Journal Articles

In vivo engineering of CAR natural killer cells for immunotherapy using ligand-directed lipid nanoparticles

Chen, Monkovic, and Mughal et al. used NK cell receptor-specific scFvs to generate NK cell-targeted LNPs (NtLNPs) that transfected up to 80% of circulating and tissue NK cells, with minimal off-target effects. NtLNP delivery of CAR mRNAs generated NK cells that specifically depleted B cells in healthy mice, restored AST/ALT to baseline levels in an autoimmune hepatitis model, and cleared antigen+ melanoma cells in a solid tumor model. NtLNPs targeting NKG2a boosted efficacy in solid tumor models by mitigating Qa-1b mediated immunosuppression. In vivo and ex vivo, NtLNP-generated human CAR NK cells induced regression in humanized lymphoma and T cell leukemia mouse models.

Contributed by Paula Hochman

Chen, Monkovic, and Mughal et al. used NK cell receptor-specific scFvs to generate NK cell-targeted LNPs (NtLNPs) that transfected up to 80% of circulating and tissue NK cells, with minimal off-target effects. NtLNP delivery of CAR mRNAs generated NK cells that specifically depleted B cells in healthy mice, restored AST/ALT to baseline levels in an autoimmune hepatitis model, and cleared antigen+ melanoma cells in a solid tumor model. NtLNPs targeting NKG2a boosted efficacy in solid tumor models by mitigating Qa-1b mediated immunosuppression. In vivo and ex vivo, NtLNP-generated human CAR NK cells induced regression in humanized lymphoma and T cell leukemia mouse models.

Contributed by Paula Hochman

ABSTRACT: Targeted lipid nanoparticles (LNPs) offer a nonviral approach for in vivo cell engineering but efficient programming of natural killer (NK) cells remains underexplored. Here we develop NK cell-targeted LNPs (NtLNPs) using engineered biparatopic NK cell ligands to drive receptor-specific uptake and mRNA delivery. Lead NtLNPs achieve up to ~80% NK cell transfection with minimal off-target effects in mice. Systemic delivery of chimeric antigen receptor (CAR) mRNA using NtLNPs generates CAR NK cells in vivo. CD19-targeted CAR NKs completely deplete circulating B cells in healthy mice and CD3-targeted CAR NKs eliminate nearly all autoreactive T cells in an autoimmune hepatitis model, highlighting a potential advantage over CAR T therapies for T cell-driven disease. In solid tumors, NtLNPs targeting the inhibitory checkpoint NKG2A enhance antitumor efficacy by relieving Qa-1(b)-mediated immunosuppression. Lastly, we optimized NtLNPs for in vivo engineering of human CAR NK cells, enabling regression of two humanized hematologic malignancy models and establishing NtLNPs as a modular immunotherapy system.

Author Info: (1) David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA, USA. Department of Chemical Engineering, Massachusetts Institute

Author Info: (1) David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA, USA. Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, MA, USA. (2) David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA, USA. Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, MA, USA. (3) David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA, USA. Department of Chemistry, Massachusetts Institute of Technology, Cambridge, MA, USA. (4) David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA, USA. Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, MA, USA. (5) David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA, USA. Harvard and MIT Division of Health Sciences and Technology, Massachusetts Institute of Technology, Cambridge, MA, USA. (6) David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA, USA. Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, MA, USA. Department of Anesthesiology, Critical Care and Pain Medicine, Boston Children's Hospital, Boston, MA, USA. (7) David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA, USA. Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, MA, USA. (8) David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA, USA. Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, MA, USA. (9) David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA, USA. Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, MA, USA. (10) David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA, USA. Department of Chemistry, Massachusetts Institute of Technology, Cambridge, MA, USA. (11) David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA, USA. Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, MA, USA. (12) David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA, USA. Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA, USA. (13) David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA, USA. Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA, USA. (14) David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA, USA. (15) David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA, USA. Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, MA, USA. Department of Chemistry, Massachusetts Institute of Technology, Cambridge, MA, USA. Harvard and MIT Division of Health Sciences and Technology, Massachusetts Institute of Technology, Cambridge, MA, USA. Department of Anesthesiology, Critical Care and Pain Medicine, Boston Children's Hospital, Boston, MA, USA. Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA, USA. Institute for Medical Engineering and Science, Massachusetts Institute of Technology, Cambridge, MA, USA. (16) David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA, USA. dgander@mit.edu. Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, MA, USA. dgander@mit.edu. Harvard and MIT Division of Health Sciences and Technology, Massachusetts Institute of Technology, Cambridge, MA, USA. dgander@mit.edu. Department of Anesthesiology, Critical Care and Pain Medicine, Boston Children's Hospital, Boston, MA, USA. dgander@mit.edu. Institute for Medical Engineering and Science, Massachusetts Institute of Technology, Cambridge, MA, USA. dgander@mit.edu.

Cullin-5 RING E3 ligase limits CD8⁺ T-cell persistence and effector responses during chronic antigen stimulation

Two recent back-to-back publications by Bachl et al. and Saco et al. used CRISPR-Cas9 knockout screens in primary human T cells exposed to repetitive antigen challenge and uncovered the enrichment of Cullin-RING Ligase 5 (CRL5) complex members over time with chronic antigen exposure. Knockouts of CRL5 components resulted in maintenance of antitumor effector function and delayed dysfunction. KO of these components in CAR-T or TCR-T cells improved tumor control in mice.

Two recent back-to-back publications by Bachl et al. and Saco et al. used CRISPR-Cas9 knockout screens in primary human T cells exposed to repetitive antigen challenge and uncovered the enrichment of Cullin-RING Ligase 5 (CRL5) complex members over time with chronic antigen exposure. Knockouts of CRL5 components resulted in maintenance of antitumor effector function and delayed dysfunction. KO of these components in CAR-T or TCR-T cells improved tumor control in mice.

ABSTRACT: T-cell exhaustion limits durable antitumor responses and remains a barrier to adoptive cell therapies (ACT) in solid tumors. To identify regulators of T-cell persistence, we performed genome-wide CRISPR knockout screens in primary human CD8_ T cells expressing an NY-ESO-1-specific T-cell receptor and subjected them to repeated tumor cell rechallenge. These screens revealed enrichment of single-guide RNAs targeting components of Cullin-RING E3 ligases, particularly Cullin-5 (CRL5) assemblies comprising CUL5, RNF7, and ARIH2. Disruption of CRL5 genes in T cells preserved cytotoxic activity and enhanced cytokine production while sustaining effector-associated transcriptional programs and suppressing exhaustion-related signatures. Pharmacologic inhibition of cullin neddylation with the NEDD8-activating enzyme inhibitor MLN4924 recapitulated CRL5 loss and enhanced T-cell effector function in vitro. In a human melanoma xenograft model, ACT with CRL5-deficient T cells improved tumor control. Together, these findings identify CRL5 as a negative regulator of T-cell fitness and a potential therapeutic target to enhance ACT durability in solid tumors.

