Browse Articles

Cell-Intrinsic Role of Immune-Responsive Gene 1 (Irg1) in CD4+ T Cells: Implications for CNS Autoimmunity 2259561

Abstract Introduction CD4+ Th17 cells are central drivers of multiple sclerosis (MS) pathology, yet mechanisms regulating their differentiation and effector functions is not fully understood. We recently discovered that Immune-Responsive Gene 1 (Irg1), a mitochondrial enzyme previously characterized in myeloid cells, is expressed in CD4+ T cells during experimental autoimmune encephalomyelitis (EAE). Methods Our published studies showed that global Irg1 knockout (Irg1KO) mice develop more severe EAE with expansion of pathogenic IL-17A+GM-CSF+IFN-γ+ CD4+ T cells. scRNA-seq analysis revealed enrichment of Th17 differentiation, IL-17 signaling, TCR, NF-κB, and cytokine—cytokine interaction pathways, consistent with heightened Th17 pathogenicity. qPCR confirmed elevated Il17a, Ifng, and Csf2 expression in Irg1 KO CD4+ T cells. Adoptive transfer of Irg1 KO CD4+ T cells induced severe EAE, confirming a cell-intrinsic pathogenic program. To directly test this, we generated an inducible CD4+ T cell-specific conditional Irg1 knockout mouse model. Results The conditional Irg1 knockout mouse model exhibited earlier onset and higher disease severity. Flow cytometry revealed robust CNS infiltration of triple-positive Th17 cells (IL-17A+GM-CSF+IFN-γ+), further supporting an intrinsic regulatory role of Irg1 in CD4+ T cells. Single cell RNA-seq revealed upregulation of Stat3, Irf4, and Batf in Irg1-deficient CD4+ T cells, suggesting activation of a proinflammatory Th17 transcriptional network. Ongoing studies demonstrate that Irg1 modulates chromatin accessibility and histone modifications at Th17-associated loci, implicating epigenetic control as a mechanism of regulation. Conclusion Collectively, our findings identify Irg1 as a critical metabolic-epigenetic checkpoint that restrains Th17-driven neuroinflammation and represents a promising therapeutic target for MS. Funding Source n/a Topic Categories Therapeutic Approaches to Autoimmunity (THER)

Overcoming tumor-intrinsic immunotherapy resistance with hypofractionated radiotherapy 2309668

Abstract Introduction Immune checkpoint blockers (ICB, e.g., anti-CTLA-4/D-1) have become a major pillar of cancer care for patients with advanced solid tumors. However, therapeutic resistance has become a bottleneck issue, limiting their overall efficacy to a fraction of patients. Concerted efforts from others and us have identified loss of IFN-g signaling as a major tumor-intrinsic resistance to ICBs in various types of tumors (melanoma, bladder cancer, and colorectal cancer). Therefore, identifying approaches to overcome this resistance will expand the patient population that can benefit from ICBs, a poorly explored area. Given the immunomodulatory effects of radiation (RT) and its ability to re-sensitize tumors to ICBs independent of IFN-g-driven tumor killing, we reason that RT represents an effective strategy to treat ICB-resistant tumors lacking IFN-g signaling (IFNgR1-KO). Methods We formulated an immunogenic hypofractionated RT regimen (hypo-RT) to treat mice bearing IFNgR1-KO tumors, +/- ICBs. To achieve long-term cure, we employed Ruxolitinib (Ruxo), a targeted therapy for IFNgR1-KO tumors and a radiosensitizer that we previously described. Isolated TILs were analyzed by multi-color flow cytometry to shed light on the underlying mechanisms. Results Hypo-RT enhanced T cell infiltration, increased cytokine production, and when combined with anti-CTLA-4, improved the progression-free survival compared to RT alone, suggesting re-sensitization of IFNgR1-KO tumors to ICB. Hypo-RT significantly boosted therapeutic effects of Ruxolitinib, a targeted therapy for IFNgR1-KO tumors (2022 Nat. Comms.) Vice versa, Ruxolitinib enhanced RT-driven tumor suppression (P = 0.08). Interestingly, hypo-RT depleted FoxP3+ Treg in wild-type but not IFNgR1-KO tumors, revealing a heretofore unknown role of tumor IFNgR1 signaling in RT-driven Treg depletion, which can be overcome by Ruxolitinib. Conclusion Hypo-RT is an effective strategy to bypass tumor-intrinsic ICB resistance in IFNgR1-KO tumors. Funding Source National Institutes of Health grants (1R21CA230475-01A1, 1R21CA259721-01A1, 1R01CA279849-01A1); V Foundation Scholar Award (V2018-023), DoD-CDMRP (ME210108), and CRI-CLIP Grant (CRI4342). Topic Categories Tumor Immunology: Cellular Responses and Tumor Microevironment (TIME)

