Journal Articles

Neoadjuvant immunotherapy promotes the formation of mature tertiary lymphoid structures in a remodeled pancreatic tumor microenvironment

Pancreatic adenocarcinoma (PDAC) is a rapidly progressing cancer that responds poorly to immunotherapies. Intratumoral tertiary lymphoid structures (TLS) have been associated with rare long-term PDAC survivors, but the role of TLS in PDAC and their spatial relationships within the context of the broader tumor microenvironment remain unknown. Herein, we report the generation of a spatial multi-omics atlas of PDAC tumors and tumor-adjacent lymph nodes from patients treated with combination neoadjuvant immunotherapies. Using machine learning-enabled hematoxylin and eosin image classification models, imaging mass cytometry, and unsupervised gene expression matrix factorization methods for spatial transcriptomics, we characterized cellular states within and adjacent to TLS spanning across distinct spatial niches and pathologic responses. Unsupervised learning identified TLS-specific spatial gene expression signatures that significantly associated with improved survival in PDAC patients. We identified spatial features of pathologic immune responses, including intratumoral TLS-associated B-cell maturation colocalizing with IgG dissemination and extracellular matrix remodeling. Our findings offer insights into the cellular and molecular landscape of TLS in PDACs during immunotherapy treatment.

Author Info: (1) Johns Hopkins Medicine, Baltimore, United States. (2) Johns Hopkins Medicine, Baltimore, MD, United States. (3) Johns Hopkins Medicine, United States. (4) Johns Hopkins Univers

Author Info: (1) Johns Hopkins Medicine, Baltimore, United States. (2) Johns Hopkins Medicine, Baltimore, MD, United States. (3) Johns Hopkins Medicine, United States. (4) Johns Hopkins University, United States. (5) Johns Hopkins Medicine, United States. (6) Johns Hopkins Medicine, Baltimore, Maryland, United States. (7) Johns Hopkins University, United States. (8) University of Maryland Medical Center, Baltimore, MD, United States. (9) Johns Hopkins University, Baltimore, MD, United States. (10) Johns Hopkins Medicine, Baltimore, MD, United States. (11) Johns Hopkins University, Baltimore, MD, United States. (12) Johns Hopkins University, United States. (13) Johns Hopkins University, United States. (14) Johns Hopkins University, Baltimore, Maryland, United States. (15) Johns Hopkins Medicine, Baltimore, Maryland, United States. (16) Johns Hopkins Medicine, Baltimore, United States. (17) Johns Hopkins University, Baltimore, United States. (18) Johns Hopkins Medicine, Baltimore, MD, United States. (19) Johns Hopkins Sidney Kimmel Comprehensive Cancer Center, Baltimore, MD, United States. (20) Johns Hopkins Medicine, Baltimore, MARYLAND, United States. (21) Johns Hopkins University, Baltimore, MD, United States. (22) Johns Hopkins University, Baltimore, MD, United States. (23) Johns Hopkins University, Baltimore, MD, United States. (24) Johns Hopkins University, Baltimore, MD, United States. (25) Johns Hopkins Medicine, Baltimore, Maryland, United States. (26) Johns Hopkins University, Baltimore, MD, United States. (27) Johns Hopkins Medicine, Baltimore, United States. (28) University of Maryland, Baltimore, Baltimore, Maryland, United States. (29) Johns Hopkins University, Baltimore, MD, United States.

Reprogramming CD8+ T-cell branched N-glycosylation limits exhaustion, enhancing cytotoxicity and tumor killing

T-cell therapies have transformed cancer treatment. While surface glycans have been shown to play critical roles in regulating T-cell development and function, whether and how the glycome influences T cell-mediated tumor immunity remains an area of active investigation. In this study, we show that the intratumoral T-cell glycome is altered early in human colorectal cancer, with substantial changes in branched N-glycans. We demonstrated that CD8+ T cells expressing _1,6-GlcNAc branched N-glycans adopted an exhausted phenotype, marked by increased PD1 and Tim3 expression. CRISPR/Cas9 deletion of key branching glycosyltransferase genes revealed that Mgat5 played a prominent role in T-cell exhaustion. In culture-based assays and tumor studies, Mgat5 deletion in CD8+ T cells resulted in improved cancer cell killing. These findings prompted assessment of whether MGAT5 deletion in anti-CD19 chimeric-antigen receptor (CAR) T cells could enable this therapeutic modality in a solid tumor setting. We showed that MGAT5 KO anti-CD19-CAR T cells inhibited the growth of CD19-transduced tumors. Together, these findings show that MGAT5-mediated branched N-glycans regulate CD8+ T-cell function in cancer and provide a strategy to enhance antitumor activity of native and CAR T cells.

Author Info: (1) i3S - Instituto de Investiga‹o e Inova‹o em Saœde, Universidade do Porto, Porto, Portugal. (2) University of Pittsburgh, Pittsbrugh, PA, United States. (3) University of Pitt

Author Info: (1) i3S - Instituto de Investiga‹o e Inova‹o em Saœde, Universidade do Porto, Porto, Portugal. (2) University of Pittsburgh, Pittsbrugh, PA, United States. (3) University of Pittsburgh, Pittsburgh, PA, United States. (4) University of Pittsburgh, Pittsburgh, PA, United States. (5) i3S - Instituto de Investiga‹o e Inova‹o em Saœde, Universidade do Porto, Porto, Porto, Portugal. (6) Hospital de Santo Ant—nio, Porto, Porto, Portugal. (7) University of Pittsburgh, Pittsburgh, PA, United States. (8) University of Pittsburgh, Pittsburgh, PA, United States. (9) i3S - Instituto de Investiga‹o e Inova‹o em Saœde, Universidade do Porto, Porto, Porto, Portugal. (10) University of Pittsburgh, Pittsburgh, PA, United States. (11) i3S - Instituto de Investiga‹o e Inova‹o em Saœde, Universidade do Porto, Porto, Portugal. (12) Complutense University of Madrid, Madrid, Spain. (13) Instituto de Investigaci—n Sanitaria del Hospital Cl’nico San Carlos, Madrid, Spain. (14) Hospital Cl’nico San Carlos, Madrid, Spain. (15) Department of Gastroenterology, Centro Hospitalar Universit‡rio do Porto, 4050-313, Porto, Portugal, Portugal. (16) i3S-Institute for Research & Innovation in Health, University of Porto, 4200-135 Porto, Portugal, Porto, Portugal. (17) University of Pittsburgh, Pittsburgh, PA, United States. (18) i3S - Instituto de Investiga‹o e Inova‹o em Saœde, Universidade do Porto, Porto, Porto, Portugal.

