Browse Articles

Immunological targeting of the endogenous retroviral protein ERVMER34-1 in carcinomas promotes epitope spread and enhances response to PD-L1 blockade 2306293

Abstract Introduction Endogenous retroviruses (ERVs) are remnants of germline retroviral infections that occurred over evolution and make up 5—8% of the human genome. Although ERVs are typically epigenetically silenced in healthy adult tissues, they are overexpressed in carcinomas and may represent a novel pool of immunotherapeutic targets. This study characterizes the ERV envelope protein ERVMER34-1 as a therapeutic target in cancer. Methods The expression of ERVMER34-1 in healthy adult and cancer tissues was assessed by IHC. Its immunogenicity in human PBMCs was evaluated by ELISPOT assays, and the lytic ability of specific T cells was assessed by live-cell imaging assays. In addition, a rationally designed ERVMER34-1—targeted therapeutic vaccine was tested for its ability to induce tumor clearance in two murine carcinoma models, as monotherapy or in combination with immuno-oncology agents. Results The ERVMER34-1 protein is overexpressed in several human carcinomas while being absent in most healthy adult tissues. ERVMER34-1—specific T cells were detected in PBMCs of cancer patients but not healthy donors after overnight stimulation, yet reactive T cells expand readily from both groups after 7 days of in-vitro stimulation. These T cells selectively kill human carcinoma cell lines expressing ERVMER34-1. A rationally designed therapeutic vaccine targeting ERVMER34-1 mediated tumor control in two syngeneic murine tumors. When combined with checkpoint blockade, the vaccine induced tumor regression and promoted the expansion of neoepitope-reactive T cells, whose function was further enhanced by an FDA-approved IL-15 superagonist. The strong neoepitope response associates with an inflamed tumor microenvironment marked by increased CD8+ T-cell infiltration and improved tumor control. Conclusion This study supports the clinical development of a therapeutic cancer vaccine targeting the retroviral envelope protein ERVMER34-1, which would represent a new class of therapeutic cancer vaccine targeting ERVs. Funding Source This study was supported by the Intramural Research Program of the Center for Cancer Research, NCI, NIH Topic Categories Tumor Immunology: Checkpoints, Prevention, and Treatment (TIPT)

Strain-specific variation drives CD8+ T cell activation responses 2305504

Abstract Introduction In the rapidly evolving immune system, genetic variation between individuals is a major driver of differential responses to infection. Previous studies in human and mouse have linked gene expression changes within immune cells to variable immune response between individuals. However, the regulatory impacts of genetic variation on genes driving immune cell activation are not well understood. Here, we used eight recombinant inbred mice strains (which capture nearly 90% of the genetic variation across the mouse genome) and the Diversity Outbred (DO) mice as models of genetic variation to study strain-specific differences in the activation of CD8+ T cells. By activating CD8+ T cells through both TCR stimulation and bystander activation, we intend to characterize the genes driving differences in immune activation. Methods Using CD8+ T cells from the eight mice strains that underlie the DO mice, we 1) generated single-cell RNA-seq data from control, TCR stimulation, and bystander activation culture conditions, and 2) characterized proliferation and activation phenotypes before and after TCR stimulation and bystander activation. We also performed GWAS analyses on 90 DO mice using measures of activation and cell surface protein expression in CD8+ T cells to further link genotypes to activation phenotypes. Results We identified hundreds of differentially expressed genes across both TCR stimulation and bystander activation that also varied by strain in CD8+ T cells. Transcription factors with key roles in CD8+ T cell activation such as AP-1 family members Jun and Jund were differentially expressed across strains, alongside receptors such as Il7r and Il18r1. Our preliminary GWAS analyses on DO mice corroborated strain-specific expression of Il18r1 and provided further loci for investigation. Conclusion For the first time, we have identified hundreds of genes that differ across activation and strain underlying TCR stimulation and bystander activation in CD8+ T cells across eight mice strains. Funding Source F31AI183775 (NIAID F31 to Jason Chobirko); R01AI110613 (NIAID R01 to Andrew Grimson); U24AI152176 (NIAID U24 to Andrew Grimson) Topic Categories Computational and Systems Immunology (COMP)