Targeting Pros1 to improve chemotherapy efficacy and immune anti-tumor response 2309304

Abstract Introduction Resistance to chemotherapy is a major challenge in the treatment of advanced melanoma. Targeting the innate immune response to improve chemotherapy efficacy is a promising yet understudied area. We identified Pros1 as an innate immune checkpoint utilized by tumors to suppress the macrophage pro-inflammatory response. Secreted Pros1 prevents macrophage response to tumor damage-associated molecular patterns (DAMPs) released during chemotherapy. However, the effect of Pros1 on other immune system cells and the molecular mechanism that triggers its expression remain unclear. Methods To determine how Pros1 affects chemotherapy efficacy and immune activation, we generated a melanoma cell line (B16F10) lacking Pros1 using CRISPR-Cas9 and tested it in a syngeneic murine preclinical model. Immune infiltration was assessed by single-cell RNA-seq in end-stage tumors. Results Mice bearing Pros1-deficient tumors showed a 63±10% (mean ± SEM) decrease in tumor growth after cisplatin treatment and a 5-fold increase in immune infiltration when compared to wild type. B cells were the most enriched group in Pros1-deficient tumors, with increased communication with T cells. Using bulk RNA-seq, we determined that Ifnγ stimulates Pros1 expression in melanoma cells as well as other immune suppressors, including Ido1, Pd-l1 and Tgfb1. Ifnγ-dependent Pros1 expression was conserved among several cancer cell types, including melanoma, lung, breast, and pancreatic cancer, suggesting its role as a global immune suppressor. By using siRNA, we determined that Ifngr1/Ifngr2 and Stat1 are key modulators of Ifnγ-dependent Pros1 expression, but not Stat3. Conclusion We identified a novel mechanism through which cancer cells promote Pros1 expression in response to chemotherapy. A better understanding of the mechanisms that regulate Pros1 expression may allow us to increase the efficacy of conventional therapies in melanoma patients. Funding Source NIH grant R01CA262241 Topic Categories Tumor Immunology: Cellular Responses and Tumor Microevironment (TIME)

Ratiometric Mycotoxin Detection in Living Plants With Dual‐Emissive Nanosensors (Adv. Mater. 47/2026)

Multi‐State Memory in 2D Magnets via Thickness‐Engineered Growth

ABSTRACT Two‐dimensional (2D) magnets offer substantial potential for high‐density spintronic memory due to their tunable magnetic states, yet a robust, scalable strategy for controlling domain configurations for 2D magnets remains elusive. Here, a selective non‐uniform nucleation strategy via chemical vapor deposition is proposed to achieve controlled, non‐homogeneous growth of room‐temperature ferromagnetic CrTe nanoflakes. This enables bottom‐up control of domain evolution by leveraging the strong correlation between the thickness profile and magnetization reversal. The stepwise magnetization reversal in multi‐thickness nanoflakes endows CrTe with multiple magnetic states. Utilizing such multi‐thickness CrTe nanoflake, a tunable multi‐state magnetoresistance is successfully realized in vertical spin valve devices. The controlled synthesis of multi‐thickness CrTe nanoflakes signifies a breakthrough in domain‐state control in 2D magnet, and establishes a robust material foundation for potential applications in multi‐state storage and spin encryption communication.