Engineered bacteria launch and control an oncolytic virus

The ability of bacteria and viruses to selectively replicate in tumours has led to synthetic engineering of new microbial therapies. Here we design a cooperative strategy whereby Salmonella typhimurium bacteria transcribe and deliver the Senecavirus A RNA genome inside host cells, launching a potent oncolytic viral infection. 'Encapsidated' by bacteria, the viral genome can further bypass circulating antiviral antibodies to reach the tumour and initiate replication and spread within immune mice. Finally, we engineer the virus to require a bacterially delivered protease to achieve virion maturation, demonstrating bacterial control over the virus. Together, we refer to this platform as 'CAPPSID' for Coordinated Activity of Prokaryote and Picornavirus for Safe Intracellular Delivery. This work extends bacterially delivered therapeutics to viral genomes, and shows how a consortium of microbes can achieve a cooperative aim.

Author Info: (1) Department of Biomedical Engineering, Columbia University, New York, NY, USA. Laboratory of Virology and Infectious Disease, The Rockefeller University, New York, NY, USA. (2)

Author Info: (1) Department of Biomedical Engineering, Columbia University, New York, NY, USA. Laboratory of Virology and Infectious Disease, The Rockefeller University, New York, NY, USA. (2) Department of Biomedical Engineering, Columbia University, New York, NY, USA. (3) Department of Biomedical Engineering, Columbia University, New York, NY, USA. (4) Department of Biomedical Engineering, Columbia University, New York, NY, USA. (5) Department of Biomedical Engineering, Columbia University, New York, NY, USA. (6) Department of Biomedical Engineering, Columbia University, New York, NY, USA. (7) Department of Biomedical Engineering, Columbia University, New York, NY, USA. (8) Department of Biomedical Engineering, Columbia University, New York, NY, USA. (9) Laboratory of Virology and Infectious Disease, The Rockefeller University, New York, NY, USA. (10) Department of Biomedical Engineering, Columbia University, New York, NY, USA. tal.danino@columbia.edu. Herbert Irving Comprehensive Cancer Center, Columbia University, New York, NY, USA. tal.danino@columbia.edu. Data Science Institute, Columbia University, New York, NY, USA. tal.danino@columbia.edu.

First-line sacituzumab tirumotecan with tagitanlimab in advanced non-small-cell lung cancer: a phase 2 trial

Sacituzumab tirumotecan (sac-TMT, also known as MK-2870 or SKB264) is an antibody-drug conjugate targeting trophoblast cell surface antigen 2. We report the initial findings from the ongoing phase 2 OptiTROP-Lung01 study, evaluating the combination of sac-TMT and tagitanlimab (KL-A167), an anti-PD-L1 antibody, as first-line therapy in patients with advanced or metastatic non-small-cell lung cancer who lack actionable genomic alterations (cohorts 1A and 1B). Cohort 1A received sac-TMT (5_mg_kg(-1), every 3_weeks) plus tagitanlimab (1,200_mg, every 3_weeks) in each 3-week cycle, whereas cohort 1B was treated with sac-TMT (5_mg_kg(-1), every 2_weeks) plus tagitanlimab (900_mg, every 2 weeks) in each 4-week cycle, in a nonrandomized manner until disease progression or unacceptable toxicity. The primary endpoints included safety and objective response rate. This study was not powered for formal hypothesis testing. A total of 40 and 63 patients were enrolled in cohorts 1A and 1B, respectively. The median age was 63_years in both cohorts. An Eastern Cooperative Oncology Group performance status of 1 was observed in 97.5% and 85.7% of patients in cohorts 1A and 1B, respectively. In cohorts 1A and 1B, the most common grade ³3 treatment-related adverse events were decreased neutrophil count (30.0% and 34.9%), decreased white blood cell count (5.0% and 19.0%) and anemia (5.0% and 19.0%). No treatment-related deaths were observed. After median follow-ups of 19.3_months for cohort 1A and 13.0_months for cohort 1B, the confirmed objective response rate in the full analysis set was 40.0% (16 of 40) and 66.7% (42 of 63), the disease control rate was 85.0% and 92.1% and median progression-free survival was 15.4_months (95% confidence interval 6.7-17.9) and not reached for cohorts 1A and 1B, respectively. sac-TMT plus tagitanlimab showed promising efficacy as a first-line treatment for advanced or metastatic non-small-cell lung cancer, with a manageable safety profile. ClinicalTrials.gov registration: NCT05351788 .

Author Info: (1) Sun Yat-sen University Cancer Center, State Key Laboratory of Oncology in South China, Collaborative Innovation Center for Cancer Medicine, Sun Yat-sen University, Guangzhou, C

Author Info: (1) Sun Yat-sen University Cancer Center, State Key Laboratory of Oncology in South China, Collaborative Innovation Center for Cancer Medicine, Sun Yat-sen University, Guangzhou, China. (2) The Affiliated Cancer Hospital of Zhengzhou University, Zhengzhou, China. Henan Cancer Hospital, Zhengzhou, China. Institute of Cancer Research, Henan Academy of Innovations in Medical Science, Zhengzhou, China. (3) Jilin Cancer Hospital, Changchun, China. (4) Hunan Cancer Hospital, Changsha, China. (5) The First Hospital of China Medical University, Shenyang, China. (6) Shanxi Cancer Hospital, Taiyuan, China. (7) West China Hospital of Sichuan University, Chengdu, China. (8) The First Affiliated Hospital of Zhengzhou University, Zhengzhou, China. (9) Hunan Cancer Hospital, Changsha, China. (10) Harbin Medical University Cancer Hospital, Harbin, China. (11) Hubei Cancer Hospital, Wuhan, China. (12) Chongqing University Cancer Hospital, Chongqing, China. (13) The Second Affiliated Hospital of Nanchang University, Nanchang, China. (14) Shandong Cancer Hospital, Jinan, China. (15) Zhejiang Cancer Hospital, Hangzhou, China. (16) The First Affiliated Hospital of Xiamen University, Xiamen, China. (17) Jiangsu Province Hospital, Nanjing, China. (18) Beijing Cancer Hospital, Beijing, China. (19) Sichuan Kelun-Biotech Biopharmaceutical Co Ltd, Chengdu, China. (20) Sichuan Kelun-Biotech Biopharmaceutical Co Ltd, Chengdu, China. (21) Sichuan Kelun-Biotech Biopharmaceutical Co Ltd, Chengdu, China. National Engineering Research Center of Targeted Biologics, Chengdu, China. (22) Sun Yat-sen University Cancer Center, State Key Laboratory of Oncology in South China, Collaborative Innovation Center for Cancer Medicine, Sun Yat-sen University, Guangzhou, China. zhangli@sysucc.org.cn. (23) Sun Yat-sen University Cancer Center, State Key Laboratory of Oncology in South China, Collaborative Innovation Center for Cancer Medicine, Sun Yat-sen University, Guangzhou, China. fangwf@sysucc.org.cn.