Author Info: (1) University of California, Los Angeles Los Angeles, CA United States. ROR: https://ror.org/046rm7j60 (2) University of California, Los Angeles LA, CA United States. ROR: https:/

Author Info: (1) University of California, Los Angeles Los Angeles, CA United States. ROR: https://ror.org/046rm7j60 (2) University of California, Los Angeles LA, CA United States. ROR: https://ror.org/046rm7j60 (3) University of California, Los Angeles Los Angeles, CA United States. ROR: https://ror.org/046rm7j60 (4) Gladstone Institutes San Francisco, California United States. ROR: https://ror.org/038321296 (5) Gladstone Institutes San Francisco, California United States. ROR: https://ror.org/038321296 (6) University of California, San Francisco San Francisco, CA United States. ROR: https://ror.org/043mz5j54 (7) University of California, Los Angeles Los Angeles, CA United States. ROR: https://ror.org/046rm7j60 (8) University of California, Los Angeles Los Angeles, CA United States. ROR: https://ror.org/046rm7j60 (9) University of California, Los Angeles Los Angeles, CA United States. ROR: https://ror.org/046rm7j60 (10) University of California, Los Angeles Los Angeles, CA United States. ROR: https://ror.org/046rm7j60 (11) Dana-Farber Cancer Institute Boston United States. ROR: https://ror.org/02jzgtq86 (12) Max Planck Institute of Biochemistry Martinsried Germany. ROR: https://ror.org/04py35477 (13) University of California, Los Angeles Los Angeles, CA United States. ROR: https://ror.org/046rm7j60 (14) University of California, San Francisco San Francisco, CA United States. ROR: https://ror.org/043mz5j54 (15) University of California, Los Angeles Los Angeles, CA United States. ROR: https://ror.org/046rm7j60 (16) University of California, Los Angeles Los Angeles, CA United States. ROR: https://ror.org/046rm7j60

Disruption of the CRL5 ubiquitin ligase complex enhances engineered T-cell persistence under chronic tumor antigen exposure

Two recent back-to-back publications by Bachl et al. and Saco et al. used CRISPR-Cas9 knockout screens in primary human T cells exposed to repetitive antigen challenge and uncovered the enrichment of Cullin-RING Ligase 5 (CRL5) complex members over time with chronic antigen exposure. Knockouts of CRL5 components resulted in maintenance of antitumor effector function and delayed dysfunction. KO of these components in CAR-T or TCR-T cells improved tumor control in mice.

Two recent back-to-back publications by Bachl et al. and Saco et al. used CRISPR-Cas9 knockout screens in primary human T cells exposed to repetitive antigen challenge and uncovered the enrichment of Cullin-RING Ligase 5 (CRL5) complex members over time with chronic antigen exposure. Knockouts of CRL5 components resulted in maintenance of antitumor effector function and delayed dysfunction. KO of these components in CAR-T or TCR-T cells improved tumor control in mice.

ABSTRACT: Engineered T-cell therapies are limited by progressive dysfunction under chronic tumor antigen exposure, and genetic perturbation of negative regulators has emerged as a promising strategy to sustain effector activity. We reanalyzed a genome-wide CRISPR knockout screen in primary human T cells under repetitive tumor stimulation; protein-protein interaction mapping revealed seven members of the Cullin-RING Ligase 5 (CRL5) complex as an enriched hit cluster. Knockout of any of these members (CUL5, ARIH2, RNF7, UBE2F, UBE2L3, CISH, DCUN1D3) enhanced tumor serial killing, and CUL5, the complex scaffold, emerged as the strongest target. CUL5 knockout T cells exhibited sustained cytotoxicity, elevated effector cytokine production, and fewer exhaustion marker-positive cells while retaining antigen specificity and cytokine dependence. Quantitative ubiquitylation proteomics in resting, and acutely and chronically stimulated T cells mapped CRL5-dependent candidate substrates beyond the canonical JAK/STAT axis, including nutrient transporters, receptor trafficking proteins, and TCR signaling components. The benefit of CUL5 knockout extended across CARs targeting CD19, BCMA, and HER2 with different costimulatory domains, and to 1XX-modified CAR and HLA-independent TCR architectures. Combining CUL5 ablation with a tumor-induced IL-2 circuit produced tumor control across nine rounds of repetitive stimulation, whereas either modification alone eventually failed. In vivo, CUL5 knockout BCMA CAR T cells were enriched in the bone marrow following repeated tumor rechallenge and exhibited enhanced ex vivo cytotoxicity after nearly 90 days in tumor-bearing mice. These findings position CUL5 perturbation as a strategy to improve T-cell persistence and the efficacy of adoptive cell therapies, potentially with engineered cytokine support.

Author Info: (1) Gladstone Institutes San Francisco, California United States. ROR: https://ror.org/038321296 (2) Gladstone Institutes San Francisco, California United States. ROR: https://ror.

Author Info: (1) Gladstone Institutes San Francisco, California United States. ROR: https://ror.org/038321296 (2) Gladstone Institutes San Francisco, California United States. ROR: https://ror.org/038321296 (3) H™pital Saint-Louis Paris France. ROR: https://ror.org/049am9t04 (4) University of California San Francisco Medical Center United States. ROR: https://ror.org/01t8svj65 (5) University of California, San Francisco San Francisco, CA United States. ROR: https://ror.org/043mz5j54 (6) University of California, San Francisco San Francisco, CA United States. ROR: https://ror.org/043mz5j54 (7) University of California, San Francisco San Francisco, CA United States. ROR: https://ror.org/043mz5j54 (8) Gladstone Institutes United States. ROR: https://ror.org/038321296 (9) University of California, San Francisco San Francisco, CA United States. ROR: https://ror.org/043mz5j54 (10) University of California, San Francisco San Francisco, CA United States. ROR: https://ror.org/043mz5j54 (11) University of California, San Francisco San Francisco, CA United States. ROR: https://ror.org/043mz5j54 (12) University of California, San Francisco San Francisco, CA United States. ROR: https://ror.org/043mz5j54 (13) UCSF San Francisco, CA United States. (14) Tango Therapeutics (United States) Boston, Massachusetts United States. ROR: https://ror.org/003x6t567 (15) University of California, San Francisco San Francisco, California United States. ROR: https://ror.org/043mz5j54 (16) University of California, San Francisco San Francisco, California United States. ROR: https://ror.org/043mz5j54 (17) University of California, San Francisco San Francisco, CA United States. ROR: https://ror.org/043mz5j54 (18) University of California, San Francisco United States. ROR: https://ror.org/043mz5j54 (19) Gladstone Institutes San Francisco, California United States. ROR: https://ror.org/038321296 (20) UCSF San Francisco, CA United States. (21) University of California, San Francisco San Francisco, CA United States. ROR: https://ror.org/043mz5j54

Systemic antitumor responses to hypofractionated radiotherapy plus Poly(I:C) in advanced hepatocellular carcinoma: A phase 2 trial

In a phase 2 clinical trial in 37 patients with advanced HCC, Qiu et al. observed that adding s.c. Poly(I:C) to hypofractionated radiotherapy (HFRT) was well tolerated, and evaluable patients had better overall responses (54.5% vs. 21.2% with HRFT alone), abscopal responses, 6-month disease control, PFS, and OS, with even more pronounced benefits in subset of patients treated with a TKI (mostly lenvatinib, but also soratenib). Systemic responses were associated with increases in circulating CD28+CD8+ T cells. In mice, CD8+ T cells and CD28 co-stimulation were required for abscopal effects, and lenvatinib enhanced T cell infiltration by normalizing the tumor vasculature.