Seasonal migration patterns of Atlantic bluefin tuna (Thunnus thynnus) occupying the eastern Mediterranean Sea

Generation of plasma cell targeting OR-gated CAR T cells for the treatment of autoimmune disease 2257520

Abstract Introduction Chimeric antigen receptor (CAR) T cells targeting autoreactive memory B cells show promise in autoimmune disease, but long-lived plasma cells may also drive pathology. To achieve a more durable immune reset, we designed a novel OR-gated CAR T cell that utilizes a single chain variable fragment (scFv) targeting both memory B and plasma cells. Methods Antigen profiling of healthy donor blood and bone marrow samples identified B cell maturation antigen (BCMA) and transmembrane activator and CAML interactor (TACI) as B cell restricted targets with low risk of target related toxicity. Despite low sequence homology, the extracellular domains of BCMA and TACI share structural similarity in their ligand-binding domains, enabling the development of cross-reactive antibodies. Results An antibody display library panned with TACI protein yielded antibodies binding to TACI or BCMA with varying affinities. CAR T cells engineered from select antibodies exhibit robust cytolytic function, proliferation, and cytokine production in response to BCMA- or TACI-expressing cell lines in vitro. BCMA/TACI CAR T cells effectively control a plasma-cell derived tumor cell line expressing both antigens in vivo. Furthermore, we demonstrate B cell depletion by BCMA/TACI CAR T cells using autoimmune patient samples. Conclusion In summary, we have generated potent CAR T cells expressing a novel, single scFv recognizing both BCMA and TACI that can be utilized to target plasma and memory B cells more widely for treatment of autoimmune diseases. Funding Source n/a Topic Categories Therapeutic Approaches to Autoimmunity (THER)

The CARD11-BCL10-MALT1 (CBM) Complex Regulates Nutrient Transport and mTORC1 Activation to Shape CD4+ T Cell Differentiation 2252439

Abstract Introduction The precise mechanisms linking TCR-dependent nutrient import and metabolic reprogramming to CD4+ T helper (Th) fate and function remain elusive. We recently demonstrated that the CBM complex facilitates TCR-induced expression of the glutamine transporter ASCT2 and the glucose transporter GLUT1 on human CD4+ T cells. Notably, patient T cells carrying deleterious dominant-negative (DN) CARD11 mutations exhibit impaired activation-induced upregulation of both transporters, which we hypothesize contributes to the abnormal Th2 skewing and severe atopy observed in this cohort. Methods To test if observed phenotypes resulted from decreased nutrient transport, we employed siRNA-mediated knockdown and pharmacological inhibition of ASCT2/GLUT1 in primary human T cells. Nutrient uptake and mTORC1 function were quantified in healthy and CARD11 DN samples using QUAS-R/2-NBDG import assays, flow cytometry, and immunoblotting. Th differentiation was assessed by spectral flow cytometry and high-sensitivity electrochemiluminescent immunoassays. Results Targeted disruption of ASCT2 and GLUT1 reduced glutamine and glucose import respectively and decreased mTORC1 activity, including protein translation. These defects were linked to MALT1 protease function and sustained by reduced nutrient import. Consequently, we observed reduced production of IFNγ and augmented secretion of Th2 (IL-4) and Tfh (IL-21) cytokines consistent with nutrient-dependent shifts in lineage-defining transcription factors, mirroring programs in CARD11 DN patient cells. Supplementing CARD11 DN T cells in vitro with glutamine and/or glucose yielded rebalanced differentiation towards a Th1 phenotype. Conclusion Collectively, our results show that the CBM signalosome governs nutrient transport and mTORC1 activation to support Th1 differentiation of human T cells. Enhancing ASCT2/GLUT1 expression and/or nutrient import represents a viable therapeutic avenue for treating patients with CARD11 DN mutations and other Th2-driven pathologies. Funding Source NIAID 5R01AI168295 Topic Categories Lymphocyte Differentiation and Peripheral Maintenance (LYM)

Co-clustering of CD16 and NKG2D receptors augments degranulation of NK cells 2259276

Abstract Introduction It has been shown that CD16 and NKG2D receptors act synergistically to activate resting NK cells’ cytotoxicity and cytokine secretion. The molecular mechanism underlying the communication between the two receptors remains to be elucidated. Methods To study cooperativity between CD16 and NKG2D receptors, we exposed human primary polyclonal NK cells with CD16 176V/V-genotype to planar lipid bilayers presenting ICAM-1 adhesion molecules with activating Herceptin and/or NKG2D ligands. The interface formation, receptor clustering, recruitment of signaling molecules, MTOC polarization, and release of cytolytic granules were analyzed using confocal, TIRF, and super-resolution imaging. Results Exposure of NK cells to the bilayer containing only one ligand induced the emergence of Herceptin- or anti-NKG2D microclusters. Simultaneous engagement of the two receptors resulted in merging of Herceptin and anti-NKG2D clusters into mixed co-clusters. These mixed co-clusters have been found to accumulate PLC gamma-2 but not PLC-gamma-1 isoform. Our results demonstrated that co-clustering of the receptors resulted in a decrease of the motility of NK cells that was accompanied by the formation of a classical immunological synapse and a sustained Ca2+ influx, which led to augmented degranulation. Conclusion These data led us to propose a molecular mechanism that regulates the synergy between CD16 and NKG2D receptors. Funding Source UO1AI148117 NIH grant to Yuri Sykulev and Kerry S Campbell Topic Categories Innate Immune Responses and Host Defense: Molecular Mechanisms (INM)

