute, London, UK; Cancer Evolution and Genome Instability Lab, The Francis Crick Institute, London, UK; CRUK Lung Cancer Centre of Excellence, University College London Cancer Institute, London, UK. Electronic address: r.vendramin@ucl.ac.uk. (2) The Tumor Immunogenomics and Immunosurveillance Lab, University College London Cancer Institute, London, UK; CRUK Lung Cancer Centre of Excellence, University College London Cancer Institute, London, UK; Pre-Cancer Immunology Lab, University College London Cancer Institute, London, UK. (3) The Tumor Immunogenomics and Immunosurveillance Lab, University College London Cancer Institute, London, UK; CRUK Lung Cancer Centre of Excellence, University College London Cancer Institute, London, UK; Organelle Dynamics Lab, School of Cancer & Pharmaceutical Sciences, King's College London, London, UK; Organelle Dynamics Lab, the Francis Crick Institute, London, UK. (4) The Tumor Immunogenomics and Immunosurveillance Lab, University College London Cancer Institute, London, UK; CRUK Lung Cancer Centre of Excellence, University College London Cancer Institute, London, UK; Department of Thoracic Surgery, Shanghai Chest Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China. (5) The Tumor Immunogenomics and Immunosurveillance Lab, University College London Cancer Institute, London, UK; CRUK Lung Cancer Centre of Excellence, University College London Cancer Institute, London, UK. (6) The Tumor Immunogenomics and Immunosurveillance Lab, University College London Cancer Institute, London, UK; CRUK Lung Cancer Centre of Excellence, University College London Cancer Institute, London, UK. (7) Department of Molecular Cell Biology, Weizmann Institute of Science, Rehovot, Israel. (8) Department of Molecular Cell Biology, Weizmann Institute of Science, Rehovot, Israel. (9) Department of Molecular Cell Biology, Weizmann Institute of Science, Rehovot, Israel. (10) The Tumor Immunogenomics and Immunosurveillance Lab, University College London Cancer Institute, London, UK; CRUK Lung Cancer Centre of Excellence, University College London Cancer Institute, London, UK. (11) The Tumor Immunogenomics and Immunosurveillance Lab, University College London Cancer Institute, London, UK; Cancer Evolution and Genome Instability Lab, The Francis Crick Institute, London, UK; CRUK Lung Cancer Centre of Excellence, University College London Cancer Institute, London, UK. (12) The Tumor Immunogenomics and Immunosurveillance Lab, University College London Cancer Institute, London, UK; CRUK Lung Cancer Centre of Excellence, University College London Cancer Institute, London, UK; Drug Discovery Technology Laboratories, Ono Pharmaceutical Co. Ltd., Osaka, Japan. (13) Cancer Evolution and Genome Instability Lab, The Francis Crick Institute, London, UK; CRUK Lung Cancer Centre of Excellence, University College London Cancer Institute, London, UK; Department of Oncology, Medical Sciences Division, University of Oxford, Oxford, UK. (14) The Tumor Immunogenomics and Immunosurveillance Lab, University College London Cancer Institute, London, UK; CRUK Lung Cancer Centre of Excellence, University College London Cancer Institute, London, UK. (15) Department of Molecular Cell Biology, Weizmann Institute of Science, Rehovot, Israel. (16) CRUK City of London Centre Single Cell Genomics Facility, University College London Cancer Institute, London, UK; Bioinformatics Hub, University College London Cancer Institute, London, UK. (17) Proteomics Research Translational Technology Platform, University College London Cancer Institute, London, UK. (18) CRUK Lung Cancer Centre of Excellence, University College London Cancer Institute, London, UK; Barcelona Supercomputing Center (BSC), Barcelona, Spain; Cancer Genome Evolution Research Group, University College London Cancer Institute, London, UK. (19) CRUK Lung Cancer Centre of Excellence, University College London Cancer Institute, London, UK; Immune Regulation and Tumor Immunotherapy Group, University College London Cancer Institute, London, UK. (20) The Tumor Immunogenomics and Immunosurveillance Lab, University College London Cancer Institute, London, UK; Cancer Evolution and Genome Instability Lab, The Francis Crick Institute, London, UK; CRUK Lung Cancer Centre of Excellence, University College London Cancer Institute, London, UK; Division of Medicine, University College London, London, UK. (21) Cancer Evolution and Genome Instability Lab, The Francis Crick Institute, London, UK; CRUK Lung Cancer Centre of Excellence, University College London Cancer Institute, London, UK; Department of Molecular Oncology and Immunology, the Netherlands Cancer Institute, Amsterdam, the Netherlands; Oncode Institute, Utrecht, the Netherlands. (22) The Tumor Immunogenomics and Immunosurveillance Lab, University College London Cancer Institute, London, UK; CRUK Lung Cancer Centre of Excellence, University College London Cancer Institute, London, UK. (23) The Tumor Immunogenomics and Immunosurveillance Lab, University College London Cancer Institute, London, UK; CRUK Lung Cancer Centre of Excellence, University College London Cancer Institute, London, UK. (24) Research Department of Haematology, University College London Cancer Institute, London, UK. (25) CRUK Lung Cancer Centre of Excellence, University College London Cancer Institute, London, UK; Immune Regulation and Tumor Immunotherapy Group, University College London Cancer Institute, London, UK; Immune Regulation Lab, Centre for Immuno-Oncology, Nuffield Department of Medicine, University of Oxford, Oxford, UK. (26) Organoid Translational Technology Platform, University College London Cancer Institute, London, UK. (27) Proteomics Research Translational Technology Platform, University College London Cancer Institute, London, UK. (28) Department of Research Pathology, University College London Cancer Institute, London, UK. (29) CRUK City of London Explant and Patient-Derived Xenograft Core, London, UK. (30) CRUK Lung Cancer Centre of Excellence, University College London Cancer Institute, London, UK; Cancer Genome Evolution Research Group, University College London Cancer Institute, London, UK. (31) Organelle Dynamics Lab, School of Cancer & Pharmaceutical Sciences, King's College London, London, UK; Organelle Dynamics Lab, the Francis Crick Institute, London, UK. (32) Cancer Evolution and Genome Instability Lab, The Francis Crick Institute, London, UK. (33) CRUK Lung Cancer Centre of Excellence, University College London Cancer Institute, London, UK; Pre-Cancer Immunology Lab, University College London Cancer Institute, London, UK. (34) Department of Molecular Cell Biology, Weizmann Institute of Science, Rehovot, Israel. (35) Cancer Evolution and Genome Instability Lab, The Francis Crick Institute, London, UK; CRUK Lung Cancer Centre of Excellence, University College London Cancer Institute, London, UK. Electronic address: charles.swanton@crick.ac.uk. (36) CRUK Lung Cancer Centre of Excellence, University College London Cancer Institute, London, UK; Immune Regulation and Tumor Immunotherapy Group, University College London Cancer Institute, London, UK. Electronic address: s.quezada@ucl.ac.uk. (37) The Tumor Immunogenomics and Immunosurveillance Lab, University College London Cancer Institute, London, UK; CRUK Lung Cancer Centre of Excellence, University College London Cancer Institute, London, UK. Electronic address: k.litchfield@ucl.ac.uk.