Lysosomal permeabilization by Group A Streptococcus releases proteins into the macrophage cytosol 2256825

Abstract Introduction Although macrophages are important for controlling Group A Streptococcus (GAS) infection, we and others have demonstrated that GAS can persist in macrophages by perforating the phagolysosome using the pore-forming toxin streptolysin O (SLO). In this study, we identified lysosomal and bacterial proteins released into the cytosol as a consequence of phagosomal perforation. Methods We prepared cytosolic preparations from macrophages infected with either wild-type (WT) or SLO-deficient (α”SLO) bacteria and uninfected controls and verified lysosomal or bacterial proteins were present using IL-1β as a measure of intracellular pathogen detection. Proteomic analysis revealed distinct cytosolic protein profiles in both WT- and α”SLO-infected macrophages. Results Bacterial M1 protein was detected only in the cytosol of WT-infected macrophages and corresponded with an IL-1β response, indicating SLO-mediated release of M1 protein from the phagosome, and providing a mechanism for cytosolic recognition of this virulence factor. Unexpectedly, cytosolic extracts of both WT- and α”SLO-infected macrophages contained all histone proteins, suggesting that nucleosomal complexes are released into the cytosol during GAS infection. We confirmed the presence of histones and the absence of contaminating nuclei in the cytosolic fraction by Western blot. DNA was not detected in the cytosol of GAS-infected cells, but histones were secreted into the extracellular medium. We are currently exploring the mechanism and purpose of this histone release. Conclusion Our data both confirms host cell detection of bacterial proteins after phagosomal perforation, as well as reveals the surprising profile of proteins altered during GAS infection. Funding Source NIH R15AI176429, AHA 17GRNT33410851, Occidental College Topic Categories Microbial, Parasitic, and Fungal Immunology (MPF)

Lattice‐Matched 2D Template Enables Efficient Cesium Tin Halide Perovskite Solar Cells (Adv. Mater. 45/2026)

Co‐Assembled Hybrid Interlayer Engineering for Enhanced Upper Interface Stability in Inverted Perovskite Solar Cells

ABSTRACT Engineering electron transport layer (ETL) interface is critical for high‐efficiency and long‐term stability in inverted perovskite solar cells (PSCs), yet co‐assembled hybrid interlayer are rarely explored for this upper interface. This work integrates 4‐aminobenzoate acid hydrochloride (4AA) with a dibenzo‐18‐crown‐6 (DB18C6) to construct a hybrid interlayer at ETL interface. The 4AA molecules intercalate into DB18C6 aggregates, homogenizing the monolayer and boosting surface coverage (from 0.57 to 0.79) and strengthening the interfacial dipole moment (from 2 to 7 Debye). This interlayer provides dual passivation, in which the ─NH 3 + and ─COOH groups of 4AA neutralize ionic defects, while DB18C6 optimizes perovskite crystallinity and energy level alignment. Therefore, modified devices achieve an efficiency of 26.33% (exceeding 22.92% of the control) with high open‐circuit voltage (V OC ) of 1.167 V and fill factor (FF) of 86.05% (compared to 1.130 V and 80.38% of the control). More importantly, the co‐assembled hybrid interlayer serves as a barrier against environmental and ionic degradation. The unencapsulated device demonstrates outstanding operational stability, retaining 93.2% of initial efficiency after 1000 h of maximum power point tracking. This work demonstrates a co‐assembly strategy to address efficiency and stability challenges at ETL interface, paving a reliable path toward high‐performance and stable inverted PSCs.

Prognostic accuracy of qsofa for in-hospital mortality among adult patients presenting with suspected infection to the Emergency Department in the Middle East

AI/ML-Accelerated Discovery of Oral α4β7 Integrin Inhibitors for Treating Inflammatory Bowel Disease 2327698

