Figure S1: CCR6 and CCL20 expression is increased in multiple tumors.

Figure S4: Ccr6-/- Tregs have reduced glycolysis compared to WT Tregs.

BACKGROUND:Ustekinumab is a monoclonal antibody therapy targeting interleukin-12 and interleukin-23 for the treatment of inflammatory bowel diseases, including ulcerative colitis (UC). While these pathways remain quite attractive for UC therapy, response to ustekinumab can be variable. There is an urgent need to better understand the underlying mucosal immune alterations associated with treatment to guide therapy decisions. This study aims to examine the mucosal immune signatures in individuals with UC with variable treatment response to ustekinumab. METHODS:Sigmoid colon tissue from individuals treated with ustekinumab were analyzed using a multimodal approach. Single-cell RNA and T cell receptor sequencing was performed on mucosal biopsies. In a subset of these patients, multiparameter flow cytometry and spatial transcriptomics was also completed on matched pre- and post-treatment tissue samples. Key findings were also validated on a larger cohort using immunohistochemistry. RESULTS:Ustekinumab significantly altered the frequency and phenotype of mucosal regulatory T cells (Tregs). Nonresponders to ustekinumab had a higher frequency of Tregs that expressed OX40 and GITR, which is associated with decreased suppressive abilities. In contrast, responders had Tregs with elevated GPR15 and reduced expression of the kinase PIM2, which can alter Treg stability and function. Additionally, T helper 17 cells in nonresponders demonstrated an enhanced proinflammatory gene expression profile. CONCLUSION:Nonresponse to ustekinumab in UC is linked to a mucosal immune environment enriched with proinflammatory T cell phenotypes and impaired regulatory T cell function. These findings suggest that Tregs are both targets and potential biomarkers of ustekinumab response, with their phenotypic and transcriptional features providing insight into mechanisms of therapeutic resistance.

Mutation-associated neoantigens (MANAs) are highly cancer-specific targets for immunotherapy where peptides derived from intracellular mutant proteins are presented on the cell surface via HLA molecules. T cell-engaging bispecific antibodies and CAR T cells can target MANAs to eliminate cancer cells via T cell activation. However, the low antigen density of MANAs on the cell surface can limit therapeutic efficacy. Here, we investigated whether increasing the affinity of the H2 single-chain variable fragment (scFv) targeting the p53 R175H MANA (HMTEVVRHC presented on HLA-A*02:01) improves its therapeutic effect. We identified higher-affinity H2 variants via phage biopanning and a thiocyanate elution method. Increasing bispecific antibody affinity to the low nanomolar range increased cancer cell killing and tumor control in mouse xenograft models without sacrificing antigen specificity. We next asked how increasing scFv affinity impacts CAR T cell function - a matter of debate. We appended each variant scFv to a CD28z CAR, CD3γ, or the T cell receptor. In striking contrast to the bispecific antibody results, increasing CAR affinity decreased function in each CAR format due to lower T cell activation upon interaction with target cancer cells. These results have important implications for the design of future immunotherapeutic approaches targeting low-density antigens.

Lethal toxins could become potent therapies against cancer, but their clinical utility is limited by adverse events upon systemic administration. These could be reduced if the toxins were delivered by effector cells that specifically infiltrate cancers, thereby releasing toxins locally into the tumor microenvironment. One of the challenges underlying this strategy is that cells delivering toxins would have to be resistant to them. We address this obstacle by showing that effectors derived from transformed human cell lines genetically engineered for resistance to bacterial adenosine diphos-phate ribosylating toxins (ADPRTs), including Pseudomonas aeruginosa exotoxin A (PE), can produce targeted immunotoxins that specifically kill cancer cells express-ing cognate tumor-associated antigens. Resistance to immunotoxins was achieved by knockout of genes in the diphthamide biosynthesis pathway (DPH1-4) required for the posttranslational modification of eukaryotic elongation factor 2 (EEF2) that is the target of ADPRTs or by mutation of EEF2 itself. We show that engineering resist-ance to ADPRTs, one of the most potent toxins acting on human cells, is essential to achieve robust function of armored effector cell lines. This work establishes a critical step on the path to equip effector cells with the ability to deliver powerful toxins to cancer cells and introduces a platform to investigate extension to primary autologous or allogeneic therapeutic cell types.

Figure S3: Ccr6-/- Tregs are transcriptionally and metabolically distinct from WT Tregs.