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.

Lymphodepleting preconditioning impairs host antitumor immunity induced by adoptive T cell therapy in mouse models

Spotlight 

Figueroa et al. demonstrated that lasting efficacy of adoptive T cell therapy (ACT) against solid tumors relied not only on the antitumor activity of transferred T cells, but also on their ability to expand host CD8+ T cells in a TNF- and cDC1-dependent manner. Host CD8+ T cells protected against rechallenge with ACT-resistant antigen-negative tumor cells. Lymphodepleting preconditioning promoted transferred T cell expansion, but impaired host immunity against antigen-loss variants. In patients with melanoma, enrichment of cDC1, TNF signaling, Tpex and Tex gene signatures correlated with clinical responses to ACT and better overall survival.

Contributed by Ute Burkhardt

Figueroa et al. demonstrated that lasting efficacy of adoptive T cell therapy (ACT) against solid tumors relied not only on the antitumor activity of transferred T cells, but also on their ability to expand host CD8+ T cells in a TNF- and cDC1-dependent manner. Host CD8+ T cells protected against rechallenge with ACT-resistant antigen-negative tumor cells. Lymphodepleting preconditioning promoted transferred T cell expansion, but impaired host immunity against antigen-loss variants. In patients with melanoma, enrichment of cDC1, TNF signaling, Tpex and Tex gene signatures correlated with clinical responses to ACT and better overall survival.