Contributed by Lauren Hitchings

In a phase 2 clinical trial in 37 patients with advanced HCC, Qiu et al. observed that adding s.c. Poly(I:C) to hypofractionated radiotherapy (HFRT) was well tolerated, and evaluable patients had better overall responses (54.5% vs. 21.2% with HRFT alone), abscopal responses, 6-month disease control, PFS, and OS, with even more pronounced benefits in subset of patients treated with a TKI (mostly lenvatinib, but also soratenib). Systemic responses were associated with increases in circulating CD28+CD8+ T cells. In mice, CD8+ T cells and CD28 co-stimulation were required for abscopal effects, and lenvatinib enhanced T cell infiltration by normalizing the tumor vasculature.

Contributed by Lauren Hitchings

BACKGROUND: The abscopal effect, regression of unirradiated tumors after localized radiotherapy, is rare. Whether hypofractionated radiotherapy (HFRT) plus the innate immune agonist polyinosinic-polycytidylic acid [Poly(I:C)] amplifies systemic antitumor immunity in advanced hepatocellular carcinoma is unclear. METHODS: In this single-arm, phase 2 trial (ChiCTR2100053441), 37 patients received HFRT plus subcutaneous Poly(I:C); 33 were response evaluable. Outcomes were compared with 33 propensity score-matched patients treated with HFRT alone. The primary endpoint was the objective response rate per RECIST v.1.1; secondary endpoints included progression-free survival, overall survival, and safety; and abscopal response was prespecified. Mechanisms were explored by single-cell RNA sequencing and flow cytometry in patients and by CD8 depletion and CD28 blockade in mice. FINDINGS: HFRT plus Poly(I:C) was well tolerated; it was associated with a higher objective response rate (54.5% vs. 21.2%; p = 0.011) and abscopal response rate (45.0% vs. 10.5%; p = 0.042) as well as longer progression-free survival (8.4 vs. 4.6 months; adjusted hazard ratio = 0.542; p = 0.042) and overall survival (not reached vs. 13.6 months; adjusted hazard ratio = 0.386; p = 0.011) vs. HFRT alone. The progression-free survival benefit was also higher in patients concurrently receiving tyrosine kinase inhibitors (p for interaction = 0.013). Peripheral CD8(+)CD28(+) T cell expansion correlated with abscopal tumor control; in mice, CD8(+) T cells and CD28 co-stimulation were required for this effect. CONCLUSIONS: HFRT plus Poly(I:C) showed promising systemic antitumor activity with manageable safety in advanced hepatocellular carcinoma, with CD8(+)CD28(+) T cells as an immune correlate of response. These exploratory findings warrant confirmation in randomized trials. FUNDING: The National Natural Science Foundation of China.

Author Info: (1) The United Innovation of Mengchao Hepatobiliary Technology Key Laboratory of Fujian Province, Mengchao Hepatobiliary Hospital of Fujian Medical University, Fuzhou, Fujian 35002

Author Info: (1) The United Innovation of Mengchao Hepatobiliary Technology Key Laboratory of Fujian Province, Mengchao Hepatobiliary Hospital of Fujian Medical University, Fuzhou, Fujian 350025, China; The Liver Center of Fujian Province, Fujian Medical University, Fuzhou, Fujian 350025, China. (2) Department of Radiotherapy, Mengchao Hepatobiliary Hospital of Fujian Medical University, Fuzhou, Fujian 350025, China. (3) The United Innovation of Mengchao Hepatobiliary Technology Key Laboratory of Fujian Province, Mengchao Hepatobiliary Hospital of Fujian Medical University, Fuzhou, Fujian 350025, China; The Liver Center of Fujian Province, Fujian Medical University, Fuzhou, Fujian 350025, China. (4) The United Innovation of Mengchao Hepatobiliary Technology Key Laboratory of Fujian Province, Mengchao Hepatobiliary Hospital of Fujian Medical University, Fuzhou, Fujian 350025, China; The Liver Center of Fujian Province, Fujian Medical University, Fuzhou, Fujian 350025, China. (5) Department of Radiotherapy, Mengchao Hepatobiliary Hospital of Fujian Medical University, Fuzhou, Fujian 350025, China. (6) Department of Radiotherapy, Mengchao Hepatobiliary Hospital of Fujian Medical University, Fuzhou, Fujian 350025, China. (7) The United Innovation of Mengchao Hepatobiliary Technology Key Laboratory of Fujian Province, Mengchao Hepatobiliary Hospital of Fujian Medical University, Fuzhou, Fujian 350025, China; The Liver Center of Fujian Province, Fujian Medical University, Fuzhou, Fujian 350025, China. (8) The United Innovation of Mengchao Hepatobiliary Technology Key Laboratory of Fujian Province, Mengchao Hepatobiliary Hospital of Fujian Medical University, Fuzhou, Fujian 350025, China; The Liver Center of Fujian Province, Fujian Medical University, Fuzhou, Fujian 350025, China. (9) The United Innovation of Mengchao Hepatobiliary Technology Key Laboratory of Fujian Province, Mengchao Hepatobiliary Hospital of Fujian Medical University, Fuzhou, Fujian 350025, China; The Liver Center of Fujian Province, Fujian Medical University, Fuzhou, Fujian 350025, China. (10) The United Innovation of Mengchao Hepatobiliary Technology Key Laboratory of Fujian Province, Mengchao Hepatobiliary Hospital of Fujian Medical University, Fuzhou, Fujian 350025, China; The Liver Center of Fujian Province, Fujian Medical University, Fuzhou, Fujian 350025, China. (11) The United Innovation of Mengchao Hepatobiliary Technology Key Laboratory of Fujian Province, Mengchao Hepatobiliary Hospital of Fujian Medical University, Fuzhou, Fujian 350025, China; The Liver Center of Fujian Province, Fujian Medical University, Fuzhou, Fujian 350025, China. (12) The United Innovation of Mengchao Hepatobiliary Technology Key Laboratory of Fujian Province, Mengchao Hepatobiliary Hospital of Fujian Medical University, Fuzhou, Fujian 350025, China; The Liver Center of Fujian Province, Fujian Medical University, Fuzhou, Fujian 350025, China. (13) Department of Radiotherapy, Mengchao Hepatobiliary Hospital of Fujian Medical University, Fuzhou, Fujian 350025, China. Electronic address: fjjhb8@sina.com. (14) The United Innovation of Mengchao Hepatobiliary Technology Key Laboratory of Fujian Province, Mengchao Hepatobiliary Hospital of Fujian Medical University, Fuzhou, Fujian 350025, China; The Liver Center of Fujian Province, Fujian Medical University, Fuzhou, Fujian 350025, China. Electronic address: caizhixiong1985@163.com. (15) The United Innovation of Mengchao Hepatobiliary Technology Key Laboratory of Fujian Province, Mengchao Hepatobiliary Hospital of Fujian Medical University, Fuzhou, Fujian 350025, China; The Liver Center of Fujian Province, Fujian Medical University, Fuzhou, Fujian 350025, China. Electronic address: xiaoloong.liu@gmail.com.