Tissue-resident, circulating and plaque-homing CD4+ T cells in human atherosclerosis 2309197

Abstract Introduction Subsets of T cells have been shown to home to atherosclerotic plaque in atherosclerosis patients. In mice, CXCR6 and CCR5 play roles in mediating T cell recruitment to atherosclerotic lesions. We hypothesized that human T cell subsets possess the capacity to traffic between blood and atherosclerotic plaque. CD4+ regulatory T cells (Tregs) are known to give rise to exTregs, but their differentiation in atherosclerosis remains unclear. To address these gaps, we integrated CITE-seq datasets of PBMC and plaque from patients, focusing on αβ CD4+ T cells composition, migratory signatures, and differentiation between blood and plaque. Methods We analyzed CITE-seq datasets from a total of 12 patients aged over 69 years (6 PBMCs and 6 plaque). Data was integrated using LIGER. αβ CD4+ T cells were subset for re-clustering. TCR-seq data was used to assess clonal expansion across CD4+ T cell subsets. Trajectory analyses were used to infer differentiation relationships. pySCENIC was preformed to infer transcription factor activity. Results Integrated analysis showed enrichment of mast cells and NKT cells in plaque, while NK cells, classical monocytes, and CD4+ T cells predominated in blood. CD4+ T cell re-clustering identified naive, effector, memory, cytotoxic, and regulatory subsets. Naive and effector CD4+ T cells were blood-enriched, whereas cytotoxic effector memory CD4+ T cells and FoxP3hi Tregs were enriched in plaque. Cytotoxic CD4+ T cells expressed CCL5, GZMA, and GZMK, consistent with cytotoxic exTregs. FoxP3hi Tregs showed an activated phenotype distinct from FoxP3low Tregs. CXCR6 and CCR5 marked plaque-homing CD4+ T cells, with CCR7 supporting inter-compartment trafficking; CCR6 emerged as an additional pathway. Trajectory and TCR analyses supported differentiation from naive CD4+ T cells through unstable Tregs toward cytotoxic states, driven by ETV7 and GFI1 regulons. Conclusion Our study showed CD4+ T cell subset composition and differentiation between blood and plaque. Funding Source n/a Topic Categories Immune Mechanisms of Human Disease (HUM)

Identification of Functional Subsets within Epidermal γδ T Cells in Imiquimod-induced Psoriasis 2259187

Abstract Introduction Approximately 3% of Americans suffer from psoriasis, an autoimmune skin disorder that causes inflamed lesions on the skin. Psoriasis pathogenesis involves a complex interplay between a variety of immune cell types, including skin-resident T cells. We have recently shown that a subset of CCR6+ epidermal γδ T cells produce IL-17 during psoriasis; however, the function of other epidermal γδ T cell subsets in disease remains unknown. We have identified and characterized five distinct functional subsets of epidermal γδ T cells in a murine model of imiquimod (IMQ)-induced psoriasis. Reanalysis of single-cell RNA sequencing data from IMQ-treated mice identifies five transcriptionally distinct epidermal γδ T cell subsets with separate functions. We have defined these subsets as: Th1, neuronal, regulatory, proliferative, and Th17. We further characterized these subsets in IMQ-induced psoriasis using immunofluorescent microscopy and flow cytometry to quantify the populations and interrogate their function. Elucidating functional epidermal γδ T cell populations in psoriasis provides a deeper understanding of skin immune regulation and the identification of potential therapeutic targets for psoriasis and related skin inflammation. Methods Epidermal sheets were isolated from imiquimod-treated murine ears, stained with γδTCR-APC, CD94-FITC, and CD24-PE, and imaged using immunofluorescence microscopy to identify and quantify functional γδ T cell subsets. Results Immunofluorescence imaging revealed epidermal γδ T cells co-expressing γγTCR-APC and CD24-PE during imiquimod-induced inflammation. Conclusion These findings suggest that distinct epidermal γδ T cell populations can be identified by CD24 expression, providing insight into their potential roles in cutaneous immune responses. Funding Source Beckman Scholars Program Topic Categories Innate Immune Responses and Host Defense: Molecular Mechanisms (INM)