Contributed by Ute Burkhardt

ABSTRACT: Adoptive T cell therapy (ACT) is effective against hematologic cancers, but the mechanisms underlying durable responses in solid tumors remain unclear. We show that adoptively transferred CD8(+) T cells that eradicate established murine tumors promote expansion of host CD8(+) T cells exhibiting tumor-reactive and tissue-resident phenotypes that contribute to tumor elimination. Mechanistically, tumor necrosis factor (TNF) from transferred cells induces dendritic cell (DC)-dependent expansion of host CD8(+) T cells, conferring protection against ACT-resistant tumor cells lacking the targeted antigen. Lymphodepleting preconditioning promotes expansion of transferred cells and primary tumor eradication but impairs host antitumor immunity and abrogates protection against ACT-resistant tumors. In human tumors, increased TNF/DC/CD8(+) T cell profiles correlate with favorable ACT responses and improved survival. These findings reveal a TNF-dependent interplay between transferred and host CD8(+) T cells underlying durable antitumor immunity that is impaired by lymphodepleting preconditioning in mouse models, suggesting an underappreciated mechanism of ACT resistance.

Author Info: (1) Centro Basal Ciencia & Vida, Fundaci—n Ciencia & Vida, Santiago, Chile. (2) Centro Basal Ciencia & Vida, Fundaci—n Ciencia & Vida, Santiago, Chile. (3) Centro Basal Ciencia & V

Author Info: (1) Centro Basal Ciencia & Vida, Fundaci—n Ciencia & Vida, Santiago, Chile. (2) Centro Basal Ciencia & Vida, Fundaci—n Ciencia & Vida, Santiago, Chile. (3) Centro Basal Ciencia & Vida, Fundaci—n Ciencia & Vida, Santiago, Chile. Centro de Investigaci—n e Innovaci—n en C‡ncer, Fundaci—n Arturo L—pez PŽrez OECI Cancer Center, Santiago, Chile. (4) Centro Basal Ciencia & Vida, Fundaci—n Ciencia & Vida, Santiago, Chile. (5) Centro Basal Ciencia & Vida, Fundaci—n Ciencia & Vida, Santiago, Chile. Centro de Investigaci—n e Innovaci—n en C‡ncer, Fundaci—n Arturo L—pez PŽrez OECI Cancer Center, Santiago, Chile. (6) Centro Basal Ciencia & Vida, Fundaci—n Ciencia & Vida, Santiago, Chile. (7) Centro Basal Ciencia & Vida, Fundaci—n Ciencia & Vida, Santiago, Chile. (8) Centro Basal Ciencia & Vida, Fundaci—n Ciencia & Vida, Santiago, Chile. (9) Centro Basal Ciencia & Vida, Fundaci—n Ciencia & Vida, Santiago, Chile. (10) Laboratory of Immunology and Cellular Stress, Facultad de Medicina, Universidad de Chile, Santiago, Chile. (11) Laboratory of Immune Regulation, NDM Centre for Immuno-Oncology, University of Oxford, Oxford, UK. (12) Centro Basal Ciencia & Vida, Fundaci—n Ciencia & Vida, Santiago, Chile. Department of Anatomy, University of California San Francisco, San Francisco, CA, USA. (13) Centro Basal Ciencia & Vida, Fundaci—n Ciencia & Vida, Santiago, Chile. Centro de Biolog’a Celular y Biomedicina (CEBICEM), Facultad de Ciencias, Universidad San Sebasti‡n, Santiago, Chile. (14) Centro Basal Ciencia & Vida, Fundaci—n Ciencia & Vida, Santiago, Chile. Laboratory of Immunology, Facultad de Ciencias, Universidad de Chile, Santiago, Chile. (15) Centro Basal Ciencia & Vida, Fundaci—n Ciencia & Vida, Santiago, Chile. Laboratory of Immunology and Cellular Stress, Facultad de Medicina, Universidad de Chile, Santiago, Chile. (16) Centro Basal Ciencia & Vida, Fundaci—n Ciencia & Vida, Santiago, Chile. vincenzo.borgna@uss.cl. Servicio de Urolog’a, Hospital Barros Luco Trudeau, Santiago, Chile. vincenzo.borgna@uss.cl. Facultad de Medicina, Universidad San Sebasti‡n, Santiago, Chile. vincenzo.borgna@uss.cl. (17) Centro Basal Ciencia & Vida, Fundaci—n Ciencia & Vida, Santiago, Chile. alladser@cienciavida.org. Facultad de Medicina, Universidad San Sebasti‡n, Santiago, Chile. alladser@cienciavida.org.