Abstract Introduction Integrins are essential cell-adhesion receptors; α4β7 mediates gastrointestinal homing of lymphocytes via MAdCAM-1 on gut endothelium. Inflammatory bowel disease (IBD) involves pathogenic infiltration of α4β7-positive T cells. Blocking α4β7 reduces intestinal inflammation, as demonstrated by the clinical efficacy of the therapeutic antibody vedolizumab. However, limitations of antibody therapy underscore the need for selective, orally available small-molecule α4β7 inhibitors. Methods We leveraged deep disease expertise and our AI/ML-driven drug discovery platform to accelerate progress toward the discovery of novel oral α4β7 inhibitors for IBD. Our modular, highly configurable platform orchestrates commercial and proprietary tools within a high-performance computing infrastructure and integrates optimized design—make—test—analyze cycles for compound optimization. These cycles are supported by a comprehensive screening cascade comprising multiple tiers of target- and indication-relevant assays, including non-cellular, in vitro assays, translational and in vivo models. Results Leveraging extensive literature data, and predictive modeling, we advanced from target identification to novel lead compounds in under 18 months. To maximize success and accelerate progress, we implemented multiple parallel and complementary strategies. The resulting promising compounds demonstrated potency and high selectivity for α4β7 over α4β1 in translational assays and in vivo models. Conclusion Our AI/ML-driven drug discovery platform enabled rapid progression to advanced oral α4β7 inhibitor leads with strong preclinical validation. These results demonstrate the power of our integrated approach to significantly accelerate timelines in small-molecule discovery. This strategy is broadly applicable beyond α4β7, offering a versatile solution for delivering innovative therapies. Funding Source n/a Topic Categories Therapeutic Approaches to Autoimmunity (THER)

Age-associated B cells predict coronary events in humans and aggravate murine atherosclerosis 2257113

Abstract Introduction Aging is a major risk factor for cardiovascular disease (CVD) and is linked to a functional decline of the immune system. B cells contribute to atherosclerosis progression by antibody secretion, antigen presentation and T cell regulation. During aging, CD11c+CD21low B cells accumulate, but it is unclear whether these age-associated B cells (ABCs) are linked with incident cardiovascular events in humans and if they contribute to atherosclerosis progression. Methods To investigate associations with first-time coronary events, circulating B cells were phenotyped with flow cytometry in a case-control study (N = 604) nested in the Malmö Diet and Cancer Study cohort with a median of 14 years follow up. Splenic B cells in young (5 months) and aged (21 months) female Ldlr-/- mice were characterized with single-cell RNA-sequencing coupled with BCR-sequencing. Atherogenicity was determined by transfer of ABCs to Ldlr-/- and Ldlr-/-Rag1-/- mice. Results ABCs were distinctly expanded in older (≥66 years), compared to middle-aged (≤52 years) individuals (P = 0.002) and accumulated in aged atherosclerotic mice. Coronary event cases had higher ABC counts than controls and regression analysis revealed an association to incident coronary events, independent of cardiovascular risk factors [odds ratio: 1.80 (95% CI 1.05-3.07), P = 0.032, comparing the 4th vs 1st quartile]. Adoptive transfer of ABCs promoted atherosclerotic lesion development. In Ldlr-/- Rag1-/- mice, ABCs also increased necrotic cores (P < 0.05). After transfer, B cells recovered from recipient mice expressed CD138 and secreted antibodies. Accordingly, we demonstrated that ABCs expressed plasma cell differentiation genes and showed the greatest clonal expansion within the B cell compartment, with extensive clonal overlap with plasma cells. Conclusion We show that ABCs predict first-time coronary events in humans and contribute to atherosclerosis in mice. ABCs may serve as potential prognostic and therapeutic targets for CVD. Funding Source Fondation Leducq Network (CHECKPOINT ATHERO); European Union (B-specific; Grant Agreement No. 101115159); ERA-CVD B-eatATHERO consortium, Dutch Heart Foundation (2019T107) and ERA4HEALTH MARGINALIZE MI consortium. Topic Categories Lymphocyte Differentiation and Peripheral Maintenance (LYM)

Inflammation in aging and Alzheimer’s progression is marked by dysfunctional T-regulatory cell states 2257412