Myeloid cell-intrinsic TAX1BP1 restrains spontaneous heart valve inflammation 2259602

Abstract Introduction Tax1 binding protein 1 (TAX1BP1) is a selective autophagy receptor that inhibits activation of tumor necrosis factor (TNF)- and lipopolysaccharide (LPS)-induced NF-kB activation to suppress inflammation. TAX1BP1 has recently been shown to promote cell survival by degrading the kinase RIPK1 to inhibit tumor necrosis factor receptor 1 (TNFR1)-mediated necroptosis. However, the physiological roles and tissue specific functions of TAX1BP1 in the regulation of cell death and inflammation are poorly understood. Methods In this study, we utilized the Cre-loxP system to generate TAX1BP1 conditional knockout mice crossed with LysM-Cre mice for a myeloid cell-specific knockout (Tax1bp1fl/fl LysMCre mice). These mice were necropsied for pathological analysis. The mice were challenged with a low dose (2.5 mg/kg) of LPS to determine their susceptibility to inflammatory stimuli. Bone marrow-derived macrophages (BMDMs) and thioglycollate-elicited peritoneal macrophages (PMs) were also generated from Tax1bp1fl/fl LysMCre mice for in vitro studies. Results Tax1bp1fl/fl LysMCre mice develop splenomegaly and spontaneous heart valve inflammation and necrosis. They also exhibit increased pro-inflammatory cytokine production and sepsis upon intraperitoneal administration of a low dose of LPS. BMDMs and PMs from Tax1bp1fl/fl LysMCre BMDMs and PMs undergo spontaneous lytic cell death that can be rescued with a kinase inactive RIPK1 D138N allele. Conclusion These findings highlight TAX1BP1 as a critical regulator of myeloid cell homeostasis by suppressing NF-kB signaling and inflammation while promoting cell survival. Additionally, the severe cardiac valvulitis that spontaneously develops in Tax1bp1fl/fl LysMCre mice indicates that TAX1BP1 expression in cardiac resident macrophages is critical for the homeostasis of these cells and inhibition of inflammation and necrosis in heart valves. Funding Source NIH/NIAID R01AI162815 Topic Categories Innate Immune Responses and Host Defense: Molecular Mechanisms (INM)

Beyond Transcription: T cell-Intrinsic IRF5 Regulates Metabolism and Post-Transcriptional Responses in Autoimmunity 2309706

Abstract Introduction T cell activation is a meticulously orchestrated process that demands changes in cellular physiology, encompassing extensive transcriptional, translational, and metabolic reprogramming. While these layers of cellular regulation are crucial for T cell differentiation and function, the precise regulatory mechanisms that functionally link them remain incompletely defined. We investigated if T cell-intrinsic IRF5 exerts control beyond transcription and integrates with metabolic pathways. Methods T cells from WT and Irf5-/- mice were activated in vitro (anti-CD3/CD28); activation (CD25, CD69, proliferation) was assessed by flow cytometry. CD40L mRNA and protein were measured. Metabolism was characterized via targeted metabolomics and nutrient transporter expression. Single-cell flow/scRNA-seq analyzed global T cell population shifts. In vivo, EAE models (Irf5-/-, T cell conditional Irf5-/-, adoptive transfer of MOG-specific Irf5-/- T cells) were used and EAE progression scored. Mechanistic analyses included mTORC1 activity, CD40L, glutamine transporters, and inflammatory cytokines in EAE mice. Results IRF5 is a novel T cell-intrinsic regulator of mTORC1 signaling and glutamine metabolism. Irf5 ablation impaired CD40L protein expression despite preserved mRNA induction. Loss of Irf5 shifted T cell metabolic programs and cellular bioenergetics. Single-cell analysis showed Irf5 deletion skewed T cell populations, altering differentiation. In vivo, conditional Irf5 deletion protected against EAE. Protection was due to suppressed mTORC1 activity, reduced CD40L, dysregulated glutamine transporter, and reduced inflammatory cytokines. Conclusion IRF5 integrates TCR signaling, metabolic reprogramming, and effector function. It regulates transcription and critical post-transcriptional events, influencing mTORC1 signaling, CD40L expression, and glutamine metabolism. Modulating IRF5 may offer a strategy to manipulate aberrant T cell responses and mitigate T cell-mediated autoimmune conditions. Funding Source Lupus Foundation of America Gina M. Finzi Fellowship (ZB), National Institutes of health grant 1R01AR076242 (BJB), National Institutes of health grant R03TR004623 (BJB), Department of Defense (DoD) CDMRP Lupus Research Program grant W81XWH-18-1-0674 (BJB), Th Topic Categories Therapeutic Approaches to Autoimmunity (THER)