Acute and chronic infections drive distinct trajectories in human memory CD4+ T cell formation

Spotlight 

Comparing CD4+ T cells generated during acute or chronic hepatitis C virus (HCV) infection, Reinscheid and Weisser et al. evaluated patient samples and found that acute infection generated various subsets of progenitor CD4+ T cells, including subsets also observed in chronic infection. In chronic infection, a subset of stem-like/resting Bcl-2+ CD4+ T cells likely gave rise to a subset of T-bet+ effector CD4+ T cells. In patients treated with DAA to clear the virus, the effector subset essentially disappeared, while the stem-like subset formed a functional long-term memory pool that was distinct from the memory pools that formed after spontaneous HCV clearance.

Contributed by Lauren Hitchings

Comparing CD4+ T cells generated during acute or chronic hepatitis C virus (HCV) infection, Reinscheid and Weisser et al. evaluated patient samples and found that acute infection generated various subsets of progenitor CD4+ T cells, including subsets also observed in chronic infection. In chronic infection, a subset of stem-like/resting Bcl-2+ CD4+ T cells likely gave rise to a subset of T-bet+ effector CD4+ T cells. In patients treated with DAA to clear the virus, the effector subset essentially disappeared, while the stem-like subset formed a functional long-term memory pool that was distinct from the memory pools that formed after spontaneous HCV clearance.

Contributed by Lauren Hitchings

ABSTRACT: Virus-specific CD4(+) T cells are essential for coordinating adaptive immunity during infection, but their differentiation and maintenance in chronic infection remain unclear. Using human hepatitis C virus (HCV) infection as a model, we assessed the determinants of virus-specific CD4(+) T cell immunity in acute, spontaneously cleared, chronic, and therapeutically cured infections. During acute infection, multiple subsets of progenitor CD4(+) T cells emerged, including subsets that are also found in chronic infection. In chronic infection, stem-like Bcl-2(+) CD4(+) T cells and T-bet(+) effector CD4(+) T cells existed in a progenitor/progeny relationship. Following therapy-mediated HCV cure, these cells retained their chronic signature but formed a stable memory pool that persisted for years and was distinct from HCV-specific CD4(+) T cell memory after spontaneous clearance. Collectively, our findings highlight differences in CD4(+) T cell fates that depend on infection outcomes and reveal common principles of CD4(+) and exhausted CD8(+) T cell maintenance during and after chronic infection.

Author Info: (1) Department of Medicine II, Medical Center - University of Freiburg, Hugstetter Str. 55, D-79106 Freiburg, Germany; Faculty of Biology, University of Freiburg, Freiburg, Germany

