Citations

Abstract Recent evidence has highlighted the critical requirement for “immune triads” of CD8+ T cells, CD4+ T cells and dendritic cells (DC) for tumor elimination in response to checkpoint inhibition (CPI) immunotherapy. However, the mechanistic underpinnings of this cross-talk are poorly understood. Immune triads are often located in peripherally-distributed DC-rich niches within the tumor stroma. We have hypothesized that stromal remodeling in response to tumor expansion generates homeostatic signals that trigger an adaptive immune response that may ultimately fail through effector exhaustion or apoptosis. In particular, stromal proteolysis of the large matrix proteoglycan versican (VCAN) has been associated with T-cell infiltration across multiple tumor types. The objective of this study was to interrogate a mechanistic link between stromal remodeling, immune triad formation and response to CPI. We used HALO image analysis to determine the spatial relationship of CD4+ and CD8+ T cells in a panel of 95 human non-small cell lung cancers. Each tumor was VCAN proteolysis-scored based on a binning system measuring staining intensity for a neoepitope exposed through site-specific VCAN proteolysis. Tumors displaying VCAN proteolysis intensity of zero (0) had an average of 0.122 CD8+ T cells located with 10um of a CD4+ T cell vs. 0.216 in tumors with any VCAN proteolysis (intensity 1, 2, 3 combined); p=0.04. Similar results were obtained when measuring proximity of CD4+ to CD8+ T cells (0.124 vs 0.223, p=0.02). These data demonstrate that remodeling stroma promotes CD4+:CD8+ crosstalk. Site-specific VCAN proteolysis releases a 441 N-terminal bioactive fragment, versikine. Versikine constitutes a classical “matrikine”, i.e., a bioactive matrix fragment possessing novel functions compared to its parent macromolecule. To determine the mechanisms by which versikine may promote immune triad niches, we generated several transplantable syngeneic tumor cell lines engineered to stably secrete versikine into the TME. One CPI-refractory tumor cell line (Lewis Lung Carcinoma, LLC) and two partially CPI-responsive transplantable tumor lines (CT26 and MC38 colorectal carcinoma) were extensively characterized through flow cytometry, spatial immunofluorescence and scRNAseq. Our results demonstrate that in all contexts, versikine results in expansion of Tfh-like CD4+ T cells expressing Cd40lg and Icos. This expansion is associated with specific patterns of CD8+ T cell exhaustion that will be discussed. Therapeutically, versikine reversed anti-PD-1 pathway refractoriness in the poorly-immunogenic LLC model and demonstrated superior synergy with CPI in the more immunogenic CT26 and MC38 models, leading to tumor eradication with resistance to tumor rechallenge. Our data bolster the mechanistic rationale for exploitation of stromal remodeling signals to promote CD4+T:CD8+T:DC cross-talk for enhanced CPI efficacy. Citation Format: Daniel Lagal, Duncan Hong, Athanasios Papadas, Alicia Gibbons, Yun Huang, Yaling Dou, Alexander Cicala, Elsa Molina, Kristina Matkowskyj, Dustin Deming, Fotis Asimakopoulos. Stromal remodeling promotes immune triad formation in the tumor microenvironment (TME) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 2588.