Lifestyle-induced regulatory T cell fragility primes adverse events after immune checkpoint blockade

Patra et al. fed mice a low-fiber, high-fat Western diet (inducing dysbiosis and outgrowth of Akkermansia) and treated them with anti-PD-1 to obtain the lifestyle-induced susceptibility to immune-related adverse events (irAEs) mouse model, LISA. LISA recapitulated the ICB toxicity profile affecting many patients, and was influenced by known demographic factors. Cytotoxic tissue-resident memory CD8+ T cells (TRM) and Tregs were increased in affected tissues. Tregs were fragile, less suppressive, and secreted IFNγ. Treg-specific disruption of IFNγ production or signaling and dietary reversal reduced irAE severity, but not antitumor efficacy. Treg fragility and cytotoxic TRM were also present in human irColitis.

Contributed by Ute Burkhardt

Patra et al. fed mice a low-fiber, high-fat Western diet (inducing dysbiosis and outgrowth of Akkermansia) and treated them with anti-PD-1 to obtain the lifestyle-induced susceptibility to immune-related adverse events (irAEs) mouse model, LISA. LISA recapitulated the ICB toxicity profile affecting many patients, and was influenced by known demographic factors. Cytotoxic tissue-resident memory CD8+ T cells (TRM) and Tregs were increased in affected tissues. Tregs were fragile, less suppressive, and secreted IFNγ. Treg-specific disruption of IFNγ production or signaling and dietary reversal reduced irAE severity, but not antitumor efficacy. Treg fragility and cytotoxic TRM were also present in human irColitis.

Contributed by Ute Burkhardt

ABSTRACT: Immune checkpoint blockade has transformed cancer therapy, but immune-related adverse events (irAEs) remain a major, sometimes fatal, limitation, and the lack of robust preclinical models has hindered mechanistic insight. We present the lifestyle-induced susceptibility to irAEs (LISA) model, in which diet-driven metabolic and microbial alterations recreate human-like susceptibility to anti-PD-1 toxicity. LISA recapitulates multi-organ irAEs with immune infiltration and cytokine production, severity depending on sex, age, treatment modality, and immunotherapy response. Mechanistically, dysbiosis marked by Akkermansia muciniphila enrichment accompanies aberrant immune activation, driving regulatory T cell (Treg) fragility and CD8+ cytotoxic effector populations, both observed in patients with irColitis. Dietary reversal, metabolite supplementation, and blocking interferon (IFN)-γ production or signaling in Tregs restored Treg stability and resolved inflammation. Human-to-mouse fecal microbiome transfer interacts with recipient diet to shape susceptibility. Together, these findings identify dietary, microbial, and immune drivers of irAEs, which can be leveraged to improve immunotherapy responses while minimizing toxicity.

Author Info: (1) UPMC Hillman Cancer Center, Pittsburgh, PA 15206, USA; Department of Immunology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15206, USA; Tumor Microenvironment

Author Info: (1) UPMC Hillman Cancer Center, Pittsburgh, PA 15206, USA; Department of Immunology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15206, USA; Tumor Microenvironment Center, UPMC Hillman Cancer Center, Pittsburgh, PA 15206, USA. (2) UPMC Hillman Cancer Center, Pittsburgh, PA 15206, USA; Department of Immunology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15206, USA; Tumor Microenvironment Center, UPMC Hillman Cancer Center, Pittsburgh, PA 15206, USA; Department of Pharmacology and Chemical Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15213, USA. (3) UPMC Hillman Cancer Center, Pittsburgh, PA 15206, USA; Department of Immunology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15206, USA; Tumor Microenvironment Center, UPMC Hillman Cancer Center, Pittsburgh, PA 15206, USA; Program in Microbiology and Immunology, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA. (4) Division of Laboratory Animal Resources, UPMC Hillman Cancer Center, Pittsburgh, PA 15213, USA. (5) UPMC Hillman Cancer Center, Pittsburgh, PA 15206, USA; Department of Immunology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15206, USA; Tumor Microenvironment Center, UPMC Hillman Cancer Center, Pittsburgh, PA 15206, USA; Department of Pharmacology and Chemical Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15213, USA. (6) UPMC Hillman Cancer Center, Pittsburgh, PA 15206, USA; Department of Immunology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15206, USA; Tumor Microenvironment Center, UPMC Hillman Cancer Center, Pittsburgh, PA 15206, USA. (7) UPMC Hillman Cancer Center, Pittsburgh, PA 15206, USA; Department of Immunology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15206, USA; Tumor Microenvironment Center, UPMC Hillman Cancer Center, Pittsburgh, PA 15206, USA; Program in Microbiology and Immunology, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA. (8) UPMC Hillman Cancer Center, Pittsburgh, PA 15206, USA; Department of Immunology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15206, USA; Tumor Microenvironment Center, UPMC Hillman Cancer Center, Pittsburgh, PA 15206, USA. (9) UPMC Hillman Cancer Center, Pittsburgh, PA 15206, USA. (10) UPMC Hillman Cancer Center, Pittsburgh, PA 15206, USA; Department of Immunology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15206, USA; Tumor Microenvironment Center, UPMC Hillman Cancer Center, Pittsburgh, PA 15206, USA. (11) Department of Pharmacology and Chemical Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15213, USA; Health Sciences Mass Spectrometry Core, University of Pittsburgh, Pittsburgh, PA 15213, USA. (12) UPMC Hillman Cancer Center, Pittsburgh, PA 15206, USA; Department of Immunology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15206, USA; Tumor Microenvironment Center, UPMC Hillman Cancer Center, Pittsburgh, PA 15206, USA; Cellular and Molecular Pathology Program, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA. (13) Department of Pharmacology and Chemical Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15213, USA; Health Sciences Mass Spectrometry Core, University of Pittsburgh, Pittsburgh, PA 15213, USA. (14) UPMC Hillman Cancer Center, Pittsburgh, PA 15206, USA; Department of Immunology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15206, USA; Tumor Microenvironment Center, UPMC Hillman Cancer Center, Pittsburgh, PA 15206, USA. (15) UPMC Hillman Cancer Center, Pittsburgh, PA 15206, USA. (16) UPMC Hillman Cancer Center, Pittsburgh, PA 15206, USA; Department of Immunology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15206, USA; Tumor Microenvironment Center, UPMC Hillman Cancer Center, Pittsburgh, PA 15206, USA; Center for Systems Immunology, Department of Immunology, University of Pittsburgh, Pittsburgh, PA 15206, USA. (17) UPMC Hillman Cancer Center, Pittsburgh, PA 15206, USA; Department of Immunology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15206, USA; Tumor Microenvironment Center, UPMC Hillman Cancer Center, Pittsburgh, PA 15206, USA. (18) UPMC Hillman Cancer Center, Pittsburgh, PA 15206, USA; Department of Immunology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15206, USA; Tumor Microenvironment Center, UPMC Hillman Cancer Center, Pittsburgh, PA 15206, USA. Electronic address: overacre@pitt.edu.

Perforin triggers iron-induced mitochondrial damage to drive ferroptosis and anti-tumor immunity

Bell et al. discovered a previously unrecognized, granzyme-independent function of perforin (Prf1) in CTLs and NK cells that showed that Prf1 can directly kill tumor cells via ferroptosis. Mechanistically, Prf1 induced transferrin receptor (CD71) endocytosis, leading to increased cellular iron uptake. Via endosomal–mitochondrial contacts, this subsequently elevated mitochondrial iron and lipid peroxidation, and tumor cell ferroptosis. In murine studies, dietary iron supplementation potentiated antitumor immunity in a Prf1-dependent manner, while in patients with cancer, higher Prf1 expression correlated with longer survival and improved responses to ICB.