Glycan shielding enables TCR-sufficient allogeneic CAR-T therapy Spotlight 

Deletion of the glycan regulator SPPL3 improved allo-CAR-T fitness by reducing T cell, NK cell, and FasL-mediated killing. SPPL3 KO increased TCR/CD3 glycosylation, reduced TCR detection and antigen recognition, and diminished GvHD in humanized mice, but did not affect the CAR molecule or functionality. In 10 patients with r/r B-NHL, SPPL3KO TCRKO allo-CAR-T were safe and efficacious, without severe GvHD. However, TCR- CAR-T persistence was limited compared to TCR+ CAR-T cells. Based on this result and further preclinical studies, 3 patients received SPPL3KO TCR+ allo-CAR-T, which also showed safety and efficacy, despite TCR competence.

Contributed by Alex Najibi

Deletion of the glycan regulator SPPL3 improved allo-CAR-T fitness by reducing T cell, NK cell, and FasL-mediated killing. SPPL3 KO increased TCR/CD3 glycosylation, reduced TCR detection and antigen recognition, and diminished GvHD in humanized mice, but did not affect the CAR molecule or functionality. In 10 patients with r/r B-NHL, SPPL3KO TCRKO allo-CAR-T were safe and efficacious, without severe GvHD. However, TCR- CAR-T persistence was limited compared to TCR+ CAR-T cells. Based on this result and further preclinical studies, 3 patients received SPPL3KO TCR+ allo-CAR-T, which also showed safety and efficacy, despite TCR competence.

Contributed by Alex Najibi

ABSTRACT: Despite the success of autologous chimeric antigen receptor (CAR)-T cell therapy, achieving persistence and avoiding rejection in allogeneic settings remains challenging. We showed that signal peptide peptidase-like 3 (SPPL3) deletion enabled glycan-mediated immune evasion in primary T cells. SPPL3 deletion modified glycan profiles on T cells, restricted ligand accessibility, and reduced allogeneic immunity without compromising the functionality of anti-CD19 CAR molecules. In a phase I clinical trial, SPPL3-null, T cell receptor (TCR)-deficient anti-CD19 allogeneic CAR-T cells reached the safety primary endpoint, with grade 3 or higher cytokine release syndrome (CRS) observed in 3 out of 9 patients with relapsed/refractory B cell non-Hodgkin lymphoma (B-NHL) (ClinicalTrials.gov: NCT06014073). Reverse translational research highlighted the pivotal role of TCR in sustaining T cell persistence. We therefore evaluated the safety of SPPL3-null, TCR-sufficient CAR-T therapy on three patients with lymphoma or leukemia for compassionate care and observed no clinical signs of graft-versus-host disease. Our findings suggest glycan shielding by SPPL3 deletion is a promising direction for optimizing universal CAR-T therapies.

Author Info: (1) Biomedical Pioneering Innovation Center, Peking-Tsinghua Center for Life Sciences, Peking University Genome Editing Research Center, State Key Laboratory of Gene Function and M

Author Info: (1) Biomedical Pioneering Innovation Center, Peking-Tsinghua Center for Life Sciences, Peking University Genome Editing Research Center, State Key Laboratory of Gene Function and Modulation Research, School of Life Sciences, Peking University, Beijing 100871, China; Changping Laboratory, Beijing 102206, China. (2) School of Medicine, Nankai University, Tianjin 300071, China; Department of Bio-Therapeutic, the First Medical Center, Chinese PLA General Hospital, Beijing 100039, China. (3) Biomedical Pioneering Innovation Center, Peking-Tsinghua Center for Life Sciences, Peking University Genome Editing Research Center, State Key Laboratory of Gene Function and Modulation Research, School of Life Sciences, Peking University, Beijing 100871, China; Changping Laboratory, Beijing 102206, China. (4) Biomedical Pioneering Innovation Center, Peking-Tsinghua Center for Life Sciences, Peking University Genome Editing Research Center, State Key Laboratory of Gene Function and Modulation Research, School of Life Sciences, Peking University, Beijing 100871, China. (5) Biomedical Pioneering Innovation Center, Peking-Tsinghua Center for Life Sciences, Peking University Genome Editing Research Center, State Key Laboratory of Gene Function and Modulation Research, School of Life Sciences, Peking University, Beijing 100871, China. (6) Department of Bio-Therapeutic, the First Medical Center, Chinese PLA General Hospital, Beijing 100039, China. (7) EdiGene Inc., Life Science Park, Changping District, Beijing 102206, China. (8) School of Pharmacy, University of Wisconsin-Madison, Madison, WI 53705, USA; First School of Clinical Medicine, Peking University, Beijing 100871, China. (9) Biomedical Pioneering Innovation Center, Peking-Tsinghua Center for Life Sciences, Peking University Genome Editing Research Center, State Key Laboratory of Gene Function and Modulation Research, School of Life Sciences, Peking University, Beijing 100871, China. (10) State Key Laboratory of Experimental Hematology, National Clinical Research Center for Blood Diseases, Haihe Laboratory of Cell Ecosystem, Institute of Hematology & Blood Diseases Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Tianjin 300020, China; Tianjin Institutes of Health Science, Tianjin 301600, China. (11) Department of Bio-Therapeutic, the First Medical Center, Chinese PLA General Hospital, Beijing 100039, China. (12) Department of Bio-Therapeutic, the First Medical Center, Chinese PLA General Hospital, Beijing 100039, China. (13) Department of Bio-Therapeutic, the First Medical Center, Chinese PLA General Hospital, Beijing 100039, China. (14) EdiGene Inc., Life Science Park, Changping District, Beijing 102206, China. (15) EdiGene Inc., Life Science Park, Changping District, Beijing 102206, China. (16) EdiGene Inc., Life Science Park, Changping District, Beijing 102206, China. (17) EdiGene Inc., Life Science Park, Changping District, Beijing 102206, China. (18) EdiGene Inc., Life Science Park, Changping District, Beijing 102206, China. (19) EdiGene Inc., Life Science Park, Changping District, Beijing 102206, China. (20) Changping Laboratory, Beijing 102206, China. (21) Changping Laboratory, Beijing 102206, China. (22) Changping Laboratory, Beijing 102206, China. (23) Biomedical Pioneering Innovation Center, Peking-Tsinghua Center for Life Sciences, Peking University Genome Editing Research Center, State Key Laboratory of Gene Function and Modulation Research, School of Life Sciences, Peking University, Beijing 100871, China. (24) Biomedical Pioneering Innovation Center, Peking-Tsinghua Center for Life Sciences, Peking University Genome Editing Research Center, State Key Laboratory of Gene Function and Modulation Research, School of Life Sciences, Peking University, Beijing 100871, China. (25) EdiGene Inc., Life Science Park, Changping District, Beijing 102206, China. (26) Biomedical Pioneering Innovation Center, Peking-Tsinghua Center for Life Sciences, Peking University Genome Editing Research Center, State Key Laboratory of Gene Function and Modulation Research, School of Life Sciences, Peking University, Beijing 100871, China. (27) EdiGene Inc., Life Science Park, Changping District, Beijing 102206, China. (28) Department of Bio-Therapeutic, the First Medical Center, Chinese PLA General Hospital, Beijing 100039, China. (29) Department of Bio-Therapeutic, the First Medical Center, Chinese PLA General Hospital, Beijing 100039, China. (30) First School of Clinical Medicine, Peking University, Beijing 100871, China; State Key Laboratory of Complex, Severe and Rare Diseases, Clinical Research Institute, Peking Union Medical College Hospital, Chinese Academy of Medical Science & Peking Union Medical College, Beijing 100730, China; Tsinghua-Peking University Joint Center for Life Sciences, Tsinghua University, Beijing 100084, China. (31) EdiGene Inc., Life Science Park, Changping District, Beijing 102206, China. (32) EdiGene Inc., Life Science Park, Changping District, Beijing 102206, China. Electronic address: pfyuan@edigene.com. (33) Changping Laboratory, Beijing 102206, China; School of Medicine, Nankai University, Tianjin 300071, China; Department of Bio-Therapeutic, the First Medical Center, Chinese PLA General Hospital, Beijing 100039, China. Electronic address: hanwdrsw@163.com. (34) Biomedical Pioneering Innovation Center, Peking-Tsinghua Center for Life Sciences, Peking University Genome Editing Research Center, State Key Laboratory of Gene Function and Modulation Research, School of Life Sciences, Peking University, Beijing 100871, China; Changping Laboratory, Beijing 102206, China. Electronic address: wswei@pku.edu.cn.