Abstract Chimeric antigen receptor T (CART) cell therapy has shown clinical successes in B cell hematological malignancies. Two B cell maturation antigen (BCMA)-directed CART cell therapies treating multiple myeloma (MM) were approved by FDA but most patients relapse within 1-2 years. Ex vivo-expanded antigen specific T cells as cancer treatment rely on TCR-mediated killing of tumors. We hypothesized that arming ex vivo enriched tumor-specific T cells with CARs targeting a different antigen improves T cell functions. First, we expanded mucin 1 (MUC1)-activated T cells from patients with MM through ex vivo stimulation by MUC1 peptides-loaded autologous PBMCs for 19 days. Then, we engineered MUC1-activated T cells with 4-1BB-co-stimulated BCMA CAR to generate MUC1 TCR+BCMA CAR+ T cells. Baseline T cells without MUC1 stimulation were engineered with BCMA CAR as control. Baseline T cells, MUC1 T cells, baseline BCMA CART cells, or MUC1 BCMA CART cells were cocultured with luc+ RPMI-8226, a MUC1+BCMA+ MM cell line, for 3 days. MUC1 BCMA CART cells showed stronger tumor killing than BCMA CART or MUC1 T cells in all 3 patients (p<0.0001). MUC1 BCMA CART showed improved proliferation than BCMA CART or MUC1 T cells (p=0.0765, p<0.0001, respectively) after a 5-day stimulation by RPMI-8226. To exclude possibility of metabolic alterations by 19-day PBMC incubation leading to better T cell functions in MUC1 BCMA CART, baseline and MUC1-stimulated T cells from 3 healthy donors were processed for the same assays. Day-19 BCMA CART cells showed inferior tumor killing compared to baseline BCMA CART in all 3 donors (p<0.0001). Lower T cell proliferation was also observed in day-19 BCMA CART (p=0.0838). Our data indicate that improved T cell functions in MUC1 BCMA CART were unlikely due to metabolic changes and required stimulation by tumor antigens prior to CAR engineering likely through MUC1 TCR. Additionally, MUC1 T cells, BCMA CART, or MUC1 BCMA CART cells from tested patients were stimulated by RPMI-8226 for bulk RNA sequencing. Both BCMA CART and MUC1 T cells had distinct transcriptomic features from MUC1 BCMA CART cells. Differential gene expression analysis showed upregulation of T cell function-associated genes (RYR2, ITGA1, GNLY, GZMA, etc.) in MUC1 BCMA CART vs BCMA CART. Enrichment of T cell function-associated pathways was also observed in MUC1 BCMA CART. Analysis between MUC1 T cell and MUC1 BCMA CART showed upregulation of T cell function-associated genes (CSF2, CRTAM, IFNG, GZMB, etc.) and enrichment of immune response pathways in MUC1 BCMA CART. Our findings suggest that equipping ex vivo-enriched tumor-specific T cells with CARs targeting a different antigen improves T cell functions in vitro. Importantly, this provides a rationale for a therapeutic regimen where patients receive ex vivo-enriched tumor-induced T cells followed by CAR engineering. Persistence of the engineered T cells is being investigated Citation Format: Kun Yun, R. Leo Sakemura, Ismail Can, Omar Gutierrez Ruiz, Ekene J. Ogbodo, Truc N. Huynh, Claudia Manriquez Roman, Olivia Sirpilla, Carli M. Stewart, Jennifer M. Feigin, Grace E. DeFranco, Sophia Y. Goldberg, Dominic A. Skeele, Mariam Stein, Dara Missan, Latha Pathangey, Long Mai, Hong Xia, Brooke Kimball, Lionel A. Kankeu Fonkoua, Mehrdad Hefazi, Michael W. Ruff, Elizabeth L. Siegler, Leif Bergsagel, Michael Gustafson, Sandra Gendler, Saad S. Kenderian. BCMA CAR engineering arms T cells with superior functions post ex vivo stimulation by MUC1 peptide-loaded autologous PBMCs [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 6103.

Background and Aims: Immunotherapy has emerged as an effective treatment for advanced HCC. We aimed to investigate the real-world effectiveness of immunotherapy compared to lenvatinib in HCC. Approach and Results: From the TriNetX database, we used a target trial emulation framework and identified patients with HCC who received first-line treatment with immunotherapy (atezolizumab/bevacizumab or tremelimumab/durvalumab) or lenvatinib between or between August 2018 and December 2023. OS was compared using Kaplan-Meier analysis and Cox proportional hazards regression. After propensity score matching, 1203 patients were included in each group. Immunotherapy was associated with improved OS versus lenvatinib (median survival: 545 vs. 425 d; HR: 0.86, 95% CI: 0.76–0.97). Regarding treatment type, atezolizumab plus bevacizumab showed improved survival compared to lenvatinib (n=1070 in each group; HR: 0.87, 95% CI: 0.77–0.99), while the point estimate favored durvalumab plus tremelimumab versus lenvatinib (HR: 0.81, 95% CI: 0.59–1.12), though this difference was not statistically significant, likely due to small sample size. Regarding etiology, immunotherapy had improved OS compared to lenvatinib in viral hepatitis (n=510 in each group; HR: 0.74, 95% CI: 0.61–0.89) and alcohol-associated liver disease (n=190 in each group; HR: 0.65, 95% CI: 0.49–0.87), but not in metabolic dysfunction–associated steatotic liver diseases (n=156 in each group; HR: 0.96, 95% CI: 0.70–1.31). Conclusions: In this real-world analysis, immunotherapy was associated with improved OS compared to lenvatinib in advanced HCC, with consistent benefit across most subgroups. These findings support the use of immunotherapy as a first-line treatment for advanced HCC.

