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Reprogramming Mesenchymal Plasticity to Restore Thymus Growth and Vascularization 2263454
Abstract Introduction Thymus organogenesis depends on coordinated communication between stromal and hematopoietic cells. We found that thymic mesenchyme contains a specialized stromal subset (CD45⁻Ter119⁻PDGFRA+PDGFRB+CD90+) distinct from mesenchyme of the pharyngeal, submandibular, tongue, and lung regions, which uniquely signals to thymic epithelial cells. Methods We compared stromal competence across organs using flow cytometry, single-cell transcriptomics, and recombinant reaggregate thymic organoid culture (ReRTOC). Pharmacologic reprogramming was tested in Tbx1neo2/neo2 embryos (∼36% normal Tbx1), a model of 22q11.2 deletion syndrome, using the FDA-approved drug Minoxidil. Results ReRTOC assays showed that pharyngeal and submandibular mesenchyme retain partial ability (∼60% thymic growth) to support epithelial expansion and thymopoiesis, revealing inherent plasticity. In Tbx1neo2/neo2 mutants, mesenchyme exhibited a shift from Bmp4 to Bmp5—Sox9 signaling, driving chondrogenic differentiation (Col2a1, Col9a1, Col11a1), loss of stromal organization, and reduced vascular branching with a lower SMA/Cx40 ratio. Minoxidil restored Bmp4 activity, suppressed Sox9/Bmp5 expression, and normalized thymic size and vascular complexity. CellChat analysis of wild-type thymic stroma revealed perivascular mesenchymal subsets acting as dominant signaling hubs, communicating with endothelial cells through the LAMININ, SPP1, BSP, and WNT pathways, which maintain basement membrane stability and endothelial anchoring. Conclusion Our results demonstrate that mesenchymal—endothelial communication is essential for thymic vascular patterning and that targeted mesenchymal reprogramming–through pharmacological or growth factor cues–can restore thymic vascularization and function, even during aging. Funding Source This work was supported, in part by grants from the National Institutes of Health Grant R01 AI114523 (Nicolai S.C. van Oers) and Jeffrey Modell Foundation (Christian A. Wysocki) Topic Categories Hematopoiesis and Immune System Development (HEM)
Modulation of the bone marrow landscape by vaccine adjuvants 2246451
Abstract Introduction Vaccination against influenza virus is currently the best strategy to lessen disease burden. The low immunogenicity of the subunit vaccines and variation in efficacy highlight the need for vaccine adjuvants. Rational vaccine design over the past decades has produced novel adjuvants, such as TLR7/8 agonists, aimed at eliciting Type 1 immune responses, like host responses against viral infections. TLR7/8 agonists induce type 1 IFNs which can have profound effects on hematopoietic stem and progenitor cells (HSPCs) in bone marrow. Methods We aimed to evaluate how the TLR7/8 agonist, IMDQ-PC, designed to adjuvant the inactivated influenza vaccine, affects HSPCs and hematopoietic output. C57BL/6 mice were vaccinated via the intramuscular route with the quadrivalent inactivated influenza vaccine (QIV) with or without IMDQ-PC or with PBS, and HSPCs in bone marrow were characterized using flow cytometry. Results Vaccination with IMDQ-PC significantly lowers the frequency of monocytes in bone marrow while increasing their frequency in the blood at day 5, indicative of increased egress into the periphery. IMDQ-PC also increased the number of phenotypic LSKs and upregulates Sca-1 expression on downstream progenitors for up to 14 days post vaccination, suggesting type 1 IFN signaling in the bone marrow. Furthermore, mice vaccinated with IMDQ-PC displayed a transient drop in phenotypically quiescent long-term HSCs compared to QIV or PBS vaccinated mice, potentially caused by a burst of increased differentiation. Conclusion These results suggest that IMDQ-PC triggers a type 1 IFN response that reaches HSPCs in the bone marrow, altering surface marker expression and inducing emergency myelopoiesis. Whether IMDQ-PC itself enters the bone marrow and whether HSPCs are being directly or indirectly signaled by type 1 IFNs is the focus of ongoing research. These findings provide insight into the working mechanism of vaccine adjuvants and their potential safety considerations. Funding Source 5T32AI078892-17 Topic Categories Vaccines and Immunotherapy (VAC)