Contributed by Katherine Turner

Bell et al. discovered a previously unrecognized, granzyme-independent function of perforin (Prf1) in CTLs and NK cells that showed that Prf1 can directly kill tumor cells via ferroptosis. Mechanistically, Prf1 induced transferrin receptor (CD71) endocytosis, leading to increased cellular iron uptake. Via endosomal–mitochondrial contacts, this subsequently elevated mitochondrial iron and lipid peroxidation, and tumor cell ferroptosis. In murine studies, dietary iron supplementation potentiated antitumor immunity in a Prf1-dependent manner, while in patients with cancer, higher Prf1 expression correlated with longer survival and improved responses to ICB.

Contributed by Katherine Turner

ABSTRACT: Cytotoxic CD8(+) T cells and natural killer cells are critical effectors of anti-tumor immunity. Cytotoxic lymphocytes eliminate tumor cells by releasing perforin (Prf1), a pore-forming protein that enables granzymes (Gzms) to enter target cells and trigger both caspase-dependent and -independent cell death. Here, we uncover a previously unrecognized, Gzm-independent function of Prf1. We find that Prf1 directly induces tumoral ferroptosis, an iron- and lipid-dependent form of cell death. Mechanistically, Prf1-mediated pore formation drives endocytosis of the transferrin receptor, which increases cellular iron uptake. Prf1 promotes the formation of endosomal-mitochondrial contacts, which leads to mitochondrial iron accumulation, mitochondrial dysfunction, and tumor cell ferroptosis. Dietary iron supplementation suppresses tumor progression in murine tumor models in a Prf1-dependent manner. Higher Prf1 expression correlates with improved survival and enhanced response to immune checkpoint blockade in patients with cancer. These findings define a Gzm-independent function of Prf1 as a direct executioner of cancer cell death through ferroptosis.

Author Info: (1) Department of Surgery, University of Michigan School of Medicine, Ann Arbor, MI, USA; Center of Excellence for Cancer Immunology and Immunotherapy, University of Michigan Rogel

Author Info: (1) Department of Surgery, University of Michigan School of Medicine, Ann Arbor, MI, USA; Center of Excellence for Cancer Immunology and Immunotherapy, University of Michigan Rogel Cancer Center, Ann Arbor, MI, USA. (2) Department of Molecular and Cellular Physiology, Albany Medical College, Albany, NY, USA. (3) Department of Surgery, University of Michigan School of Medicine, Ann Arbor, MI, USA; Center of Excellence for Cancer Immunology and Immunotherapy, University of Michigan Rogel Cancer Center, Ann Arbor, MI, USA; Graduate Program in Immunology, University of Michigan, Ann Arbor, MI, USA. (4) Department of Surgery, University of Michigan School of Medicine, Ann Arbor, MI, USA; Center of Excellence for Cancer Immunology and Immunotherapy, University of Michigan Rogel Cancer Center, Ann Arbor, MI, USA. (5) Department of Computational Medicine and Bioinformatics, University of Michigan, Ann Arbor, MI, USA. (6) Department of Molecular & Physiology, University of Michigan, Ann Arbor, MI, USA. (7) Program in Cellular and Molecular Medicine, Boston Children's Hospital and Department of Pediatrics, Harvard Medical School, Boston, MA 02115, USA. (8) Program in Cellular and Molecular Medicine, Boston Children's Hospital and Department of Pediatrics, Harvard Medical School, Boston, MA 02115, USA. (9) Department of Computational Medicine and Bioinformatics, University of Michigan, Ann Arbor, MI, USA. (10) Department of Surgery, University of Michigan School of Medicine, Ann Arbor, MI, USA; Center of Excellence for Cancer Immunology and Immunotherapy, University of Michigan Rogel Cancer Center, Ann Arbor, MI, USA. (11) Department of Surgery, University of Michigan School of Medicine, Ann Arbor, MI, USA; Center of Excellence for Cancer Immunology and Immunotherapy, University of Michigan Rogel Cancer Center, Ann Arbor, MI, USA. (12) Department of Molecular & Physiology, University of Michigan, Ann Arbor, MI, USA. (13) Department of Molecular & Physiology, University of Michigan, Ann Arbor, MI, USA. (14) Program in Cellular and Molecular Medicine, Boston Children's Hospital and Department of Pediatrics, Harvard Medical School, Boston, MA 02115, USA. (15) Cancer Immunology Program, Peter MacCallum Cancer Centre, Melbourne, VIC, Australia. (16) Department of Surgery, University of Michigan School of Medicine, Ann Arbor, MI, USA; Center of Excellence for Cancer Immunology and Immunotherapy, University of Michigan Rogel Cancer Center, Ann Arbor, MI, USA. (17) Department of Surgery, University of Michigan School of Medicine, Ann Arbor, MI, USA; Center of Excellence for Cancer Immunology and Immunotherapy, University of Michigan Rogel Cancer Center, Ann Arbor, MI, USA. (18) Department of Surgery, University of Michigan School of Medicine, Ann Arbor, MI, USA; Center of Excellence for Cancer Immunology and Immunotherapy, University of Michigan Rogel Cancer Center, Ann Arbor, MI, USA; Graduate Program in Immunology, University of Michigan, Ann Arbor, MI, USA. (19) Department of Surgery, University of Michigan School of Medicine, Ann Arbor, MI, USA; Center of Excellence for Cancer Immunology and Immunotherapy, University of Michigan Rogel Cancer Center, Ann Arbor, MI, USA; Graduate Program in Immunology, University of Michigan, Ann Arbor, MI, USA. (20) Department of Surgery, University of Michigan School of Medicine, Ann Arbor, MI, USA. (21) Department of Surgery, University of Michigan School of Medicine, Ann Arbor, MI, USA. (22) Department of Surgery, University of Michigan School of Medicine, Ann Arbor, MI, USA. (23) Cayman Chemical, Ann Arbor, MI, USA. (24) Cayman Chemical, Ann Arbor, MI, USA. (25) Department of Molecular & Physiology, University of Michigan, Ann Arbor, MI, USA; Department of Internal Medicine, Division of Gastroenterology, University of Michigan, Ann Arbor, MI, USA. (26) Department of Surgery, University of Michigan School of Medicine, Ann Arbor, MI, USA; Center of Excellence for Cancer Immunology and Immunotherapy, University of Michigan Rogel Cancer Center, Ann Arbor, MI, USA; Graduate Program in Immunology, University of Michigan, Ann Arbor, MI, USA. Electronic address: ilonak@umich.edu. (27) Cancer Immunology Program, Peter MacCallum Cancer Centre, Melbourne, VIC, Australia. (28) Department of Molecular and Cellular Physiology, Albany Medical College, Albany, NY, USA. Electronic address: barrosm@amc.edu. (29) Program in Cellular and Molecular Medicine, Boston Children's Hospital and Department of Pediatrics, Harvard Medical School, Boston, MA 02115, USA. Electronic address: judy.lieberman@childrens.harvard.edu. (30) Department of Surgery, University of Michigan School of Medicine, Ann Arbor, MI, USA; Center of Excellence for Cancer Immunology and Immunotherapy, University of Michigan Rogel Cancer Center, Ann Arbor, MI, USA; Graduate Program in Immunology, University of Michigan, Ann Arbor, MI, USA. Electronic address: wzou@umich.edu.