Clinical and molecular dissection of CAR T cell resistance in pancreatic cancer Featured  

Aznar and Good et al. reported on a Phase I study assessing a mesothelin-targeting CAR T cell product (huCART-meso) in patients with advanced PDAC. Treatment was feasible and safe, but lacked efficacy. Biopsy and ascites analysis showed limited persistence of the CAR-T and remaining CAR-T upregulated transcription factors SOX4 and ID3, related to dysfunction. Murine studies showed limited effects of ID3KO in CART, while SOX4KO improved antitumor efficacy, but tumors relapsed. Double KO of ID3 and SOX4 in the CAR-T prevented relapses and improved relapse-free survival.

Aznar and Good et al. reported on a Phase I study assessing a mesothelin-targeting CAR T cell product (huCART-meso) in patients with advanced PDAC. Treatment was feasible and safe, but lacked efficacy. Biopsy and ascites analysis showed limited persistence of the CAR-T and remaining CAR-T upregulated transcription factors SOX4 and ID3, related to dysfunction. Murine studies showed limited effects of ID3KO in CART, while SOX4KO improved antitumor efficacy, but tumors relapsed. Double KO of ID3 and SOX4 in the CAR-T prevented relapses and improved relapse-free survival.

ABSTRACT: Patients with advanced pancreatic ductal adenocarcinoma (PDAC) have a median survival of less than a year, highlighting the urgent need for treatment advancements. We report on a phase 1 clinical trial assessing the safety and feasibility of intravenous and local administration of anti-mesothelin CAR T cells in patients with advanced PDAC. While therapy is well tolerated, it demonstrates limited clinical efficacy. Analyses of patient samples provide insights into mechanisms of treatment resistance. Single-cell genomic approaches reveal that post-infusion CAR T cells express exhaustion signatures, including previously identified transcription factors ID3 and SOX4, and display enrichment for a GZMK(+) phenotype. Single knockout of ID3 or SOX4 enhances efficacy in xenograft models, though with donor-dependent variability. However, single-knockout cells eventually fail. Conversely, ID3 and SOX4 double-knockout CAR T cells exhibit prolonged relapse-free survival, demonstrating a sustained therapeutic effect and a potential avenue for engineering more potent CAR T cells in PDAC. This study was registered at ClinicalTrials.gov (NCT03323944).

Author Info: (1) Center for Cellular Immunotherapies, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA 19104, USA; Department of Pathology and Laboratory Medicine, Unive