Xynapse-T — an antigen-specific scaffold for functional stimulation and expansion of T-cells 2259137
Abstract Introduction Precise and reproducible activation of antigen-specific T cells to evaluate functionality and efficient proliferation of specific cells is critical across diverse research areas, including CAR/TCR cell therapy monitoring, infectious disease response, vaccination, and cancer immunology. We previously demonstrated that MHC class I—based Xynapse-T reagents, which co-display MHC-peptide (MHCp) complexes and CD28 co-engagers, selectively stimulate antigen-specific CD8+ T cells and provide a cell-free platform for in vitro functional assessment. In this study, we extend the Xynapse-T technology to MHC class II artificial antigen-presenting cell (aAPC) scaffolds to enable targeted stimulation and expansion of antigen-specific CD4+ T cells. Methods Artificial antigen-presenting cell (aAPC) scaffolds were engineered by conjugating MHC class II peptide (MHCIIp ) complexes and CD28 co-engagers to a synthetic scaffold. These Xynapse-T reagents simultaneously engage the T cell receptor (TCR) and CD28 on CD4+ T cells, facilitating selective activation within a polyspecific T cell population. T cell activation was assessed through surface marker expression and proliferation assays. Results Xynapse-T reagents displaying MHCIIp and CD28 co-engagers effectively stimulated antigen-specific CD4+ T cells, resulting in: 1. Upregulation of activation markers (CD69, CD134) 2. Enhanced cytokine production (e.g. IFN-γ) 3. Robust T cell expansion No activation was observed with control Xynapse-T reagents displaying irrelevant MHCp. Conclusion Xynapse-T technology enables precise ex vivo stimulation, activation, and expansion of antigen-specific CD4+ T cells. This platform offers a versatile and highly specific tool for: • Potency assessment of engineered T cells • Generation of antigen-specific T cell lines • Enrichment of rare T cell populations Funding Source n/a Topic Categories Technological Innovations in Immunology (TECH)
Longitudinal Immune Responses to Radiotherapy Differentiate Progression-Free Survival Outcomes in Esophageal Cancer 2309969
Abstract Introduction We hypothesized that baseline immune composition and post-radiotherapy (RT) immune dynamics influence progression-free survival (PFS) in esophageal cancer. Methods Peripheral blood mononuclear cells collected from patients with esophageal adenocarcinoma or squamous cell carcinoma (N = 20, N = 7 progressed, median follow up: 404 days) at baseline, during and immediately post-RT, as well as ∼days 45 and 180 post-RT were analyzed by mass cytometry. Manual gating followed by cox regression, linear mixed models or unpaired t-test, with Benjamini—Hochberg correction identified associations with PFS. Results At baseline, higher proportions of total CD3+ T cells (HR = 9.66, p = 0.01) and CD4+ central memory T cells (HR = 8.55, p = 0.017) correlated with reduced PFS by cox regression. FOXP3 expression across T cell subsets was similarly linked to reduced PFS (p < 0.05). Radiotherapy induced transient lymphopenia (mean ALC decrease 0.96 K/µL) and expression of activation (HLA-DR, CD38, PD-1, CD95) and proliferation (Ki67) markers across effector, regulatory, and helper T cell subsets (all p < 0.001). Longitudinal analyses revealed that at ∼45 days post-RT, PD-1+ CD8+ T cells (HR 0.10, p = 0.042) and IFNγ CD56hi NK cells (HR 0.082, p = 0.021) were associated with improved PFS, whereas persistence