Abstract Introduction Aging and Alzheimer’s disease (AD) are marked by progressive brain inflammation. Microglia can become sensitized during aging, potentiating immune activation. T-regulatory (Treg) cells, which help restrain inflammation by suppressing microglial activation, likely play a crucial role in the progression of Alzheimer’s and aging, but their activity remains poorly understood. In this study, we elucidate mechanisms of immune activation by classifying microglia and Treg activity in aged and AD mouse models. Methods Brain sections of wild-type (WT) 2—3 (n = 5), 6—8 (n = 4), 11-12 (n = 5), and 22-month mice (n = 5) and 6 (n = 3) and 12-month (n = 4) amyloid precursor protein/presenilin 1 (APP/PS1) mice were immunostained for IBA1 to visualize microglia and skeletonized analysis was performed in FIJI. Splenic Treg populations (CD4+Foxp3+) and activation status (CD25+/-) were investigated through flow cytometry. Conventional T cells were stained with CTV and cultured with Tregs to determine Treg suppressive capacity. Results There was a significant increase in microglial activation in multiple brain regions with aging. Within the S1BF region, aging increased microglia density and promoted morphological changes such as decreased branch length, number, and end point voxels. Microglial staining was also increased with age in APP/PS1 mice. Aging increased Treg frequency and number in WT mice. Despite this increase, the frequency of activated CD25+Tregs was significantly decreased with age. Moreover, the proliferation index of Tregs was significantly higher in aged mice, suggesting a diminished suppressive response. Conclusion Age is associated with neuroinflammation. Here we show that while Tregs increase with age, the CD25+ activated subset decreases and their suppressive capacity is hampered, suggesting aging diminishes the functional capacity of Tregs. Funding Source This research was funded by a grant from the NIA. Topic Categories Neuroimmunology (NEUR)

Sex Differences in Platelet-Immune Responses in a Murine Model of Psychological Stress 2309588

Abstract Introduction Psychological stress is associated with systemic inflammation and platelet activation. Activated platelets serve important immune functions, such as recruiting leukocytes to sites of inflammation, but the responses of platelets and leukocytes to psychological stress are incompletely understood. Methods To induce psychological stress, young adult female and male C57BL/6J mice were exposed to restraint and predator scent two hours daily for three consecutive days, and compared to unmanipulated controls (n = 10 per group). Effectiveness of the paradigm was confirmed by measuring corticosterone in feces and epinephrine in urine by ELISA at the final stressor. Body weight was compared before and two days after the stress paradigm. Platelet P-selectin and MHC-I expression were measured by flow cytometry two hours after the stress paradigm. Complete blood cell counts and flow cytometric analysis of circulating monocyte CD11b and CCR5 expression were performed two days or one week after the stress paradigm. Statistical analysis was conducted by Mann-Whitney U test with significance set at alpha = 0.05. Results Stress increased corticosterone levels and induced weight loss in both male and female mice, but epinephrine was increased in male mice only. Platelet P-selectin and MHC-I expression were increased approximately twofold in stressed males, and accompanied by a threefold decrease in circulating CD11b+ and CCR5+ classical monocytes within two days of the stress paradigm. Female mice did not demonstrate platelet activation, and a decrease in circulating CD11b+ and CCR5+ classical monocytes was not noted until one week after application of stress. Conclusion These data demonstrate sex differences in platelet-immune responses to psychological stress. Given that epinephrine potentiates platelet activation, further studies will query if epinephrine-driven platelet activation in male mice contributes to redistribution of activated monocytes out of the blood in the context of psychological stress. Funding Source This research was funded by NIH T32-ES007020, P30-ES002109, and DoD W911NF-21-S-0008. Topic Categories Cellular Adhesion, Migration, and Inflammation (CAM)

Neurotrophic receptor GFRα2 drives pro-metastatic programming of Kupffer cells in liver metastases 2257034