Multi-omics profiling with InTraSeq™ uncovers novel single-cell RNA and Proteomics events in CARTs 2265562

Abstract Introduction CAR T-cell therapy has transformed treatment of blood cancers but faces persistent barriers in solid tumors. A major limitation is the absence of single-cell approaches that integrate gene expression with protein and post-translational modification (PTM) data to capture dynamic CAR signaling. Conventional scRNA-seq provides only transcriptional snapshots. We applied InTraSeq™, a multimodal single-cell platform enabling simultaneous quantification of mRNA, surface, cytoplasmic, and nuclear proteins–including PTMs–to comprehensively profile CAR activity. Methods Jurkat and anti-CD20 CAR-Jurkat cells were analyzed under four conditions: monocultures or co-cultures with CD20+ Raji cells. Each was assessed at 2 and 24 hours (eight total samples). Samples were labeled with unique Histone H3 InTraSeq™ barcodes and pooled for processing with Genomics 3’ kits. An anti-Whitlow/218 linker antibody InTraSeq™ conjugate distinguished CAR-Jurkat cells. Combined transcriptomic and proteomic data identified activation- and signaling-related changes. Results All samples were accurately demultiplexed using Histone H3 barcodes. The anti-Whitlow/218 probe confirmed CAR expression and enabled detection of engineered Jurkats. In CAR-Jurkat co-cultures, unique RNA clusters and protein signaling states emerged at both timepoints, highlighting CAR activation. Apoptosis scoring revealed elevated apoptotic protein signals specifically in Raji cells after 24-hour co-culture with CAR-Jurkats, validating targeted cytotoxic function. Conclusion InTraSeq™ provides high-resolution, multi-omic insight into CAR-T activation, integration of RNA and protein data, and effector-target interactions. This approach delivers a unified view of CAR signaling and function, offering a valuable framework for refining CAR-T therapy design and optimization. Funding Source n/a Topic Categories Technological Innovations in Immunology (TECH)

Thermally Modulated Metasurface Sensor for Dynamic and Time‐Resolved Isolation of Extracellular Vesicles (Adv. Mater. 44/2026)

Transparency‐Photothermal Efficiency Coordinated Dual‐Invisible Soft Robots

ABSTRACT Soft robots capable of seamless environmental integration and unobtrusive operation are essential for covert monitoring, camouflage, and preserving optical integrity. Near‐infrared (NIR) photo‐actuation offers an ideal route for invisible control; however, it is traditionally governed by a fundamental trade‐off where strong photo‐responses necessitate dark materials and high filler loadings that compromise transparency. Here, we report a system achieving spectral decoupling of visible transparency and photothermal responsiveness through interfacial engineering of two‐dimensional atomically thin CuS (AT–CuS) nanosheets to form a stable monolayer dispersion in the polymer matrix. Such material reaches a record‐breaking photothermal efficiency of 96.3% in the NIR‐II window. At an ultralow loading of 0.01 wt % At‐CuS, the composite maintains over 80% visible‐light transmittance while exhibiting rapid NIR responses (>130°C within 2 s). We further demonstrate “dual‐invisible” soft robots with imperceptible bodies and hidden NIR control, where the body and the NIR control signal remain imperceptible, performing agile amphibious locomotion.