of regulatory (FOXP3+) and terminally differentiated (CD57+ EMRA) phenotypes across CD4+ and CD8+ subsets was associated with progression (p < 0.05). In this initial cohort, baseline immune composition and post-RT immune remodeling distinguished patients with durable tumor control. Conclusion Patients who progressed exhibited a more regulatory immune profile at baseline that persists to ∼45 days post-RT. In contrast, immune activation across T-cell and innate populations was associated with improved PFS. Ongoing work will expand this cohort (n = 40) and compare differences in immune composition between treatment modalities. Funding Source NCI program grant (P01 CA261669), VIC Innovation Fund and VIC Education Fund Topic Categories Immune Mechanisms of Human Disease (HUM)
Mural cell-specific deletion of MHC class I antigen presentation increases immune cell infiltration into the brain during neurotropic virus infection 2257679
Abstract Introduction The H-2Db class I restricted CD8 T cell response is critical for clearance of neurotropic Theiler’s virus encephalomyelitis virus (TMEV) infection. However, the key central nervous system cell types required to present H-2Db for CD8 T cell trafficking and expansion in the brain are not known. Mural cells are a critical cellular component of the neurovascular unit for maintaining vascular integrity which also express MHC class I molecules. However, the importance of mural cell antigen-restricted engagement by CD8 T cells has not been investigated. Methods To determine the function of mural cell MHC Class I antigen presentation, we crossed the Pdgfrβ-creERT2 mouse to our novel H-2Db LoxP mouse developed by our laboratory. This transgenic mouse approach enabled us to target mural cells during neuroinflammation. Results We observed robust knockdown of H-2Db on mural cells during acute TMEV infection. Conditional H-2Db knockdown in mural cells resulted in increased brain infiltration of CD8 T cells specific to the immunodominant Db:VP2 121-130 epitope of TMEV, demonstrating an important role of mural cells for immune cell recruitment. Conclusion To our knowledge, this is the first study demonstrating MHC class I antigen presentation by mural cells impacts CD8 T cell recruitment during neuroinflammation. Funding Source R01-NS103212, T32-AI170478 Topic Categories Neuroimmunology (NEUR)
Mechanically Robust and Flexible Nitride Ceramic Nanofibers Enabling Advanced Thermal Management
ABSTRACT Nitride ceramics with high thermal conductivity can effectively dissipate localized heat and prevent hotspot formation in advanced electronics. However, their intrinsic brittleness and limited deformability render them susceptible to structural damage under multiaxial mechanical loading, disrupting the continuity of heat transfer pathways and compromising long‐term device reliability. Here, a multiscale structural optimization strategy based on organic–inorganic hybrid chains is proposed to produce high‐strength, flexible, yet highly thermally conductive nitride ceramic nanofibers. This achievement arises from an optimized grain structure with high crystallinity at the microscopic scale, coupled with a continuous, defect‐minimized fibrous architecture at the mesoscale, collectively striking an optimal balance between stress transfer and phonon scattering to achieve rapid stress dissipation and efficient heat transport. Building on this feature, the nitride ceramic nanofibers exhibit excellent flexibility and a mechanical strength of up to 528.3 MPa despite possessing high crystallinity, a characteristic that typically leads to brittleness. Meanwhile, large‐area, free‐standing aligned fiber membranes fabricated via electrospinning achieve a high thermal conductivity of 16.58 W m − 1 K − 1 and structural stability during bending. This work offers new opportunities for high‐performance fibrous materials in next‐generation electronic systems.