Abstract Introduction Gastrointestinal cancers such as colorectal and pancreatic cancers frequently metastasize to the liver, contributing significantly to cancer-related mortality and resistance to immune checkpoint inhibitors. Recent multidisciplinary studies have highlighted a functional and mechanistic interplay between nervous and immune systems in cancers, termed as neuroimmune axis. Besides, liver is known to have rich innervation. Therefore, targeting neuroimmune regulatory molecules on immune cells to facilitate liver metastasis treatment is possible. Methods Our study employed analysis of public available single-cell omics datasets from patients and donors. Single-cell sequencing, high-dimensional flow cytometry and multiplex-immunofluorescence analysis in mice and patients with liver metastasis were also applied in this study. Results Glial cell line-derived neurotrophic factor (GDNF) family receptor alpha 2 (GFRα2) was found to have elevated expression on colorectal cancer liver metastasis (CRLM) adjacent livers compared with healthy livers. It was further confirmed that GFRα2 was selectively expressed and was upregulated on Timd4+Clec4f+Kupffer cells (KCs). Notably, GFRα2+KCs were found in close proximity to liver-infiltrating nerve fibers in CRLM, whereas chemical liver denervation led to reduced expression of GFRα2, suggesting a possible neural regulation on GFRα2+KCs. Functionally, lentiviral shRNA-mediated Gfra2 knockdown significantly suppressed liver metastasis without affecting primary colorectal tumor growth, which was abolished by KCs depletion. Mechanistically, GFRα2+KCs displayed impaired antigen presentation capacity, but enhanced pro-angiogenesis and neutrophil recruitment, suggesting a reprogrammed, metastasis-promoting phenotype. Gfra2 knockdown significantly reduced neutrophil accumulation and angiogenesis, but increased MHCII+KCs. Conclusion Taken together, our findings pinpointed that GFRα2 upregulation reprograms KCs towards a pro-metastatic state, thereby facilitate liver metastasis. Funding Source This project is supported by the Hong Kong University Grants Committee through the General Research Fund (14107622, 24110323) and the Strategic Seed Funding for Collaborative Research Scheme (SSFCRS) from the Chinese University of Hong Kong. Topic Categories Tumor Immunology: Checkpoints, Prevention, and Treatment (TIPT)

Enhanced Neutrophil Depletion in a Lung Tumor Model Using Recombinant IgG2c Anti-Ly6G [1A8] Antibody 2307287

Abstract Introduction Neutrophils are key modulators of tumor progression and immune responses. Ly6G-neutralizing antibodies are widely used to study neutrophil biology, yet their long-term depletion efficiency varies. We compared two anti-Ly6G [1A8] antibody formats -rat IgG2a and recombinant mouse IgG2c- in a KP intratracheal (KPi.t.) lung tumor model to assess sustained neutrophil depletion and tissue clearance. Methods KPi.t. mice (n = 3/group) were randomized into four treatment arms, anti-Ly6G [1A8] rat IgG2a or mouse IgG2c and corresponding isotype controls, and administered intraperitoneal injections of the indicated antibodies at 200μg per mouse every three days. Peripheral blood was collected on days 0, 3, and 12 to monitor neutrophil depletion. Mice were sacrificed on day 15, and blood and lung tissues were analyzed for CD11b+ Gr-1+ cells by flow cytometry. Results The recombinant mouse IgG2c anti-Ly6G [1A8] achieved robust and sustained depletion of circulating neutrophils throughout the 15-day study. In contrast, the rat IgG2a format showed partial recovery of CD11b+ Gr-1+ cells by day 12. Lung analysis revealed near-complete neutrophil clearance with mouse IgG2c, whereas rat IgG2a-treated mice retained 15 % parenchymal neutrophil CD11b+ Gr1+. Conclusion The mouse IgG2c anti-Ly6G [1A8] chimeric antibody (ab320708) demonstrated superior long-term efficacy compared to the rat IgG2a format. This optimized approach reduces injection frequency and hands on time whilst greatly improving animal welfare. It additionally enhances reliability in neutrophil depletion studies and supports deeper insights into neutrophil biology in cancer. Funding Source n/a Topic Categories Technological Innovations in Immunology (TECH)