Rapid CAR-T manufacturing with low-intensity priming enhances CAR-T cell fitness and limits inflammatory programming in a phase 1 study Featured  

Yang and Li et al. developed a 2-day protocol called PrimeCAR in which T cells were primed ex vivo with low-intensity soluble anti-CD3 in combination with IL-7 and IL-21. Compared to a longer conventional CAR T cell manufacturing protocol, PrimeCAR T cell manufacturing yielded less activated, less differentiated CAR T cell products that maintained strong cytotoxicity, while limiting inflammatory cytokine signaling and associated toxicities. PrimeCAR T cells showed promising results for maintaining efficacy while limiting toxicity in two phase 1 trials in patients with either B-ALL or B-NHL.

Yang and Li et al. developed a 2-day protocol called PrimeCAR in which T cells were primed ex vivo with low-intensity soluble anti-CD3 in combination with IL-7 and IL-21. Compared to a longer conventional CAR T cell manufacturing protocol, PrimeCAR T cell manufacturing yielded less activated, less differentiated CAR T cell products that maintained strong cytotoxicity, while limiting inflammatory cytokine signaling and associated toxicities. PrimeCAR T cells showed promising results for maintaining efficacy while limiting toxicity in two phase 1 trials in patients with either B-ALL or B-NHL.

ABSTRACT: Conventional chimeric antigen receptor (CAR)-T manufacturing requires prolonged ex vivo activation and expansion, which may compromise T cell fitness and promote inflammatory differentiation. Here, we developed PrimeCAR, a 2-day manufacturing strategy combining low-intensity anti-CD3 priming with IL-7 and IL-21. Compared with conventionally manufactured CAR-T cells, PrimeCAR products exhibited a distinct transcriptional state characterized by preserved stemness-associated programs and a PrimeCAR-enriched primed naive T cell (Tn)-like state. Following antigen stimulation, PrimeCAR cells preferentially engaged proliferative programs while limiting exhaustion-associated regulators and inflammatory mediators. Functionally, PrimeCAR cells demonstrated enhanced expansion, persistence, and durable antitumor activity in xenograft models. In investigator-initiated phase I studies of relapsed or refractory B cell acute lymphoblastic leukemia and B cell non-Hodgkin lymphoma, PrimeCAR showed encouraging clinical activity with a favorable safety profile. Together, these findings demonstrate that rapid manufacturing with controlled early T cell priming selectively restrains inflammatory programming while preserving CAR-T cell fitness, providing a practical strategy for improving CAR-T manufacturing and therapeutic performance.

Author Info: (1) Chongqing Key Laboratory of Gene and Cell Therapy, Chongqing 400039, China; Chongqing Precision Biotech Co., Ltd., Chongqing 400039, China. Electronic address: yangzhi@precisio

Author Info: (1) Chongqing Key Laboratory of Gene and Cell Therapy, Chongqing 400039, China; Chongqing Precision Biotech Co., Ltd., Chongqing 400039, China. Electronic address: yangzhi@precision-biotech.com. (2) 920th Hospital of Joint Logistics Support Force of People's Liberationsi Army of China, Kunming 650100, Yunnan, China. (3) Chongqing Key Laboratory of Gene and Cell Therapy, Chongqing 400039, China; Chongqing Precision Biotech Co., Ltd., Chongqing 400039, China. (4) Chongqing Key Laboratory of Gene and Cell Therapy, Chongqing 400039, China; Chongqing Precision Biotech Co., Ltd., Chongqing 400039, China. (5) Chongqing Key Laboratory of Gene and Cell Therapy, Chongqing 400039, China; Chongqing Precision Biotech Co., Ltd., Chongqing 400039, China. (6) 920th Hospital of Joint Logistics Support Force of People's Liberationsi Army of China, Kunming 650100, Yunnan, China. (7) 920th Hospital of Joint Logistics Support Force of People's Liberationsi Army of China, Kunming 650100, Yunnan, China. (8) 920th Hospital of Joint Logistics Support Force of People's Liberationsi Army of China, Kunming 650100, Yunnan, China. (9) Chongqing Key Laboratory of Gene and Cell Therapy, Chongqing 400039, China; Chongqing Precision Biotech Co., Ltd., Chongqing 400039, China. (10) 920th Hospital of Joint Logistics Support Force of People's Liberationsi Army of China, Kunming 650100, Yunnan, China. (11) 920th Hospital of Joint Logistics Support Force of People's Liberationsi Army of China, Kunming 650100, Yunnan, China. (12) Chongqing Key Laboratory of Gene and Cell Therapy, Chongqing 400039, China; Chongqing Precision Biotech Co., Ltd., Chongqing 400039, China. (13) Chongqing Key Laboratory of Gene and Cell Therapy, Chongqing 400039, China; Chongqing Precision Biotech Co., Ltd., Chongqing 400039, China. (14) Chongqing Key Laboratory of Gene and Cell Therapy, Chongqing 400039, China; Chongqing Precision Biotech Co., Ltd., Chongqing 400039, China. (15) Chongqing Key Laboratory of Gene and Cell Therapy, Chongqing 400039, China; Chongqing Precision Biotech Co., Ltd., Chongqing 400039, China. (16) Chongqing Key Laboratory of Gene and Cell Therapy, Chongqing 400039, China; Chongqing Precision Biotech Co., Ltd., Chongqing 400039, China. (17) 920th Hospital of Joint Logistics Support Force of People's Liberationsi Army of China, Kunming 650100, Yunnan, China. (18) 920th Hospital of Joint Logistics Support Force of People's Liberationsi Army of China, Kunming 650100, Yunnan, China. (19) 920th Hospital of Joint Logistics Support Force of People's Liberationsi Army of China, Kunming 650100, Yunnan, China. Electronic address: sanbin1011@163.com. (20) Chongqing Key Laboratory of Gene and Cell Therapy, Chongqing 400039, China; Chongqing Precision Biotech Co., Ltd., Chongqing 400039, China. Electronic address: cqian8634@gmail.com.

NKG2D overexpression potentiates CAR-T cells through costimulation by tumor NKG2D ligands Spotlight 

Li, Han, and Zhu et al. observed that T cell activation increased NKG2D ligand (NKG2DL) expression, enabling T cell depletion by NK cells. Overexpressing (OE) full-length NKG2D in CAR T cells led to its colocalization with NKG2DL on the cell surface, reducing NKG2DL detection (by cis masking) and NK killing. NKG2D-OE also improved CAR T cell cytotoxicity by binding NKG2DL on tumor cells (in trans) and increasing CAR-T activation, memory phenotype, CAR-proximal signaling, and proliferative capacity, without affecting exhaustion. NKG2D-OE improved CAR-T efficacy in heme and solid tumor models, and boosted tumor infiltration.