Abstract The purpose of this study is to investigate the formation and maintenance of germinal centers in TLS by defining mechanisms and spatial patterns of CXCL13 chemokine gradients in tumor tissue. In secondary lymphoid tissue, it is recognized that CXCL13 is immobilized by follicular dendritic cells (FDCs), but the mechanisms of immobilization remain unclear. Furthermore, spatial patterns and mechanisms of CXCL13 immobilization in TLS are unknown. We developed a CXCL13 Immunohistochemistry /in situ hybridization (ICH/ISH) dual stain to determine which cells are responsible for CXCL13 production and display. We stained 3 formalin-fixed paraffin-embedded tonsil specimens and found that CXCL13 protein and mRNA staining have distinct localizations. Specifically, spatial analyses were performed on 10 individual follicular structures from each tonsil to compare the percent surface area of either germinal centers or mantle zones (n=30) demonstrating staining by IHC or ISH. This analysis revealed that CXCL13 protein was highly expressed in germinal centers and expressed relatively lower in mantle zones (31% vs 12% surface area, p<0.0001). However, CXCL13 ISH staining was the inverse: mRNA expression was lower in germinal centers, and higher in mantle zones (20% vs 37%, p<0.0001). Flow cytometry analysis of fresh tonsil tissue revealed stromal cells displaying CXCL13, (PDPN+CD31-CXCL13+), supporting the CXCL13 IHC findings. Similarly, spatial transcriptomics showed similar findings to the CXCL13 ISH, providing additional validation of the spatial expression pattern. Together, these results support a model where CXCL13 is primarily secreted from mantle zones, then immobilized in germinal centers. To investigate if these findings extend to TLS in tumor tissues, we applied our CXCL13 IHC/ISH dual stain to lung cancer tissue from the definitive resection specimen from patients treated with neoadjuvant anti-PD-1 immunotherapy. Spatial analyses revealed that similar to what was observed in tonsil, the density of CXCL13 mRNA is highly correlated with CXCL13 protein staining (n=3, Spearman’s r: 0.9, p<9.311e-19), though these do not immediately overlap spatially, with an average distance of 30 μm between mRNA and protein expression, suggesting capture of the diffusing CXCL13. We also observed that stromal cells in mature TLS immobilize CXCL13, but stromal cells in immature TLS do not. In addition, we found CXCL13 immobilized on elastin fibers near mRNA staining. These findings show that CXCL13 immobilization may contribute to the formation and maintenance of TLS. New therapies enhancing the formation and maturation of TLS by modulating CXCL13 may warrant exploration. Ongoing single cell RNA sequencing and in vitro studies are focused on determining the mechanisms responsible for the immobilization of CXCL13 in lymphoid and tumor tissue. Citation Format: Jonathan Rex Skidmore, Aleksandra Ogurtsova, Grant Salvucci, Victoria Jacobs, Logan Engle, Robert A. Anders, Drew M. Pardoll, Janis Marie Taube. CXCL13 secretion and immobilization show distinct geographies within secondary and tertiary lymphoid structures (TLS) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 2219.

Mucosal-associated invariant T (MAIT) cells are innate-like T cells capable of MR1-dependent immune surveillance, but how intratumoral bacteria modulate MR1 expression in human lung tumors remains unclear. We studied intratumoral MAIT cells from paired single-cell RNA and TCR sequencing datasets of tumor-infiltrating CD3 T cells isolated from non-small cell lung cancer tumors in patients receiving neoadjuvant PD-1 blockade therapy. MAIT cells were subclustered to identify conventional MAIT-associated TCR clonotypes, which were then used to examine how bacterial exposure impacts cell-surface MR1 expression and downstream MAIT TCR activation. We found that select intratumoral Enterococcus species (spp.) did not directly activate MAIT cells but enhanced MR1-dependent MAIT activation in the presence of exogenous 5-OP-RU by increasing cell-surface MR1 expression on antigen-presenting cells including dendritic cells, B cells, and mononuclear phagocytes. This increase in MR1 cell surface expression is modulated through a posttranscriptional mechanism consistent with altered intracellular processing and trafficking of MR1. These findings reveal a role for tumor-associated bacteria in modulating MR1-dependent innate-like T cell activation and provide a basis for future studies examining whether this process influences response to immune checkpoint blockade.