Vaccine Misconceptions and Healthcare Workers 2306364

Abstract Introduction Despite vaccination being one of the most effective public-health interventions, vaccine confidence has recently declined. Widespread misinformation and reduced trust in public health agencies have created challenges for those in healthcare. Perceived gaps in federal communication further complicate clinical and educational information-seeking. As institutional credibility is questioned, healthcare professionals rely on fragmented online sources that amplify uncertainty and misinformation. Polling data show that parents report trusting non-medical influencers (i.e. Joe Rogan, RFK Jr., and the Children’s Defense Fund) for critical children’s health information. The politicization of science underscores the urgent need to strengthen immunology and vaccine literacy education across clinical training and healthcare professionals. Methods We conducted an IRB-approved, anonymous assessment (n = 7711) across those employed in healthcare/science (HCW/S) and non-healthcare workers (NHCW). Results Results showed that although HCW/S generally held more accurate vaccine perceptions than NHCW, misconceptions remained common. For the statement, “There is evidence that the mercury in the measles vaccine can cause autism”, HCW had a lower weighted mean score (1.97 vs. 2.35; n = 7711; p = 0.05). However, 32% still selected strongly agree, agree, or neither agree nor disagree, despite this statement being factually incorrect. Similar analyses were observed across other statements, including 33% of HCW believing the influenza vaccine can cause influenza. Conclusion Vaccine education rooted in core immunologic concepts and communication literacy is critical amid declining trust in traditional public-health authorities. Strengthening immunology curricula, CMEs, CEUs, role playing, discussions, etc. are critical to ensure that healthcare professionals can critically evaluate evidence, identify reliable sources, and counter misinformation to sustain a scientifically informed workforce. Funding Source n/a Topic Categories Immunology Education and Communication (EDU)

Increased expression of Response Gene to Complement-32 in PBMC and kidneys of patients with lupus nephritis 2247376

Abstract Introduction We have previously shown that response gene to complement-32 (RGC-32) promoted the Th17 and Th1 dependent proinflammatory and profibrotic responses in a murine model of immune complex mediated glomerulonephritis. The expression and function of RGC-32 in PBMC and kidney biopsies from patients with lupus nephritis (LN) have not yet been investigated. Methods RGC-32 expression was evaluated by RT-PCR, WB, flow cytometry and cDNA Array in PBMC from lupus patients and controls and in human proximal tubular epithelial cells (PTEC) and by immunohistochemistry in kidney biopsies from 25 patients with LN and 11 disease controls. Results RGC-32 mRNA expression was highest in CD3 cells followed by CD14 and CD19 cells and was increased in CD14 and CD19 cells from patients with vs those without renal disease. RGC-32 expression was induced by T cell stimulation under Th17 and Treg conditions and by serum from patients with high titer anti-dsDNA Ab. In kidneys, RGC-32 immunostaining was predominant in tubules and staining intensity was significantly higher in SLE than in non-SLE specimens. RGC-32 expression was also detected in glomeruli and in inflammatory cells in the interstitium, was significantly higher in LN specimens vs. controls and correlated with the activity (r = 0.4), chronicity (r = 0.5) and interstitial fibrosis scores (r = 0.5). RGC-32 colocalized with CD4+, CD68+ cells and C5b-9 deposition. In PTEC, RGC- 32 mRNA and protein expression was upregulated by IL-1, TNFα, TGFβ, IFNγ and sublytic C5b-9. TGFβ induced mRNA production of Collagen I and collagen III by PTEC was increased in RGC-32 transfected cells. Conclusion RGC-32 is upregulated in PBMC, glomeruli and tubulointerstitium of patients with LN. RGC-32 upregulation in PTEC is mediated by proinflammatory cytokines and complement activation and may play a role in organ damage by promoting manifestations of progressive renal disease such as interstitial fibrosis. Thus, RGC-32 is a potential therapeutic target in the treatment of LN. Funding Source N/A Topic Categories Immune Mechanisms of Human Disease (HUM)

Reversibly Cross‐Linked Damage‐Tolerant Polymers Breaking the Strength–Stretchability Trade‐Off

ABSTRACT Polymers that combine exceptional stretchability with high mechanical robustness are essential for advanced applications. To ensure their reliability, it is critical to integrate damage tolerance, which suppresses crack propagation and prevents catastrophic failure under extreme deformation. However, overcoming the intrinsic trade‐off between stretchability and strength remains a formidable challenge in polymer science, particularly when damage tolerance is also required. Here, we show the scalable fabrication of super‐stretchable polymers exhibiting exceptional mechanical robustness and remarkable damage tolerance, achieved by cross‐linking soft polymer chains through synergistic urea‐based hydrogen bonding and hydrophobic interactions. These polymers exhibit record‐high elongations up to ∼100,000 times their original length while maintaining an extensional true stress of 35.0 MPa at a strain of 33.6, and an extraordinary fracture energy exceeding 374.8 kJ m −2 . The extreme stretchability of these polymers arises from the successive breakage, chain slippage, and reformation of noncovalent cross‐links. Meanwhile, mechanical robustness and pronounced strain hardening are sustained by a strain‐induced transition of urea hydrogen bonds from double to quadruple configurations, together with the progressive orientation of polymer chains. These reversibly cross‐linked polymers, featuring intrinsic self‐healing and reprocessability, open broad opportunities for extremely deformable polymer materials where robustness, reliability, and sustainability are paramount.