Importance The use of immune checkpoint inhibitors (ICIs) is increasing. Little is known about the frequency of late-onset immune-related adverse events (irAEs) and the patient-specific risk factors associated with their development. Objectives To assess the incidence of persistent or de novo late-onset irAEs requiring hospitalization and identify patient factors associated with risk of late-onset irAEs. Design, Setting, and Participants This retrospective observational cohort study conducted from January 2011 to October 2022 included patients who received ICIs and were hospitalized with irAEs at an academic medical center. Exclusion criteria included ICI therapy outside of the hospital system and no irAE diagnosis during admission. Data were analyzed from November 15, 2022, to January 8, 2025. Exposure Late-onset irAEs. Main Outcomes and Measures The main study outcomes were (1) incidence of irAE hospitalization at 0 to 6 months (early), more than 6 to 12 months (intermediate), and more than 12 months (late) after ICI initiation and (2) patient factors associated with risk of late-onset irAEs. Results Among the 795 patients hospitalized with irAEs, the median age was 67.3 years (IQR, 58.3-74.8 years); 476 (59.9%) were male. Most patients (n = 517 [65.0%]) received anti–programmed death ligand 1 (PD-L1) and anti–programmed cell death 1 monotherapy, with the most common indications being melanoma (n = 335 [42.1%]) and lung cancer (n = 167 [21.0%]). The median time from start of ICI therapy to hospital admission was 2.7 months (IQR, 1.2-6.1 months), with 14.7% of patients (n = 117 of 795) presenting 6 to 12 months after initial ICI exposure and 10.8% of patients (86 of 795) presenting more than 12 months after initial exposure. The irAEs most likely to present late included those involving the kidney (10 of 32 [31.3%]) and hematologic (5 of 23 [21.7%]) organ systems. In univariate analysis, ICI type was significantly associated with the timing of hospital admission for irAEs; of the 517 patients receiving anti–PD-L1–based therapy, 13.5% (n = 70) presented late compared with 5.4% (9 of 167) receiving dual therapy with anti–cytotoxic T-lymphocyte–associated protein 4 ( P < .001). Patients receiving perioperative ICI therapy were significantly more likely to be admitted at the intermediate interval (16 of 68 [23.5%]) compared with those with metastatic disease (87 of 678 [12.8%]) ( P = .03). Timing of irAE was also significantly associated with active ICI exposure; among the patients presenting late, 7.4% (48 of 651) had received ICI therapy within the last 60 days compared with 26.4% (38 of 144) who had not had recent ICI exposure ( P < .001). Conclusions and Relevance The findings of this retrospective observational cohort study suggest that late irAEs are possible, with a subset of patients presenting years after the start of ICI therapy. Clinicians must remain vigilant for irAEs regardless of elapsed time from ICI therapy, especially as patients live longer and ICIs become more widely used. Future investigations are needed to better understand the risk factors for late-onset irAEs and the distinct immunologic pathways that underlie such events.

Abstract Germinal center B-cell lymphomas frequently exhibit monoallelic loss-of-function mutations in the histone acetyltransferases CREBBP and p300, which contribute to lymphoma development, disrupt normal germinal center biology, and promote immune evasion. This study evaluates YF2, a histone acetyltransferase activator that increases CREBBP/p300 activity, as a potential therapeutic approach for B-cell lymphoma. YF2 binds the bromodomain of CREBBP/p300, increasing their auto-acetylation and enzymatic activity. It also induces cytotoxicity in B-cell lymphoma cell lines, with a stronger response in those carrying CREBBP / EP300 mutations. Additionally, YF2 increases the acetylation of key CREBBP/p300 substrates, including H3K27, p53, and BCL6, leading to enhanced apoptosis and altered B-cell diTerentiation. YF2 is well tolerated in vivo and extends survival in both cell line- and patient-derived xenograft mouse models. Moreover, YF2 exposure upregulates antigen-presentation markers and reshapes the immune microenvironment, strengthening responses to immune checkpoint blockade. Taken together, these findings support pharmacologic activation of CREBBP/p300 with YF2 as a compelling therapeutic strategy for B-cell lymphoma. Significance YF2 stimulates CREBBP/p300 activity to normalize B-cell diTerentiation and enhance immune recognition, oTering a potential approach to limit lymphoma progression and strengthen immunotherapy responses.

Peroxisomes are vital but often overlooked metabolic organelles. We found that excessive interferon signaling remodeled macrophage peroxisomes. This loss of peroxisomes impaired inflammation resolution and lung repair during severe respiratory viral infections. Peroxisomes were found to modulate lipid metabolism and mitochondrial health in a macrophage type-specific manner and enhanced alveolar macrophage-mediated tissue repair and alveolar regeneration after viral infection. Peroxisomes also prevented excessive macrophage inflammasome activation and IL-1β release, limiting accumulation of KRT8 high dysplastic epithelial progenitors following viral injury. Pharmacologically enhancing peroxisome biogenesis mitigated both acute symptoms and post-acute sequelae of COVID-19 (PASC) in animal models. Thus, macrophage peroxisome dysfunction contributes to chronic lung pathology and fibrosis after severe acute respiratory syndrome coronavirus 2 infection.