Author Info: (1) Center for Cellular Immunotherapies, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA 19104, USA; Department of Pathology and Laboratory Medicine, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA 19104, USA. (2) Department of Cell and Developmental Biology, Penn Epigenetics Institute, Perelman School of Medicine, Philadelphia, PA 19104, USA. (3) Center for Cellular Immunotherapies, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA 19104, USA. (4) Center for Cellular Immunotherapies, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA 19104, USA. (5) Center for Cellular Immunotherapies, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA 19104, USA. (6) Department of Biostatistics, Epidemiology and Informatics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA. (7) Department of Cell and Developmental Biology, Penn Epigenetics Institute, Perelman School of Medicine, Philadelphia, PA 19104, USA. (8) Center for Cellular Immunotherapies, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA 19104, USA. (9) Department of Cell and Developmental Biology, Penn Epigenetics Institute, Perelman School of Medicine, Philadelphia, PA 19104, USA. (10) Department of Biostatistics, Epidemiology and Informatics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA. (11) Center for Cellular Immunotherapies, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA 19104, USA. (12) Center for Cellular Immunotherapies, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA 19104, USA. (13) Center for Cellular Immunotherapies, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA 19104, USA. (14) Department of Cell and Developmental Biology, Penn Epigenetics Institute, Perelman School of Medicine, Philadelphia, PA 19104, USA; Department of Systems Pharmacology and Translational Therapeutics, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA 19104, USA; Institute for Immunology and Immune Health, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA 19104, USA. (15) Department of Cell and Developmental Biology, Penn Epigenetics Institute, Perelman School of Medicine, Philadelphia, PA 19104, USA. (16) Department of Cell and Developmental Biology, Penn Epigenetics Institute, Perelman School of Medicine, Philadelphia, PA 19104, USA. (17) Center for Cellular Immunotherapies, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA 19104, USA. (18) Center for Cellular Immunotherapies, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA 19104, USA. (19) Center for Cellular Immunotherapies, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA 19104, USA. (20) Center for Cellular Immunotherapies, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA 19104, USA; Department of Pathology and Laboratory Medicine, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA 19104, USA. (21) Abramson Cancer Center, University of Pennsylvania, Philadelphia, PA 19104, USA; Ovarian Cancer Research Center, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA. (22) Abramson Cancer Center, University of Pennsylvania, Philadelphia, PA 19104, USA; Division of Pulmonary, Allergy, and Critical Care, Department of Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA. (23) Abramson Cancer Center, University of Pennsylvania, Philadelphia, PA 19104, USA; Department of Radiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA. (24) Abramson Cancer Center, University of Pennsylvania, Philadelphia, PA 19104, USA; Department of Radiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA. (25) Center for Cellular Immunotherapies, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA 19104, USA. (26) Center for Cellular Immunotherapies, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA 19104, USA; Division of Hematology/Oncology, Department of Medicine, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA 19104, USA. (27) Center for Cellular Immunotherapies, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA 19104, USA; Department of Pathology and Laboratory Medicine, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA 19104, USA; Abramson Cancer Center, University of Pennsylvania, Philadelphia, PA 19104, USA; Department of Microbiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA. (28) Center for Cellular Immunotherapies, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA 19104, USA. (29) Center for Cellular Immunotherapies, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA 19104, USA; Department of Pathology and Laboratory Medicine, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA 19104, USA; Parker Institute for Cancer Immunotherapy, University of Pennsylvania, Philadelphia, PA 19104, USA. Electronic address: ryoung@upenn.edu. (30) Department of Cell and Developmental Biology, Penn Epigenetics Institute, Perelman School of Medicine, Philadelphia, PA 19104, USA. Electronic address: bergers@pennmedicine.upenn.edu. (31) Center for Cellular Immunotherapies, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA 19104, USA; Department of Pathology and Laboratory Medicine, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA 19104, USA; Parker Institute for Cancer Immunotherapy, University of Pennsylvania, Philadelphia, PA 19104, USA. Electronic address: cjune@upenn.edu. (32) Abramson Cancer Center, University of Pennsylvania, Philadelphia, PA 19104, USA; Department of Medicine, Division of Hematology and Oncology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA. Electronic address: mark.ohara@pennmedicine.upenn.edu.

Genome-wide CRISPR screens identify critical targets to enhance CAR-NK cell antitumor potency Spotlight 

Biederstadt and Basar et al. developed a genome-wide CRISPR screen platform for primary human NK cells, and identified MED12, ARIH2, and CCNC as critical regulators of NK cell function under repeated tumor challenge and immunosuppressive pressure. Deletion of these genes enhanced NK cell metabolic fitness, proinflammatory cytokine secretion, and expansion of both innate and CAR-NK cells, and improved antitumor potency against multiple treatment-refractory human cancers xenografts. Dual ARIH2/CCNC editing augmented CAR-NK cell proliferation, activation, and inflammatory signaling, leading to enhanced tumor clearance.

Contributed by Shishir Pant

Biederstadt and Basar et al. developed a genome-wide CRISPR screen platform for primary human NK cells, and identified MED12, ARIH2, and CCNC as critical regulators of NK cell function under repeated tumor challenge and immunosuppressive pressure. Deletion of these genes enhanced NK cell metabolic fitness, proinflammatory cytokine secretion, and expansion of both innate and CAR-NK cells, and improved antitumor potency against multiple treatment-refractory human cancers xenografts. Dual ARIH2/CCNC editing augmented CAR-NK cell proliferation, activation, and inflammatory signaling, leading to enhanced tumor clearance.

Contributed by Shishir Pant

ABSTRACT: Adoptive cell therapy using engineered natural killer (NK) cells is a promising approach for cancer treatment, with targeted gene editing offering the potential to further enhance their therapeutic efficacy. However, the spectrum of actionable genetic targets to overcome tumor and microenvironment-mediated immunosuppression remains largely unexplored. We performed multiple genome-wide CRISPR screens in primary human NK cells and identified critical checkpoints regulating resistance to immunosuppressive pressures. Ablation of MED12, ARIH2, and CCNC significantly improved NK cell antitumor activity against multiple treatment-refractory human cancers in vitro and in vivo. CRISPR editing augmented both innate and CAR-mediated NK cell function, associated with enhanced metabolic fitness, increased secretion of proinflammatory cytokines, and expansion of cytotoxic NK cell subsets. Through high-content genome-wide CRISPR screening in NK cells, this study reveals critical regulators of NK cell function and provides a valuable resource for engineering next-generation NK cell therapies with improved efficacy against cancer.

Author Info: (1) Department of Stem Cell Transplantation and Cellular Therapy, The University of Texas MD Anderson Cancer Center, Houston, TX, USA; Institute for Cell Therapy Discovery and Inno