Contributed by Alex Najibi

Li, Han, and Zhu et al. observed that T cell activation increased NKG2D ligand (NKG2DL) expression, enabling T cell depletion by NK cells. Overexpressing (OE) full-length NKG2D in CAR T cells led to its colocalization with NKG2DL on the cell surface, reducing NKG2DL detection (by cis masking) and NK killing. NKG2D-OE also improved CAR T cell cytotoxicity by binding NKG2DL on tumor cells (in trans) and increasing CAR-T activation, memory phenotype, CAR-proximal signaling, and proliferative capacity, without affecting exhaustion. NKG2D-OE improved CAR-T efficacy in heme and solid tumor models, and boosted tumor infiltration.

Contributed by Alex Najibi

ABSTRACT: Chimeric antigen receptor (CAR)-T cell therapy has achieved remarkable success in hematologic malignancies, but durable efficacy remains limited by insufficient CAR-T cell persistence. Here, we show that activated CAR-T cells upregulate multiple NKG2D ligands (NKG2DLs), increasing their susceptibility to natural killer (NK) cell-mediated CAR-T cell cytotoxicity. To address this vulnerability, we engineer CAR-T cells to overexpress full-length NKG2D (CAR-NG). NKG2D overexpression reduces the surface accessibility of endogenous NKG2DLs without decreasing their total protein abundance. In parallel, engagement of tumor-expressed NKG2DLs in trans provides additional costimulatory signals, enhancing CAR-T cell activation and proliferation while attenuating exhaustion-associated phenotypes. In hematologic and solid tumor models, CAR-NG cells exhibit reduced susceptibility to NK-cell-mediated cytotoxicity, improved tumor control, increased in vivo expansion, deeper tumor infiltration, and no evident additional toxicity compared with conventional CAR-T cells. These findings identify NKG2D overexpression as an engineering strategy that enhances CAR-T antitumor activity primarily through tumor NKG2DL-dependent trans costimulation.

Author Info: (1) Department of Biotherapy, the First Medical Center, Chinese PLA General Hospital, Beijing, China. (2) Department of Biotherapy, the First Medical Center, Chinese PLA General Ho

Author Info: (1) Department of Biotherapy, the First Medical Center, Chinese PLA General Hospital, Beijing, China. (2) Department of Biotherapy, the First Medical Center, Chinese PLA General Hospital, Beijing, China. (3) Department of Biotherapy, the First Medical Center, Chinese PLA General Hospital, Beijing, China. (4) Department of Biotherapy, the First Medical Center, Chinese PLA General Hospital, Beijing, China. (5) Department of Biotherapy, the First Medical Center, Chinese PLA General Hospital, Beijing, China. (6) Department of Biotherapy, the First Medical Center, Chinese PLA General Hospital, Beijing, China. (7) Department of Biotherapy, the First Medical Center, Chinese PLA General Hospital, Beijing, China. (8) Beijing Changping Laboratory, Beijing, China; School of Basic Medical Sciences, Tsinghua University School of Medicine, Beijing, China; Tsinghua-Peking Center for Life Sciences, Beijing, China. Electronic address: linxin307@tsinghua.edu.cn. (9) Beijing Changping Laboratory, Beijing, China. Electronic address: woshiwjs@hotmail.com. (10) Department of Biotherapy, the First Medical Center, Chinese PLA General Hospital, Beijing, China. Electronic address: hanwdrsw@163.com.

A structure-informed deep learning framework for modeling TCR-peptide-HLA interactions Spotlight 

Cao et al. developed StriMap – a prediction framework for modeling peptide–HLA presentation and TCR recognition as a coupled process based on physicochemical, sequence-context, and structure-informed representations that capture relevant constraints. StriMap was successfully used to screen bacterial peptides and identity candidates to activate TCRs associated with ankylosing spondylitis (AS), and a top candidate in AS was also confirmed in IBD, linking the two autoimmune diseases. In cancer, StriMap demonstrated efficacy for both TCR selection in adoptive cell therapy, and prioritizing potential neoepitopes for personalized cancer vaccines.

Contributed by Lauren Hitchings

Cao et al. developed StriMap – a prediction framework for modeling peptide–HLA presentation and TCR recognition as a coupled process based on physicochemical, sequence-context, and structure-informed representations that capture relevant constraints. StriMap was successfully used to screen bacterial peptides and identity candidates to activate TCRs associated with ankylosing spondylitis (AS), and a top candidate in AS was also confirmed in IBD, linking the two autoimmune diseases. In cancer, StriMap demonstrated efficacy for both TCR selection in adoptive cell therapy, and prioritizing potential neoepitopes for personalized cancer vaccines.

Contributed by Lauren Hitchings

ABSTRACT: The interaction between T cell receptors (TCRs), peptides, and human leukocyte antigens (HLAs) underlies antigen-specific T cell immunity. Despite substantial advances in peptide-HLA presentation prediction, accurate modeling of coupled TCR-peptide-HLA recognition remains underdeveloped, limiting applications such as TCR and neoepitope prioritization in cancer and antigen identification in autoimmunity. Here we present StriMap, a unified framework for predicting TCR-peptide-HLA interactions by integrating physicochemical, sequence-context, and structural features at recognition interfaces. StriMap achieves state-of-the-art performance with improved generalizability and enables applications in both cancer and autoimmunity. As a case study in ankylosing spondylitis (AS), we screened 13 million peptides derived from 43,241 bacterial proteins and identified candidate molecular mimics that were experimentally validated to activate T cells expressing an AS-associated TCR. Notably, a top validated peptide was enriched in patients with inflammatory bowel disease (IBD), suggesting potential shared microbial triggers between AS and IBD. Overall, StriMap provides a generalizable framework for rational immunotherapy design and for dissecting antigenic drivers of autoimmunity.

Author Info: (1) Broad Institute of MIT and Harvard, Cambridge, MA, United States. (2) Broad Institute of MIT and Harvard, Cambridge, MA, United States. Department of Molecular Biology, Massach

Author Info: (1) Broad Institute of MIT and Harvard, Cambridge, MA, United States. (2) Broad Institute of MIT and Harvard, Cambridge, MA, United States. Department of Molecular Biology, Massachusetts General Hospital and Harvard Medical School, Boston, MA, United States. (3) Broad Institute of MIT and Harvard, Cambridge, MA, United States. (4) Broad Institute of MIT and Harvard, Cambridge, MA, United States. (5) Broad Institute of MIT and Harvard, Cambridge, MA, United States. (6) Broad Institute of MIT and Harvard, Cambridge, MA, United States. Department of Molecular Biology, Massachusetts General Hospital and Harvard Medical School, Boston, MA, United States. (7) Broad Institute of MIT and Harvard, Cambridge, MA, United States. (8) Broad Institute of MIT and Harvard, Cambridge, MA, United States. Department of Molecular Biology, Massachusetts General Hospital and Harvard Medical School, Boston, MA, United States. (9) Broad Institute of MIT and Harvard, Cambridge, MA, United States. Klarman Cell Observatory, Broad Institute of MIT and Harvard, Cambridge, MA, United States. (10) Broad Institute of MIT and Harvard, Cambridge, MA, United States. Massachusetts Institute of Technology, Cambridge, MA, United States. (11) Broad Institute of MIT and Harvard, Cambridge, MA, United States. Department of Molecular Biology, Massachusetts General Hospital and Harvard Medical School, Boston, MA, United States. Klarman Cell Observatory, Broad Institute of MIT and Harvard, Cambridge, MA, United States. Center for the Study of Inflammatory Bowel Disease, Massachusetts General Hospital, Boston, MA, United States.