Defining How Iron Metabolism and Cycling Shape the Immune Microenvironment in Synovial Sarcoma 2253974
Abstract Introduction Synovial Sarcoma (SyS) is an aggressive soft tissue sarcoma that is unresponsive to immune checkpoint blockades or adoptive cell therapy, leaving few treatment options. The disease originates in the synovial lining of the joint space and disproportionately affects young adults between the ages of 20 and 30. Resistance to immunotherapies is due to its genetic stability and low tumor mutational burden; as a result, the current standard of care is limited to wide surgical excision and chemotherapy. To improve the efficacy of immunotherapies against SyS, we aim to elucidate how the dysregulation of iron metabolism and cycling in leukocytes drive tumor growth while simultaneously creating an immunosuppressive tumor microenvironment. Methods Utilizing single-cell RNA sequencing, we characterized the immune microenvironment in human monophasic and biphasic SyS tumors from 12 different patients. Results Preliminary data show that biphasic SyS is immunologically distinct from monophasic SyS, both functionally and compositionally. Analysis of iron metabolism and cycling within leukocytes illustrates high levels of iron storage with low levels of iron metabolism, indicating potential malfunction of metabolic pathways, including the electron transport chain. This malfunction may lead to attenuated anti-tumor responses from tumor-infiltrating leukocytes and unchecked tumor growth. Conclusion Our findings elucidate potential immunotherapeutic strategies that target iron uptake in tumor-infiltrating leukocytes, such as a CD163-blocking antibody, to alleviate their immunosuppressive phenotype and promote an anti-tumor response. This research aims to improve and expand current treatment options for patients with synovial sarcoma. Funding Source n/a Topic Categories Tumor Immunology: Cellular Responses and Tumor Microevironment (TIME)
Keratinocyte VISTA suppresses skin IFN-I and inflammatory crosstalk after UV exposure 2256619
Abstract Introduction Ultraviolet (UV) light induces type I interferons (IFN-I) that aid repair but drive pathology when excessive, as in lupus (SLE). Loss of VISTA heightens UV-triggered IFN-I response and skin injury. Here we define how keratinocyte VISTA shapes epidermal responses to UV. Methods RNAseq was performed on biopsies from healthy and SLE skin pre- and 24hr after UV. GSE186476 dataset was analyzed for VSIR expression and IFN-I response in human keratinocytes. FLEX scRNA-seq was performed from skin FFPE sections from K14creVsirfl/fl and controls at baseline and 24 hr after UVB (250mJ/cm2). Human VISTA expressing mice were treated i.d. with 200ug aVISTA or control IgG immediately after UV. Results In human skin, VSIR expression is lower in SLE vs healthy skin 24 h after UV, coincident with heightened IFN-I activity. VSIR levels are also diminished in SLE keratinocytes at baseline, particularly in activated basal cells. In mice, scRNA-seq revealed decreased follicular and expanded basal keratinocytes in K14creVsirfl/fl skin and increased IFN-I signatures in these subsets. Both keratinocyte subsets upregulated Vsir after UV. In the absence of keratinocyte VISTA, K14creVsirfl/fl epidermis showed expansion of activated basal cells, loss of follicular keratinocytes, and further elevation in IFN-I scores. IFN-I and chemokine pathways were enriched in VISTA-deficient keratinocytes, while wound-healing programs were suppressed. UV-exposed K14creVsirfl/fl skin displayed fibroblast and monocyte expansion with melanocyte loss and high IFN-I scores in fibroblasts, macrophages, and monocytes. Cell—cell communication analysis indicated reduced junctional contacts ( JAM) and increased secreted cues (CCL, ANGPTL). Targeting VISTA reduced UV-induced ISGs and prevented skin injury. Conclusion Keratinocyte VISTA constrains skin IFN-I and preserves immune—stromal homeostasis after UV. Modulating VISTA locally suppresses UV-triggered IFN-I, implicating it as a novel target in inflammatory skin disease such as lupus. Funding Source Lupus Research Alliance, CDMRP Lupus Program, and NIAMS R21AR079661-01 Topic Categories Immune Mechanisms of Human Disease (HUM)
T cell dysregulation in pediatric obesity 2266671