Immune-stromal crosstalk governs tissue fibrosis, which is marked by dysregulated extracellular matrix (ECM) production and aberrant vasculature. Here, we investigate how γδ T cell interactions with stromal cells shape fibrosis in the foreign body response. During the acute reaction, type-1 (γδIFNγ) and type-17 (γδ17) effector subsets accumulated at the implant. While γδIFNγ decreased as fibrosis progressed, activated γδ17 persisted as dominant interleukin-17 producers. The γδ17 increased with aging and high-fat diet, both factors associated with chronic inflammation and fibrosis. Co-culture with γδ17 stimulated fibroblast expression of collagen genes and intercellular communication inference linked γδ T cell ligands to activation of ECM remodeling and vascular development programs in fibroblasts and endothelial cells. Finally, genetic deletion of γδ T cells altered expression of ECM components and increased vessel size within the fibrotic matrix. Altogether, our findings implicate γδ T cells in regulating stromal behavior to modulate composition and vascularity of fibrotic tissues.

Lymphocyte activation gene 3 (LAG-3) is an immune checkpoint implicated in T cell exhaustion and a potential therapeutic target in glioblastoma (GBM). We conducted a multicenter, open-label, phase 1 study with sequential allocation to evaluate the safety and preliminary activity of the anti-LAG-3 antibody relatlimab, administered alone or with the anti-programmed cell death protein 1 (PD-1) antibody nivolumab, in patients with recurrent GBM. Forty-six patients were treated (23 per cohort). The primary endpoint of safety was met, with maximum tolerated doses of 800 mg relatlimab for monotherapy and 160 mg relatlimab/240 mg nivolumab for combination therapy. Treatment-related grade 3-4 adverse events occurred in 6 of 23 patients receiving combination therapy and were not observed with monotherapy. Neoadjuvant administration was associated with increased intratumoral CD8+ T cell infiltration for both monotherapy and combination therapy. Exploratory analyses suggested that tumors with elevated baseline interferon signaling and increased T cell clonality were enriched among patients with durable responses to combination therapy. Twelve-month overall survival was 34.8% with relatlimab alone and 52.2% with combination therapy; however, this study was not designed to assess efficacy. These findings demonstrate an acceptable safety profile and provide preliminary immunologic and clinical signals supporting further evaluation of LAG-3 blockade in GBM. ClinicalTrials.gov identifier: NCT02658981 .

Chimeric antigen receptors (CARs) can induce T cells to kill cancer cells but also to kill normal cells that express the same antigens1. Designing CARs to recognize combinations of antigens, via Boolean logic, can simultaneously expand the scope of targetable antigens and make CAR T cells more specific to cancer2. For example, one antigen may be expressed on cancer cells and normal bone marrow cells, while a second antigen may be present on the same cancer cells but only in normal lungs. If recognition of both antigens is required for T cell activation, only the cancer cells will be killed. Creating such AND-gated CAR T cells has been challenging given the need to engineer non-natural signaling mechanisms that integrate two ligand binding events into a single T cell activation stimulus3-6. Here, we design a fundamentally new AND-gated receptor called M ulti- AN tigen T riggered I mmune S ynapse (MANTIS), which leverages differences in extracellular receptor dimensions to regulate CAR signaling. MANTIS initially prevents CAR activity by steric blocking with a bulky extracellular domain. Upon engagement of the first antigen, MANTIS sheds this blocking domain, releasing a free CAR that can bind a second antigen and activate the T cell in an AND-gated manner. This work demonstrates how differences in extracellular receptor size can be leveraged to spatially regulate intracellular signaling pathways in response to antigen patterns, paving the way for new applications in synthetic biology and cell engineering.

Figure S5: Ccr6 ablation reduces Treg immunosuppression of CD8 T cells in the context of tumor growth.

Figure S2: CCR6 expression is enhanced in tumor-infiltrating Tregs co-expressing checkpoints and Ccr6 ablation reduces immunosuppressive phenotype.

Figure S6: Ccr6 ablation delays tumor growth across multiple tumor models and CCR6CCL20 expression correlates with overall survival in cancer patients.

The gut microbiota influences systemic immunity and the function of distal tissues, including the brain, liver, skin, lung, and muscle. However, the role of the gut microbiota in the foreign body response and fibrosis is largely unexplored. To investigate this connection, we perturbed the homeostasis of the murine gut microbiota via infection with the pathogenic bacterial species enterotoxigenic Bacteroides fragilis (ETBF) and implanted particulate material (mean particle size <600 μm) of the synthetic polymer polycaprolactone (PCL) into a distal muscle injury. ETBF infection in mice led to increased neutrophil and γδ T cell infiltration into the PCL implant site. ETBF infection alone promoted systemic inflammation, increased levels of neutrophils in lymphoid tissues, and altered skeletal muscle gene expression. At the PCL implant site, we found significant changes in the transcriptome of sorted stromal cells between infected and control mice, including differences related to ECM components such as proteoglycans and glycosaminoglycans. However, we did not observe ETBF-induced differences in fibrosis levels. These results demonstrate the ability of the gut microbiota to mediate long-distance effects such as immune and stromal responses to a distal biomaterial implant.