Abstract Background Morbidity from bacteremia may be exacerbated by inadequate initial antibiotic therapy. This concern however may lead to hesitance to de-escalate while awaiting pathogen identification and susceptibility results. We performed a randomized controlled trial evaluating the impact of a strategy for rapid pathogen and resistance detection directly from positive blood culture bottles (BCBs) using a rapid multiplex PCR (rmPCR) complemented with a rapid phenotypic screen (RPS) for extended-spectrum beta-lactamases and carbapenemases in a setting with moderate to high levels of antimicrobial resistance where routine direct-from-BCB disk diffusion susceptibility testing was in place Methods Patients with positive BCBs were randomized to 2 arms: 1) Standard BCB processing (including direct from BCB disk diffusion susceptibility testing - control, n=419) with antimicrobial stewardship (AS); or 2) rmPCR/RPS with AS (rmPCR/RPS, n=425). The primary outcome was time from positive blood culture result to prescription of an effective or optimal antibiotic regimen. An effective antibiotic regimen was defined as one active against the detected pathogen(s); an optimal antibiotic regimen was defined as an effective regimen which was also the most narrow spectrum and targeted one, as recommended by institutional guidelines. Mortality was analyzed as a secondary outcome. Results True bacteremia was identified in 658 patients (n=329 in both arms); 597 were monomicrobial (405 with aerobic or facultative anaerobic Gram negatives rods [AGNR]) and 61 polymicrobial (41 harboring AGNR). Time from BCB Gram stain to prescription of an effective antibiotic was 9.9 versus 3.2 hours in the control and rmPCR/RPS groups, respectively (p=0.58) and time from Gram stain to prescription of an optimal antibiotic regimen was 41.6 versus 29.4 hours in the two groups, respectively (p < 0.001). 14-day mortality was 7.6% versus 4.3% (p = 0.069) and 30-day mortality was 11.2% versus 8.8% (p=0.30) in the two groups, respectively. Conclusion Compared to control methods which included direct-from-BCB disk diffusion susceptibility testing, an intervention comprising rmPCR/RPS with AS applied to positive blood cultures improved time to optimal therapy but not time to effective therapy or mortality. Disclosures Shawn Vasoo, MBBS, MRCP, D(ABP), D(ABIM) (Inf Dis), FRCPath, bioMerieux: In-kind, for this study|Rosco Diagnostica: In-kind, for this study Robin Patel, MD, a patent on Bordetella pertussis/parapertussis PCR issued, a patent on a device/method for sonication with royalties paid by Samsung to Mayo Clinic, a: See above|MicuRx Pharmaceuticals and BIOFIRE: Grant/Research Support|PhAST, Day Zero Diagnostics, Abbott Laboratories, Sysmex, DEEPULL DIAGNOSTICS, S.L., Netflix, Oxford Nanopore Technologies and CARB-X: Advisor/Consultant|Up-to-Date and the Infectious Diseases Board Review Course.: Honoraria Paul Tambyah, MBBS (S'pore), Diplomate, American Board of Internal Medicine and Infectious Diseases, Moderna: Grant/Research Support|Sanofi-Pasteur: Grant/Research Support

Abstract Attempts to activate an anti-tumor immune response in glioblastoma (GBM) have been met with many challenges due to its inherently immunosuppressive tumor microenvironment. The degree and mechanisms by which molecularly and phenotypically diverse tumor-propagating glioma stem cells (GSCs) contribute to this state are poorly defined. In this study, our multifaceted approach combining bioinformatics analyses of clinical and experimental datasets, single-cell sequencing, and molecular and pharmacologic manipulation of patient-derived cells identified GSCs expressing immunosuppressive effectors mimicking regulatory T cells (Tregs). We show that this I mmunosuppressive T reg- L ike (ITL) GSC state is specific to the mesenchymal GSC subset and is associated with and driven specifically by TGF-β type II receptor (TGFBR2) in contrast to TGFBR1. Transgenic TGFBR2 expression in patient-derived GBM neurospheres promoted a mesenchymal transition and induced a 6-gene ITL signature consisting of CD274 (PD-L1), NT5E (CD73), ENTPD1 (CD39), LGALS1 (galectin-1), PDCD1LG2 (PD-L2), and TGFB1. This TGFBR2-driven ITL signature was identified in clinical GBM specimens, patient-derived GSCs and systemic mesenchymal malignancies. TGFBR2 High GSCs inhibited CD4+ and CD8+ T cell viability and their capacity to kill GBM cells, effects reversed by pharmacologic and shRNA-based TGFBR2 inhibition. Collectively, our data identify an immunosuppressive GSC state that is TGFBR2-dependent and susceptible to TGFBR2-targeted therapeutics.