Author Info: (1) Department of Stem Cell Transplantation and Cellular Therapy, The University of Texas MD Anderson Cancer Center, Houston, TX, USA; Institute for Cell Therapy Discovery and Innovation, The University of Texas MD Anderson Cancer Center, Houston, TX, USA; Department of Medicine III: Hematology & Oncology, School of Medicine, Technical University of Munich, Munich, Germany. (2) Department of Stem Cell Transplantation and Cellular Therapy, The University of Texas MD Anderson Cancer Center, Houston, TX, USA; Institute for Cell Therapy Discovery and Innovation, The University of Texas MD Anderson Cancer Center, Houston, TX, USA. (3) Department of Stem Cell Transplantation and Cellular Therapy, The University of Texas MD Anderson Cancer Center, Houston, TX, USA; Institute for Cell Therapy Discovery and Innovation, The University of Texas MD Anderson Cancer Center, Houston, TX, USA. (4) Department of Stem Cell Transplantation and Cellular Therapy, The University of Texas MD Anderson Cancer Center, Houston, TX, USA; Institute for Cell Therapy Discovery and Innovation, The University of Texas MD Anderson Cancer Center, Houston, TX, USA. (5) Department of Stem Cell Transplantation and Cellular Therapy, The University of Texas MD Anderson Cancer Center, Houston, TX, USA; Institute for Cell Therapy Discovery and Innovation, The University of Texas MD Anderson Cancer Center, Houston, TX, USA. (6) Department of Stem Cell Transplantation and Cellular Therapy, The University of Texas MD Anderson Cancer Center, Houston, TX, USA; Institute for Cell Therapy Discovery and Innovation, The University of Texas MD Anderson Cancer Center, Houston, TX, USA. (7) Department of Bioinformatics and Computational Biology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA. (8) Department of Stem Cell Transplantation and Cellular Therapy, The University of Texas MD Anderson Cancer Center, Houston, TX, USA; Institute for Cell Therapy Discovery and Innovation, The University of Texas MD Anderson Cancer Center, Houston, TX, USA. (9) Department of Stem Cell Transplantation and Cellular Therapy, The University of Texas MD Anderson Cancer Center, Houston, TX, USA; Institute for Cell Therapy Discovery and Innovation, The University of Texas MD Anderson Cancer Center, Houston, TX, USA. (10) Department of Stem Cell Transplantation and Cellular Therapy, The University of Texas MD Anderson Cancer Center, Houston, TX, USA; Institute for Cell Therapy Discovery and Innovation, The University of Texas MD Anderson Cancer Center, Houston, TX, USA. (11) Department of Stem Cell Transplantation and Cellular Therapy, The University of Texas MD Anderson Cancer Center, Houston, TX, USA; Institute for Cell Therapy Discovery and Innovation, The University of Texas MD Anderson Cancer Center, Houston, TX, USA. (12) Department of Stem Cell Transplantation and Cellular Therapy, The University of Texas MD Anderson Cancer Center, Houston, TX, USA; Institute for Cell Therapy Discovery and Innovation, The University of Texas MD Anderson Cancer Center, Houston, TX, USA. (13) Department of Stem Cell Transplantation and Cellular Therapy, The University of Texas MD Anderson Cancer Center, Houston, TX, USA; Institute for Cell Therapy Discovery and Innovation, The University of Texas MD Anderson Cancer Center, Houston, TX, USA. (14) Department of Stem Cell Transplantation and Cellular Therapy, The University of Texas MD Anderson Cancer Center, Houston, TX, USA; Institute for Cell Therapy Discovery and Innovation, The University of Texas MD Anderson Cancer Center, Houston, TX, USA. (15) Department of Stem Cell Transplantation and Cellular Therapy, The University of Texas MD Anderson Cancer Center, Houston, TX, USA; Institute for Cell Therapy Discovery and Innovation, The University of Texas MD Anderson Cancer Center, Houston, TX, USA. (16) Department of Stem Cell Transplantation and Cellular Therapy, The University of Texas MD Anderson Cancer Center, Houston, TX, USA; Institute for Cell Therapy Discovery and Innovation, The University of Texas MD Anderson Cancer Center, Houston, TX, USA. (17) Department of Neurology, the University of Texas McGovern Medical School at Houston, Houston, TX, USA. (18) Department of Stem Cell Transplantation and Cellular Therapy, The University of Texas MD Anderson Cancer Center, Houston, TX, USA; Institute for Cell Therapy Discovery and Innovation, The University of Texas MD Anderson Cancer Center, Houston, TX, USA. (19) Department of Stem Cell Transplantation and Cellular Therapy, The University of Texas MD Anderson Cancer Center, Houston, TX, USA; Institute for Cell Therapy Discovery and Innovation, The University of Texas MD Anderson Cancer Center, Houston, TX, USA. (20) Department of Stem Cell Transplantation and Cellular Therapy, The University of Texas MD Anderson Cancer Center, Houston, TX, USA; Institute for Cell Therapy Discovery and Innovation, The University of Texas MD Anderson Cancer Center, Houston, TX, USA. (21) Department of Stem Cell Transplantation and Cellular Therapy, The University of Texas MD Anderson Cancer Center, Houston, TX, USA; Institute for Cell Therapy Discovery and Innovation, The University of Texas MD Anderson Cancer Center, Houston, TX, USA. (22) Department of Stem Cell Transplantation and Cellular Therapy, The University of Texas MD Anderson Cancer Center, Houston, TX, USA; Institute for Cell Therapy Discovery and Innovation, The University of Texas MD Anderson Cancer Center, Houston, TX, USA. (23) Department of Stem Cell Transplantation and Cellular Therapy, The University of Texas MD Anderson Cancer Center, Houston, TX, USA; Institute for Cell Therapy Discovery and Innovation, The University of Texas MD Anderson Cancer Center, Houston, TX, USA. (24) Department of Stem Cell Transplantation and Cellular Therapy, The University of Texas MD Anderson Cancer Center, Houston, TX, USA; Institute for Cell Therapy Discovery and Innovation, The University of Texas MD Anderson Cancer Center, Houston, TX, USA. (25) Department of Stem Cell Transplantation and Cellular Therapy, The University of Texas MD Anderson Cancer Center, Houston, TX, USA; Institute for Cell Therapy Discovery and Innovation, The University of Texas MD Anderson Cancer Center, Houston, TX, USA; The University of Texas MD Anderson Cancer Center UTHealth Houston Graduate School of Biomedical Sciences, Houston, TX, USA. (26) Department of Stem Cell Transplantation and Cellular Therapy, The University of Texas MD Anderson Cancer Center, Houston, TX, USA; Institute for Cell Therapy Discovery and Innovation, The University of Texas MD Anderson Cancer Center, Houston, TX, USA. (27) Department of Stem Cell Transplantation and Cellular Therapy, The University of Texas MD Anderson Cancer Center, Houston, TX, USA; Institute for Cell Therapy Discovery and Innovation, The University of Texas MD Anderson Cancer Center, Houston, TX, USA. (28) Department of Stem Cell Transplantation and Cellular Therapy, The University of Texas MD Anderson Cancer Center, Houston, TX, USA; Institute for Cell Therapy Discovery and Innovation, The University of Texas MD Anderson Cancer Center, Houston, TX, USA. (29) Department of Stem Cell Transplantation and Cellular Therapy, The University of Texas MD Anderson Cancer Center, Houston, TX, USA; Institute for Cell Therapy Discovery and Innovation, The University of Texas MD Anderson Cancer Center, Houston, TX, USA. (30) Department of Stem Cell Transplantation and Cellular Therapy, The University of Texas MD Anderson Cancer Center, Houston, TX, USA; Institute for Cell Therapy Discovery and Innovation, The University of Texas MD Anderson Cancer Center, Houston, TX, USA. (31) Department of Stem Cell Transplantation and Cellular Therapy, The University of Texas MD Anderson Cancer Center, Houston, TX, USA; Institute for Cell Therapy Discovery and Innovation, The University of Texas MD Anderson Cancer Center, Houston, TX, USA. (32) Department of Stem Cell Transplantation and Cellular Therapy, The University of Texas MD Anderson Cancer Center, Houston, TX, USA; Institute for Cell Therapy Discovery and Innovation, The University of Texas MD Anderson Cancer Center, Houston, TX, USA. (33) Department of Stem Cell Transplantation and Cellular Therapy, The University of Texas MD Anderson Cancer Center, Houston, TX, USA. (34) Department of Stem Cell Transplantation and Cellular Therapy, The University of Texas MD Anderson Cancer Center, Houston, TX, USA; Institute for Cell Therapy Discovery and Innovation, The University of Texas MD Anderson Cancer Center, Houston, TX, USA; Department of Veterinary Medicine & Surgery, The University of Texas MD Anderson Cancer Center, Houston, TX, USA. (35) Department of Bioinformatics and Computational Biology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA. (36) Department of Stem Cell Transplantation and Cellular Therapy, The University of Texas MD Anderson Cancer Center, Houston, TX, USA; Institute for Cell Therapy Discovery and Innovation, The University of Texas MD Anderson Cancer Center, Houston, TX, USA. Electronic address: krezvani@mdanderson.org.