Enhancing productivity and soil health in pearl millet-wheat cropping system with legume based integrated nutrient management

Virus-specific CD8+ T cells in murine glioblastoma originate from brain resident and circulating memory pools and exhibit bystander activation 2259751

Abstract Introduction Tumor-infiltrating CD8+ T cells expressing tissue residency markers (CD69/CD103) correlate with positive prognosis in numerous cancers. While tumor-infiltrating lymphocytes (TILs) were once thought to be largely tumor-specific, T cells specific for common viral infections are prevalent in human tumors and often express CD69 and CD103. Many murine tumor models fail to recapitulate the T cell subsets in human tumors, as they lack both non-tumor-specific T cells and CD103+ resident memory T cells (Trm). As a result, studies of TIL migratory properties paint an incomplete picture. Indeed, the source of these non-tumor specific T cells remains unclear. Methods We established an orthotopic murine glioblastoma (GBM) model with trackable virus-specific CD8+ T cells capable of expressing CD69 and CD103. Using adoptive transfers and depletion strategies, we assessed the contribution and phenotype of circulating memory T cells (Tcircm) and pre-existing Trm to the TIL population by flow cytometry and single-cell RNA sequencing. Results We found that Tcircm infiltrate GBM tumors and upregulate CD69, while pre-existing virus-specific brain Trm constitute the CD69+/CD103+ population. Furthermore, infecting mice bearing GBM tumors showed that new infection-specific Trm can form in established tumors and express CD69 and CD103. To better understand the role of these cells in brain tumors, we evaluated activation markers at early timepoints following adoptive transfer of Tcircm into tumors. Within 24 hours, we observed increased cytotoxic molecule production and enhanced activation marker expression, suggesting that newly infiltrating virus-specific T cells in tumors are activated in a bystander, non-TCR manner. Conclusion Ongoing efforts focus on identifying the factors driving bystander activation of virus-specific TILs and their impact on tumor control. Overall, this study broadens our understanding of tumor immunosurveillance and provides insights for the development of novel T cell-based immunotherapies. Funding Source NCI R01CA269455 Topic Categories Tumor Immunology: Cellular Responses and Tumor Microevironment (TIME)

Preexisting helminth infection alters the metabolic state of tissue resident macrophages 2257375

Abstract Introduction Majority of the infections are coinfections. Previously, we showed that herpesvirus infection in mice with preexisting helminth infection increased infection and virus reactivation in tissue resident large peritoneal macrophages in a vitamin A dependent manner. The aim of this study is to determine the molecular mechanism underlying helminth-induced herpesvirus reactivation in large peritoneal macrophages. Methods Mice were first infected with Heligmosomoides polygyrus (HP), and then infected with murine gammaherpesvirus-68 (MHV68). Results With single cell RNA sequencing, we observed larger proportions of large peritoneal macrophages with HP-only infection and HP+MHV68 coinfection, which indicated expansion of these cells in these two conditions and not in uninfected or virus-only infection. Differential gene expression analysis of large peritoneal macrophages showed upregulation of metabolism pathways in coinfection and HP-only infection. Upon assessing metabolic flux in peritoneal macrophages, we observed higher mitochondrial respiration for coinfection and HP-only infection. Conclusion These observations suggest that intestinal helminth infection altered the metabolic state of resident macrophages in the peritoneal cavity, which may impact herpesvirus reactivation during coinfection. Further analysis of the mechanism is on-going. Funding Source n/a Topic Categories Microbial, Parasitic, and Fungal Immunology (MPF)

Defining Macrophage Polarization Signatures via Bioactive Recombinant Proteins and LEGENDplex™ profiling 2257843