Author Info: (1) Department of Medicine II, Medical Center - University of Freiburg, Hugstetter Str. 55, D-79106 Freiburg, Germany; Faculty of Biology, University of Freiburg, Freiburg, Germany. (2) Department of Medicine II, Medical Center - University of Freiburg, Hugstetter Str. 55, D-79106 Freiburg, Germany; Faculty of Biology, University of Freiburg, Freiburg, Germany. (3) Department of Medicine II, Medical Center - University of Freiburg, Hugstetter Str. 55, D-79106 Freiburg, Germany; Faculty of Medicine, University of Freiburg, Freiburg, Germany. (4) Department of Medicine II, Medical Center - University of Freiburg, Hugstetter Str. 55, D-79106 Freiburg, Germany; Faculty of Medicine, University of Freiburg, Freiburg, Germany. (5) Department of Medicine II, Medical Center - University of Freiburg, Hugstetter Str. 55, D-79106 Freiburg, Germany; Faculty of Medicine, University of Freiburg, Freiburg, Germany. (6) Department of Medicine II, Medical Center - University of Freiburg, Hugstetter Str. 55, D-79106 Freiburg, Germany; Faculty of Biology, University of Freiburg, Freiburg, Germany. (7) Department of Medicine II, Medical Center - University of Freiburg, Hugstetter Str. 55, D-79106 Freiburg, Germany; Faculty of Medicine, University of Freiburg, Freiburg, Germany. (8) Department of Medicine II, Medical Center - University of Freiburg, Hugstetter Str. 55, D-79106 Freiburg, Germany; Faculty of Biology, University of Freiburg, Freiburg, Germany. (9) Institute for Transfusion Medicine and Gene Therapy, Freiburg University Medical Center, Faculty of Medicine, University of Freiburg, Freiburg, Germany. (10) Institute of Virology, Medical Faculty and University Hospital DŸsseldorf, Heinrich Heine University DŸsseldorf, DŸsseldorf, Germany. (11) Institute of Virology, Medical Faculty and University Hospital DŸsseldorf, Heinrich Heine University DŸsseldorf, DŸsseldorf, Germany. (12) Department of Medicine II, Medical Center - University of Freiburg, Hugstetter Str. 55, D-79106 Freiburg, Germany; Faculty of Biology, University of Freiburg, Freiburg, Germany. (13) Institute of Virology, Medical Faculty and University Hospital DŸsseldorf, Heinrich Heine University DŸsseldorf, DŸsseldorf, Germany. (14) Institute of Molecular and Clinical Ophthalmology Basel, Basel, Switzerland. (15) Department of Medicine II, Medical Center - University of Freiburg, Hugstetter Str. 55, D-79106 Freiburg, Germany; Faculty of Medicine, University of Freiburg, Freiburg, Germany; German Cancer Consortium (DKTK), Heidelberg, Germany, partner site Freiburg, Freiburg, Germany; Signaling Research Centers BIOSS and CIBSS, Freiburg, Germany. (16) Department of Medicine II, Medical Center - University of Freiburg, Hugstetter Str. 55, D-79106 Freiburg, Germany; Faculty of Medicine, University of Freiburg, Freiburg, Germany. (17) Department of Medicine II, Medical Center - University of Freiburg, Hugstetter Str. 55, D-79106 Freiburg, Germany; Faculty of Medicine, University of Freiburg, Freiburg, Germany. Electronic address: robert.thimme@uniklinik-freiburg.de. (18) Department of Medicine II, Medical Center - University of Freiburg, Hugstetter Str. 55, D-79106 Freiburg, Germany; Faculty of Medicine, University of Freiburg, Freiburg, Germany. Electronic address: tobias.boettler@uniklinik-freiburg.de. (19) Department of Medicine II, Medical Center - University of Freiburg, Hugstetter Str. 55, D-79106 Freiburg, Germany; Faculty of Medicine, University of Freiburg, Freiburg, Germany. Electronic address: maike.hofmann@uniklinik-freiburg.de.

In vivo generation of CAR myeloid cells through erythrocyte-mediated mRNA delivery for cancer immunotherapy Spotlight 

Nie, Liu, Song, and Yao et al. developed a spleen delivery platform of mRNA-loaded lipid nanoparticles (LNPs) covalently bound to erythrocytes (mRNA-LNP-Ery), which naturally target splenic CD11b+ myeloid cells. Unlike conventional LNPs, mRNA-LNP-Ery entered cells via phagocytosis, avoiding lysosomal degradation and efficiently delivering mRNA. CAR myeloid cells (HER2 or CD19) adopted a proinflammatory antigen-presenting phenotype, migrated to tumors, and stimulated T and NK cell influx, potent antitumor activity, and systemic immunity, which was spleen-dependent. Repeated doses of mRNA-LNP-Ery resulted in superior efficacy at 1/10 the dose of LNPs.