Engineering affinity-matured variants of an anti-polysialic acid monoclonal antibody with superior cytotoxicity-mediating potency Spotlight 

Wang et al. focused on improving the potency and specificity of mAbs targeting cancer- and infection-associated carbohydrates. Using structure-based rational design and directed evolution, variants of mAb735 – a modest-affinity, polysialic acid (polySia)-specific antibody – were generated (scFv and IgG formats) with significantly increased affinity (4- to 7-fold) compared to parental mAb735. Affinity-matured mAb735 IgG variants bound more avidly to polySia-positive tumor cell lines, and demonstrated increased functional potency and tumor cell killing, including ADCC and CDC, providing a framework for enhancing the promise of anti-glycan Abs.

Contributed by Katherine Turner

Wang et al. focused on improving the potency and specificity of mAbs targeting cancer- and infection-associated carbohydrates. Using structure-based rational design and directed evolution, variants of mAb735 – a modest-affinity, polysialic acid (polySia)-specific antibody – were generated (scFv and IgG formats) with significantly increased affinity (4- to 7-fold) compared to parental mAb735. Affinity-matured mAb735 IgG variants bound more avidly to polySia-positive tumor cell lines, and demonstrated increased functional potency and tumor cell killing, including ADCC and CDC, providing a framework for enhancing the promise of anti-glycan Abs.

Contributed by Katherine Turner

ABSTRACT: Monoclonal antibodies (mAbs) that specifically recognize cell surface glycans associated with cancer and infectious disease hold tremendous value for basic research and clinical applications. However, high-quality anti-glycan mAbs with sufficiently high affinity and specificity remain scarce, highlighting the need for strategies that enable optimization of antigen-binding properties. To this end, we engineered the affinity of a polysialic acid (polySia)-specific antibody called mAb735, which possesses only modest affinity. Using a combination of rational design and directed evolution, we isolated several affinity-matured IgG variants with _5- to 7-fold stronger affinity for polySia relative to mAb735. The higher affinity IgG variants opsonized polySia-positive cancer cells more avidly and triggered greater antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). Collectively, these results demonstrate the effective application of molecular evolution techniques to an important anti-glycan antibody, providing insights into its carbohydrate recognition and uncovering variants with greater therapeutic promise due to their enhanced affinity and potency.

Author Info: (1) Robert F. Smith School of Chemical and Biomolecular Engineering, Cornell University, Olin Hall, Ithaca, NY 14853, USA. (2) Robert F. Smith School of Chemical and Biomolecular E

Author Info: (1) Robert F. Smith School of Chemical and Biomolecular Engineering, Cornell University, Olin Hall, Ithaca, NY 14853, USA. (2) Robert F. Smith School of Chemical and Biomolecular Engineering, Cornell University, Olin Hall, Ithaca, NY 14853, USA. (3) Nancy E. and Peter C. Meinig School of Biomedical Engineering School of Biomedical Engineering, Cornell University, Olin Hall, Ithaca, NY 14853, USA. (4) Robert F. Smith School of Chemical and Biomolecular Engineering, Cornell University, Olin Hall, Ithaca, NY 14853, USA. (5) Robert F. Smith School of Chemical and Biomolecular Engineering, Cornell University, Olin Hall, Ithaca, NY 14853, USA; Nancy E. and Peter C. Meinig School of Biomedical Engineering School of Biomedical Engineering, Cornell University, Olin Hall, Ithaca, NY 14853, USA; Cornell Institute of Biotechnology, Cornell University, 130 Biotechnology Building, Ithaca, NY 14853, USA. Electronic address: md255@cornell.edu.

Matrix-M adjuvant triggers inflammasome activation and enables antigen cross-presentation through induction of lysosomal membrane permeabilization Spotlight 

Zarnegar and Carow et al. showed that the bark-derived, saponin-based adjuvant Matrix-M® (M®; used in COVID-19 and malaria vaccines) – comprising Matrix-A (A) and, to a lesser extent, Matrix-C (C) nanoparticles – colocalized with antigens in lysosomes following uptake by BM-derived DCs. In vitro, M®, A, and most strongly C induced lysosomal membrane permeabilization (LMP), which is required for IL-1β and IL-18 secretion, and A most strongly matured APCs. In vivo, M®, A, and C exhibited robust adjuvant effects in the presence and absence of the NLRP3 inflammasome. LMP induced by M®, A, and most strongly C enabled antigen cross-presentation to induce CD8+ T cell responses.

Contributed by Paula Hochman

Zarnegar and Carow et al. showed that the bark-derived, saponin-based adjuvant Matrix-M® (M®; used in COVID-19 and malaria vaccines) – comprising Matrix-A (A) and, to a lesser extent, Matrix-C (C) nanoparticles – colocalized with antigens in lysosomes following uptake by BM-derived DCs. In vitro, M®, A, and most strongly C induced lysosomal membrane permeabilization (LMP), which is required for IL-1β and IL-18 secretion, and A most strongly matured APCs. In vivo, M®, A, and C exhibited robust adjuvant effects in the presence and absence of the NLRP3 inflammasome. LMP induced by M®, A, and most strongly C enabled antigen cross-presentation to induce CD8+ T cell responses.