Peripheral Blood as the Sole Source for Cancer Neoantigen and Reactive T-Cell Discovery Spotlight 

To expand the accessibility of neoantigen-based immunotherapy, Garcia-Garijo et al. integrated circulating tumor DNA (ctDNA) whole-exome sequencing (WES) to identify non-synonymous mutations (NSMs) with the isolation of neoantigen-specific T cells from circulating CD8+PD1hiCD38+ and CD4+PD1hi populations. In 8 patients with metastatic solid cancers, this strategy identified most lesion-derived NSMs (and additional neoantigens) enriched for clonal variants. In a cohort of 69 patients with metastatic solid cancers, neoantigen discovery based only on ctDNA seemed feasible in ~50% of patients (~85% of patients with colorectal cancer).

Contributed by Ute Burkhardt

To expand the accessibility of neoantigen-based immunotherapy, Garcia-Garijo et al. integrated circulating tumor DNA (ctDNA) whole-exome sequencing (WES) to identify non-synonymous mutations (NSMs) with the isolation of neoantigen-specific T cells from circulating CD8+PD1hiCD38+ and CD4+PD1hi populations. In 8 patients with metastatic solid cancers, this strategy identified most lesion-derived NSMs (and additional neoantigens) enriched for clonal variants. In a cohort of 69 patients with metastatic solid cancers, neoantigen discovery based only on ctDNA seemed feasible in ~50% of patients (~85% of patients with colorectal cancer).

Contributed by Ute Burkhardt

ABSTRACT: Neoantigen-targeted T-cell therapies require invasive tumor biopsies to identify neoantigens and reactive T cells, limiting their applicability and potentially underestimating tumor heterogeneity. We propose a novel strategy relying solely on peripheral blood to identify non-synonymous mutations via cell-free DNA whole-exome sequencing and isolate neoantigen-specific CD4+ and CD8+ T cells based on PD-1 and CD39 co-expression. Our method identified most neoantigens and increased the number of T-cell reactivities detected by standard biopsy-based techniques in an initial cohort of eight metastatic cancer patients. Critically, our approach uncovered additional neoantigens missed by tumor biopsy analysis, highlighting the potential for broader neoantigen targeting using liquid biopsies. Analysis of a cohort of 69 metastatic cancer patients revealed that approximately half could be eligible for our blood-only approach. This proof-of-concept study demonstrates the potential of using blood as a surrogate or complement for tumor biopsies to develop neoantigen-reactive T-cell therapies against a broader spectrum of neoantigens.

Author Info: (1) Vall d'Hebron Institute of Oncology Barcelona, Barcelona Spain. ROR: https://ror.org/054xx3904 (2) Vall d'Hebron Institute of Oncology Barcelona, Barcelona Spain. ROR: https://

Author Info: (1) Vall d'Hebron Institute of Oncology Barcelona, Barcelona Spain. ROR: https://ror.org/054xx3904 (2) Vall d'Hebron Institute of Oncology Barcelona, Barcelona Spain. ROR: https://ror.org/054xx3904 (3) Vall d'Hebron Institute of Oncology Barcelona, Barcelona Spain. ROR: https://ror.org/054xx3904 (4) Vall d'Hebron Institute of Oncology Spain. ROR: https://ror.org/054xx3904 (5) Vall d'Hebron Institute of Oncology Barcelona, Barcelona Spain. ROR: https://ror.org/054xx3904 (6) Vall d'Hebron Institute of Oncology Barcelona, Barcelona Spain. ROR: https://ror.org/054xx3904 (7) Vall d'Hebron Institute of Oncology Barcelona, Barcelona Spain. ROR: https://ror.org/054xx3904 (8) Universitat Aut˜noma de Barcelona Barcelona, Barcelona Spain. ROR: https://ror.org/052g8jq94 (9) Vall d'Hebron Institute of Oncology Barcelona, Barcelona Spain. ROR: https://ror.org/054xx3904 (10) Vall d'Hebron Institute of Oncology Barcelona, Barcelona Spain. ROR: https://ror.org/054xx3904 (11) Vall d'Hebron Institute of Oncology Barcelona, Barcelona Spain. ROR: https://ror.org/054xx3904 (12) Vall d'Hebron Institute of Oncology Barcelona, Barcelona Spain. ROR: https://ror.org/054xx3904 (13) National Institutes of Health United States. ROR: https://ror.org/01cwqze88 (14) Vall d'Hebron Institute of Oncology Barcelona, Barcelona Spain. ROR: https://ror.org/054xx3904 (15) Vall d'Hebron Institute of Oncology Barcelona, Barcelona Spain. ROR: https://ror.org/054xx3904 (16) Vall d'Hebron Institute of Oncology Barcelona, Barcelona Spain. ROR: https://ror.org/054xx3904 (17) Vall d'Hebron Institute of Oncology Barcelona, Barcelona Spain. ROR: https://ror.org/054xx3904 (18) Instituto de Investigaci—n Sanitaria Arag—n Zaragoza Spain. ROR: https://ror.org/03njn4610 (19) Stanford University Stanford, CA United States. ROR: https://ror.org/00f54p054 (20) Vall d'Hebron Institute of Oncology Barcelona Spain. ROR: https://ror.org/054xx3904 (21) Vall d'Hebron Institute of Oncology barcelona, barcelona, spain Spain. ROR: https://ror.org/054xx3904 (22) Vall d'Hebron Institute of Oncology (VHIO) Barcelona Spain. (23) Vall d'Hebron Institute of Oncology Barcelona, Barcelona Spain. ROR: https://ror.org/054xx3904 (24) Vall d'Hebron Institute of Oncology Barcelona Spain. ROR: https://ror.org/054xx3904 (25) Vall d'Hebron Institute of Oncology Barcelona, Barcelona Spain. ROR: https://ror.org/054xx3904 (26) Vall d'Hebron Institute of Oncology Barcelona, Barcelona Spain. ROR: https://ror.org/054xx3904 (27) Vall d'Hebron Institute of Oncology Barcelona Spain. ROR: https://ror.org/054xx3904 (28) Institut d'Investigaci— BiomŽdica de Bellvitge L'Hospitalet de Llobregat Spain. ROR: https://ror.org/0008xqs48 (29) Vall d'Hebron Institute of Oncology Barcelona Spain. ROR: https://ror.org/054xx3904 (30) Vall d'Hebron Institute of Oncology Barcelona, Barcelona Spain. ROR: https://ror.org/054xx3904 (31) Centre Hospitalier de l'UniversitŽ de MontrŽal Montreal Canada. ROR: https://ror.org/0410a8y51 (32) The Francis Crick Institute London United Kingdom. ROR: https://ror.org/04tnbqb63 (33) Vall d'Hebron Institute of Oncology Barcelona Spain. ROR: https://ror.org/054xx3904 (34) Vall d'Hebron Institute of Oncology Barcelona, Barcelona Spain. ROR: https://ror.org/054xx3904 (35) Vall d'Hebron Institute of Oncology Barcelona, Barcelona Spain. ROR: https://ror.org/054xx3904

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