Contributed by Katherine Turner

Nie, Liu, Song, and Yao et al. developed a spleen delivery platform of mRNA-loaded lipid nanoparticles (LNPs) covalently bound to erythrocytes (mRNA-LNP-Ery), which naturally target splenic CD11b+ myeloid cells. Unlike conventional LNPs, mRNA-LNP-Ery entered cells via phagocytosis, avoiding lysosomal degradation and efficiently delivering mRNA. CAR myeloid cells (HER2 or CD19) adopted a proinflammatory antigen-presenting phenotype, migrated to tumors, and stimulated T and NK cell influx, potent antitumor activity, and systemic immunity, which was spleen-dependent. Repeated doses of mRNA-LNP-Ery resulted in superior efficacy at 1/10 the dose of LNPs.

Contributed by Katherine Turner

ABSTRACT: Engineering myeloid cells with chimeric antigen receptors (CARs) holds great therapeutic promise, but their generation in vivo remains challenging. Here, we developed an erythrocyte-mediated messenger RNA (mRNA) delivery platform, termed mRNA-LNP-Ery, in which mRNA-loaded lipid nanoparticles (LNPs) are covalently anchored onto erythrocytes. Exploiting erythrocytes' intrinsic splenic homing capacity and unique biocompatibility, mRNA-LNP-Ery enables highly selective and efficient mRNA delivery to CD11b(+) myeloid cells in the spleen, with minimal uptake by hepatocytes. We also demonstrated that mRNA-LNP-Ery is internalized through phagocytosis and avoids lysosomal degradation, resulting in enhanced cytosolic mRNA translation. Delivery of mRNAs encoding CARs targeting human epidermal growth factor receptor 2 (HER2) or CD19 generated functional CAR myeloid cells in vivo that adopted a proinflammatory, antigen-presenting phenotype. These cells migrated to tumors, eliminated cancer cells, and remodeled the tumor microenvironment, leading to increased infiltration of effector T and natural killer (NK) cells. The antitumor effect was abolished in splenectomized mice and partially diminished in nude mice, indicating that therapeutic activity depends on both CAR myeloid cell formation within the spleen and their cross-talk with adaptive immunity. Furthermore, repeated administration of mRNA-LNP-Ery achieved superior antitumor efficacy to conventional mRNA-LNPs at one-tenth the mRNA dose, with minimal systemic toxicity, underscoring the high efficiency and safety of spleen-targeted delivery. Together, our findings established a clinically translatable erythrocyte-based mRNA platform that enables direct in vivo immune cell programming and advances CAR myeloid therapies for solid tumors.

Author Info: (1) Key Laboratory of Growth Regulation and Translational Research of Zhejiang Province, School of Life Sciences, Westlake University, Hangzhou, Zhejiang 310030, China. Westlake La

Author Info: (1) Key Laboratory of Growth Regulation and Translational Research of Zhejiang Province, School of Life Sciences, Westlake University, Hangzhou, Zhejiang 310030, China. Westlake Laboratory of Life Sciences and Biomedicine, Hangzhou, Zhejiang 310024, China. (2) Key Laboratory of Growth Regulation and Translational Research of Zhejiang Province, School of Life Sciences, Westlake University, Hangzhou, Zhejiang 310030, China. Westlake Laboratory of Life Sciences and Biomedicine, Hangzhou, Zhejiang 310024, China. Research Center for Industries of the Future and School of Life Sciences, Westlake University, Hangzhou, Zhejiang 310030, China. (3) Westlake Therapeutics, Hangzhou, Zhejiang 310024, China. (4) Key Laboratory of Growth Regulation and Translational Research of Zhejiang Province, School of Life Sciences, Westlake University, Hangzhou, Zhejiang 310030, China. Westlake Laboratory of Life Sciences and Biomedicine, Hangzhou, Zhejiang 310024, China. (5) Westlake Therapeutics, Hangzhou, Zhejiang 310024, China. (6) Peking-Tsinghua Center for Life Sciences, Academy for Advanced Interdisciplinary Studies, Peking University, Beijing 100871, China. Ministry of Education Key Laboratory of Cell Proliferation and Differentiation, School of Life Sciences, Peking University, Beijing 100871, China. (7) Key Laboratory of Growth Regulation and Translational Research of Zhejiang Province, School of Life Sciences, Westlake University, Hangzhou, Zhejiang 310030, China. Westlake Laboratory of Life Sciences and Biomedicine, Hangzhou, Zhejiang 310024, China. Research Center for Industries of the Future and School of Life Sciences, Westlake University, Hangzhou, Zhejiang 310030, China.

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