Abstract Introduction Macrophage polarization into pro-inflammatory M1 and anti-inflammatory M2 phenotypes, including M2a (tissue repair), M2b (immune regulation), and M2c (immune suppression and remodeling), is a critical process in immune regulation and tissue homeostasis. Understanding and controlling this polarization is essential for immunological research and therapeutic applications. This study evaluated the effectiveness of bioactive recombinant growth factors (M-CSF and GM-CSF) and cytokines (IL-4, IL-13, and IFN-γ) in directing M1 and M2 macrophage differentiation and polarization from human peripheral blood mononuclear cells. Methods M1 polarization was induced using human recombinant GM-CSF, IFN-γ, and E. coli LPS, while M2 polarization was achieved with human recombinant M-CSF, IL-4, and IL-13. Phenotypic characterization was performed via flow cytometry (FC) and immunocytochemistry (ICC) using markers specific to M1 (iNOS, CD74, CD80) and M2 (CD163, TGM2). Functional validation was conducted using LEGENDplex™ multiplex cytokine profiling with the Human Macrophages/Microglia Panel (IL-12p70, TNFα, IL-6, IL-1β, IL-12p40, IL-23, IFNγ, IP-10, IL-4, IL-10, Arginase, TARC, and IL-1RA) and the Human Tumor-Associated Macrophage (TAM) Panel (MMP-9, CXCL-9, CCL20, CCL18, CD163, MMP-2, Galectin-3, VEGF, M-CSF, PDGF-BB, G-CSF, CCL22, and YKL-40). Results Using directly conjugated antibodies in FC and ICC, the study confirmed successful differentiation of M1 and M2 macrophages through the expression of specific markers. LEGENDplex™ analysis revealed distinct cytokine signatures associated with inflammatory, anti-inflammatory, and regulatory responses, validating the functional polarization of the macrophages. Conclusion These findings demonstrate the reliability of the in-house bioactive recombinant proteins for macrophage differentiation and polarization. They also highlight the utility of specific signature markers, direct-conjugated antibodies, and LEGENDplex™ panels for downstream immunological applications. Funding Source n/a Topic Categories Innate Immune Responses and Host Defense: Cellular Mechanisms (INC)

USP22 orchestrates an NK-suppressive program for tumor immune evasion 2309931

Abstract Introduction Over the past 17 years, our laboratory has established USP22 as a key regulator of both oncogenic and immune-evasive programs. However, how USP22 drives dysfunction of natural killer (NK) cells and CD8+ T cells–the principal cytotoxic effectors of tumor control–remains poorly understood, despite their central role in tumor progression and immunotherapy resistance. Methods We performed unbiased profiling of intratumoral immune cells and delineated USP22-driven immunosuppressive networks by integrating genetic, molecular, and immunological approaches with single-cell transcriptomic, epigenomic, and proteomic analyses. Using advanced bioinformatics and AI/ML methods, we defined a USP22-associated molecular signature that robustly predicts immunotherapeutic resistance in human tumors. Results We demonstrate that tumor cell—specific inhibition of USP22 markedly increased NK cell infiltration across multiple syngeneic tumor models. Mechanistically, USP22 enforces a tumor-intrinsic immunosuppressive program by suppressing IL-15 and tumor-attracting chemokines, including CXCL9, CXCL10, and CXCL11, while promoting expression of the immune checkpoint ligand CD155. At the molecular level, USP22 drives epigenetic silencing and stabilizes key negative regulatory signaling pathways that enable tumor immune evasion. Importantly, multiplex immunophenotyping revealed that high USP22 expression was associated with reduced NK and CD8+ T-cell abundance and resistance to anti—PD-1 therapy. Notably, pharmacological inhibition of USP22 using our second-generation inhibitor overcame immune checkpoint blockade resistance in preclinical cancer models. Conclusion Our study identifies USP22 as a tumor-intrinsic suppressor of NK- and CD8+ T-cell—mediated antitumor immunity and reports a USP22-specific inhibitor with dual onco-targeting and immune-boosting activity, representing a first-in-class therapeutic strategy. IND-enabling studies are underway to advance this inhibitor toward clinical translation. Funding Source R01CA257520, R01CA284740 and CA232347 Topic Categories Tumor Immunology: Cellular Responses and Tumor Microevironment (TIME)