Contributed by Paula Hochman

ABSTRACT: Matrix-M® adjuvant, containing saponins, delivers a potent adjuvant effect and good safety profile. Given that Matrix-M is composed of Matrix-A and Matrix-C particles, comprising different saponin fractions, understanding their distinct roles can provide deeper insight into the mechanism of action of Matrix-M and guide future applications. Here, we demonstrate that the antigen and Matrix-M, Matrix-A, or Matrix-C colocalize in lysosomes following uptake by bone marrow-derived dendritic cells. Matrix-M, Matrix-A, and Matrix-C induce lysosomal membrane permeabilization (LMP), but Matrix-C shows the highest LMP potential. LMP is required for interleukin (IL)-1β and IL-18 secretion in vitro. In vivo, a robust adjuvant effect of Matrix-M, Matrix-A, and Matrix-C is observed, both in the presence and absence of the NLRP3 inflammasome. LMP induced by Matrix-M, as well as Matrix-A and Matrix-C, also enables antigen cross-presentation. Thus, Matrix-induced LMP explains the capability of Matrix-M-adjuvanted protein vaccines to induce CD8+ T-cell responses.

Author Info: (1) Novavax AB, Uppsala, Sweden. (2) Novavax AB, Uppsala, Sweden. bcarow@novavax.com. (3) Department of Medical Biochemistry and Microbiology, Uppsala University, Uppsala, Sweden.

Author Info: (1) Novavax AB, Uppsala, Sweden. (2) Novavax AB, Uppsala, Sweden. bcarow@novavax.com. (3) Department of Medical Biochemistry and Microbiology, Uppsala University, Uppsala, Sweden. (4) Novavax AB, Uppsala, Sweden. (5) Novavax AB, Uppsala, Sweden. (6) Novavax AB, Uppsala, Sweden. (7) Novavax AB, Uppsala, Sweden. (8) Novavax AB, Uppsala, Sweden. (9) Novavax AB, Uppsala, Sweden. (10) Novavax AB, Uppsala, Sweden. (11) Novavax AB, Uppsala, Sweden. (12) Novavax AB, Uppsala, Sweden. (13) Novavax AB, Uppsala, Sweden. (14) Novavax AB, Uppsala, Sweden. (15) Department of Medical Biochemistry and Microbiology, Uppsala University, Uppsala, Sweden. Science for Life Laboratory, Uppsala, Sweden. (16) Novavax AB, Uppsala, Sweden. (17) Novavax AB, Uppsala, Sweden. (18) Novavax AB, Uppsala, Sweden.

Expanding the cytokine receptor alphabet reprograms T cells into diverse states Spotlight 

Zhao et al. engineered a chimera of an IL-2 receptor domain linked to the transmembrane and intracellular domains of diverse, orthogonal (o) cytokine receptors to explore novel JAK/STAT outputs in T cells. Many o-receptors enhanced tumor control, despite divergent pSTAT/gene expression patterns. oIL-22R promoted a Tscm phenotype in vivo, which correlated with chromatin alterations, including enrichment of BACH2 (stemness)- and AP1 (cytotoxicity)-bound motifs. oIL4R induced Th2/Tc2 differentiation, with maintenance of Th1 cytokines. oGCSFR promoted myeloid gene expression and endowed phagocytic capacity, without compromising T cell identity.

Contributed by Morgan Janes

Zhao et al. engineered a chimera of an IL-2 receptor domain linked to the transmembrane and intracellular domains of diverse, orthogonal (o) cytokine receptors to explore novel JAK/STAT outputs in T cells. Many o-receptors enhanced tumor control, despite divergent pSTAT/gene expression patterns. oIL-22R promoted a Tscm phenotype in vivo, which correlated with chromatin alterations, including enrichment of BACH2 (stemness)- and AP1 (cytotoxicity)-bound motifs. oIL4R induced Th2/Tc2 differentiation, with maintenance of Th1 cytokines. oGCSFR promoted myeloid gene expression and endowed phagocytic capacity, without compromising T cell identity.

Contributed by Morgan Janes

ABSTRACT: T cells respond to cytokines through receptor dimers that have been selected over the course of evolution to activate canonical JAK-STAT signalling and gene expression programs1. However, the potential combinatorial diversity of JAK-STAT receptor pairings can be expanded by exploring the untapped biology of alternative non-natural pairings. Here we exploited the common γ chain (γc) receptor as a shared signalling hub on T cells and enforced the expression of both natural and non-natural heterodimeric JAK-STAT receptor pairings using an orthogonal cytokine receptor platform2-4 to expand the γc signalling code. We tested receptors from γc cytokines as well as interferon, IL-10 and homodimeric receptor families that do not normally pair with γc or are not naturally expressed on T cells. These receptors simulated their natural counterparts but also induced contextually unique transcriptional programs. This led to distinct T cell fates in tumours, including myeloid-like T cells with phagocytic capacity driven by orthogonal GSCFR (oGCSFR), and type 2 cytotoxic T (TC2) and helper T (TH2) cell differentiation driven by orthogonal IL-4R (o4R). T cells with orthogonal IL-22R (o22R) and oGCSFR, neither of which are natively expressed on T cells, exhibited stem-like and exhaustion-resistant transcriptional and chromatin landscapes, enhancing anti-tumour properties. Non-native receptor pairings and their resultant JAK-STAT signals open a path to diversifying T cell states beyond those induced by natural cytokines.

Author Info: (1) Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, CA, USA. (2) Department of Molecular and Cellular Physiology, Stanford Univer

Author Info: (1) Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, CA, USA. (2) Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, CA, USA. (3) Department of Radiation Oncology, Stanford University School of Medicine, Stanford, CA, USA. (4) Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, CA, USA. (5) Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, CA, USA. (6) Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, CA, USA. (7) Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, CA, USA. (8) Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, CA, USA. (9) Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, CA, USA. (10) Department of Radiation Oncology, Stanford University School of Medicine, Stanford, CA, USA. (11) Department of Radiation Oncology, Stanford University School of Medicine, Stanford, CA, USA. (12) Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, CA, USA. (13) Department of Radiation Oncology, Stanford University School of Medicine, Stanford, CA, USA. akalbasi@stanford.edu. Stanford Cancer Institute, Stanford University School of Medicine, Stanford, CA, USA. akalbasi@stanford.edu. (14) Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, CA, USA. kcgarcia@stanford.edu. Howard Hughes Medical Institute, Stanford University School of Medicine, Stanford, CA, USA. kcgarcia@stanford.edu. Department of Structural Biology, Stanford University School of Medicine, Stanford, CA, USA. kcgarcia@stanford.edu.

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