KM curvces for recurrence based on stage and RVT

Programmed cell death protein-1 (PD-1) inhibitors plus chemotherapy have been the standard of care in the first-line treatment of advanced gastric or gastroesophageal junction (G/GEJ) adenocarcinoma; however, the survival benefits are modest in patients with low programmed death ligand 1 (PD-L1) expression. Here we investigated the efficacy and safety of cadonilimab (PD-1/cytotoxic T lymphocyte antigen-4 (CTLA-4) bispecific antibody) plus chemotherapy as first-line treatment in G/GEJ adenocarcinoma. The prespecified interim analysis is reported here. This was a randomized, double-blind, placebo-controlled phase 3 study. Eligible patients were adults with untreated, unresectable, locally advanced or metastatic G/GEJ adenocarcinoma. Patients were randomized 1:1 to receive cadonilimab (10 mg kg−1 every 3 weeks) or placebo plus chemotherapy (every 3 weeks). The primary endpoint was overall survival (OS) in the intention-to-treat population (one-sided significance level, P = 0.025). Secondary endpoints included OS in patients with a PD-L1 combined positive score ≥5, progression-free survival, objective response rate, duration of response and safety. As of 18 August 2023, 610 patients from 75 study centers were randomized to cadonilimab (n = 305) or placebo (n = 305). With a median follow-up of 18.7 months, the cadonilimab group had a significantly longer median OS (14.1 versus 11.1 months; hazard ratio (HR) 0.66; 95 NCT05008783 . In a prespecified interim analysis of the randomized, double-blind phase 3 COMPASSION-15 trial, patients with advanced HER2-negative gastric/GEJ cancer treated with the anti-PD-L1/CTLA-4 bispecific Ab cadonilimab plus chemotherapy showed significantly improved overall survival compared with patients treated with placebo plus chemotherapy as first-line treatment.

Immune checkpoint blockade (ICB) is standard of care in advanced diffuse pleural mesothelioma (DPM), but its role in the perioperative management of DPM is unclear. In tandem, circulating tumor DNA (ctDNA) ultra-sensitive residual disease detection has shown promise in providing a molecular readout of ICB efficacy across resectable cancers. This phase 2 trial investigated neoadjuvant nivolumab and nivolumab/ipilimumab in resectable DPM along with tumor-informed liquid biopsy residual disease assessments. Patients with resectable epithelioid/biphasic DPM enrolled sequentially to nivolumab 240 mg every 2 weeks (q2w) for three cycles (Arm A, n = 16) or nivolumab 3 mg kg-1 q2w for three cycles plus ipilimumab 1 mg kg-1 on cycle 1 (Arm B, n = 14), followed by surgery, optional chemotherapy and/or radiotherapy, and nivolumab 480 mg q4w for 1 year. Co-primary endpoints included safety and feasibility; key exploratory endpoints included progression-free survival (PFS), overall survival (OS) and ctDNA analyses. The trial met its primary endpoints, and, in Arms A and B, 81.3% and 85.7% of patients proceeded to surgery, respectively. Treatment was safe, with a single dose-limiting toxicity in each arm. In Arm A, median PFS and OS were 9.6 months (95% confidence interval (CI): 2.5-27.7) and 19.3 months (95% CI: 14.9-34.7), respectively. In Arm B, median PFS and OS were 19.8 months (7.1-not reached) and 28.6 months (20.4-not reached), respectively. Persistent ctDNA was detected during neoadjuvant therapy in patients who did not undergo complete surgical resection due to disease progression (Fisher's exact test, P = 0.00013). Patients with detectable ctDNA on cycle 3 and pre-surgery had shorter PFS (log-rank test, P = 0.027 and P = 0.0059, respectively); this association was more pronounced when quantitative ctDNA changes were considered (log-rank test, P = 1.8 × 10-6). Our findings support the feasibility of neoadjuvant ICB and the clinical utility of ctDNA analyses to capture residual disease in resectable DPM. ClinicalTrials.gov identifier: NCT03918252 .