Composite Liquid Marble Templated Millimetric Capsule With Tunable Rigidity, Porosity, and Thermal Reconfigurability Toward 3D Cell Culture

ABSTRACT The extensive reliance on animal models in biomedical research motivates the development of advanced in vitro systems that recapitulate physiological complexity while minimizing animal use. Large cellular spheroids can mimic native tissue architecture; however, scalable fabrication of spheroids exceeding millimeter dimensions remains challenging. Here, we introduce a rigid porous capsule (RPC) in millimeter scale, a mechanically robust yet highly permeable platform that imposes external geometric confinement to enable cell–cell aggregation and three‐dimensional proliferation. The RPC is fabricated by thermally processing LMs composed of a binary mixture of superhydrophobic particles: meltable poly(octadecyl acrylate) (PODAc) microparticles and non‐meltable bovine serum albumin (BSA) nanoparticles. Selective melting of PODAc induces a transformation of the initially fragile, porous LM shell into a rigid and macroporous architecture (pore size in µm scale). By modulating the binary mixture composition, the shell stiffness and porosity are precisely tuned to balance mechanical stability with efficient nutrient transport. The resulting RPCs retain a highly spherical geometry and support 3D cell culture for at least 14 days, enabling the formation of viable, scaffold‐free spheroids on a millimetric scale. This RPC establishes a physiologically relevant system for advanced tissue modeling and drug screening.

Nonlacunar covert brain infarcts on noncontrast CT as sentinel imaging markers of stroke etiology and outcome

Exploring gut host—microbiome interactions associated with RhCMV/SIV vaccine efficacy against SIV in rhesus macaques 2257552

Abstract Introduction A live-attenuated rhesus cytomegalovirus (RhCMV)-based vaccine protects ∼59% of rhesus macaques (RM) against simian immunodeficiency virus (SIV) via CD8+ T cell—mediated control, but the exact mechanism of non-protection in ∼41% remain unclear. We previously identified gut microbial features linked to RhCMV/SIV vaccine protection and now aim to validate these signatures and investigate host—microbiome interactions influencing vaccine efficacy. Methods A new cohort of 14 RMs was vaccinated with prime and boost doses of 68-1 RhCMV/SIV vector. At week 79, they were challenged with SIVmac239, and 8 of 14 (57%) were protected. Full-length 16S rRNA and total RNA sequencing were performed on rectal swabs collected at three time points before and after vaccination. Gut microbiome was profiled using full-length 16S sequencing, while gut microbial meta-transcriptome was identified by total RNA-sequencing. Total RNA-seq reads mapped to the RM genome also characterized host immune responses in the gut. Multi-omics data have been processed, and downstream analyses are ongoing. Results From the total RNA-seq data we obtained ∼60 million reads per sample. In our preliminary findings, both 16S and total RNA-seq data contained a high proportion of reads assigned to unknown species, as well as a substantial fraction of unmapped reads. These phenomena present challenges for analyzing host and microbial composition and function in the RM model. Conclusion We will continue to develop the multi-omics analysis pipeline to achieve our aims and present the results at the presentation. The findings will contribute to the optimization of future RhCMV-based vaccine strategies by improving protection prediction and guiding vaccine adjuvants. Funding Source NIAID, NIH, HHS R21AI120713 and Contract No. HHSN272201800008C Topic Categories Mucosal and Regional Immunology (MUC)

Distinct Treg subsets in human peripheral blood are defined by unique transcriptomic signatures 2252633

Abstract Introduction Regulatory CD4+ T cells (Tregs) are critical for maintaining tolerance to self. Chronic inflammation can cause Treg instability which is defined by loss of CD25 and the lineage-defining transcription factor FOXP3, resulting in exTreg cells. Methods To better understand the factors driving Treg to exTreg conversion, we analyzed transcriptomes of Treg subsets expressing varied levels of CD25 and FOXP3 in human peripheral blood CD4+ T cells. Results We found differential expression of genes related to inflammatory signaling (IFNG, TNF), cytotoxicity (PRF1, GZMB) and cellular stress (HSPA5). This was confirmed at the protein level by flow cytometry. Distinct Treg populations were also defined by unique metabolic profiles suggesting a role of cell-intrinsic metabolic programs in maintaining Treg stability. Conclusion Our findings start to define novel cellular pathways contributing to Treg homeostasis and instability in human peripheral blood. Funding Source We acknowledge the support from National Institutes of Health (awards P01 HL136275 and R35 HL145241) to K.L. Topic Categories Lymphocyte Differentiation and Peripheral Maintenance (LYM)