Abstract Introduction The prevalence of obesity has increased worldwide over the last three decades, including severe obesity increasing amongst US children in recent years. Unfortunately, most children with obesity become adults with obesity, highlighting the complexities of significant weight loss. To date, the mechanisms by which obesity impacts immune function is not well understood. Here, we assessed the peripheral immune landscape of two distinct groups: 1) healthy children across ages and body mass indices (BMI) at baseline and 2) the impact of weight loss in adolescent patients before and 6-12 months following bariatric surgery. Methods We integrated spectral flow cytometry, in vitro T cell assays, and single-cell RNA-sequencing of PBMCs to evaluate the impact of obesity in otherwise healthy children and the impact of weight loss in adolescents with severe obesity. Results Children living with obesity have dysregulated non-naive CD8+ T cells (Tnn), including increased expression of activation markers (e.g., PD-1, CD69, CD95) and production of effector cytokines (IL-2, IFN-γ, TNF-α, which were significantly associated with BMI. Surgically induced weight loss had relatively minor impacts on immune phenotype and function. Transcriptionally, CD8+ T cells were enriched for cytokine signaling pathways in children with increased BMI, including amplified NF-kB signaling. Conclusion These data demonstrate a pattern of CD8+ T cell dysregulation in obesity that is only somewhat normalized by surgical weight loss. Funding Source Chan Zuckerberg Initiative; Burroughs Wellcome Fund Topic Categories Immune Mechanisms of Human Disease (HUM)
Aging-Associated Nrf2 Loss in Microglia Exacerbates Neuroinflammation and Trained Immunity 2249338
Abstract Introduction Aging is the greatest risk factor for Alzheimer’s disease (AD) and related dementias, with chronic neuroinflammation playing a central role in disease progression. Microglia–the brain’s resident immune cells–undergo age-related changes that exacerbate neuroimmune dysfunction. The transcription factor Nuclear Factor Erythroid 2—Related Factor 2 (Nrf2), a key regulator of cellular stress responses, has an unclear role in microglial aging. Methods To investigate the role of Nrf2 in aging, we employed both Nrf2-deficient (Nrf2⁻/⁻) mice and microglia-specific Nrf2 knockout (MG-Nrf2-KO) models. Immune profiles and cognitive functions were assessed using flow cytometry and behavioral testing, respectively. Results Our results show that Nrf2 expression declines in aged microglia, coinciding with increased neuroinflammation. Nrf2⁻/⁻ mice exhibit heightened microglial activation, elevated MHC class II expression, and infiltration of peripheral immune cells, including CD4+ T cells. Flow cytometry confirms that these infiltrating cells localize within the brain parenchyma rather than remaining in circulation. Moreover, Nrf2⁻/⁻ microglia adopt a disease-associated microglia (DAM)-like phenotype, marked by upregulation of activation markers and altered gene expression profiles. Loss of Nrf2 impairs motor learning and leads to cognitive deficits in aging mice. Mechanistically, Nrf2-deficient microglia display enhanced trained immunity, characterized by exaggerated inflammatory responses to systemic immune challenges. In MG-Nrf2-KO mice, repeated lipopolysaccharide (LPS) exposure results in elevated IL-1β production, indicating that Nrf2 modulates microglial immune memory. Conclusion These findings suggest that targeting Nrf2 signaling may offer a promising therapeutic strategy to reduce microglial hyperactivation, chronic neuroinflammation, and cognitive decline associated with aging and neurodegenerative diseases. Funding Source NIH R21AG070971, NIH R01NS102449 Topic Categories Neuroimmunology (NEUR)
Dysregulated IL-33/ST2 axis in hemochromatotic hosts aggravates susceptibility to the Yersinia enterocolitica infection 2259344
Abstract Introduction Iron is essential for host metabolism but becomes toxic in excess, disrupting immune balance. Hemochromatosis (iron overload) heightens susceptibility to severe bacterial infections. Although IL-33 maintains mucosal homeostasis via the IL-33/ST2 axis on Tregs, it’s regulation under hemochromatosis remains unclear. Here, we show that excess iron rewires IL-33 responses, converting it from a regulatory cytokine to a pathogenic inflammation driver during Yersinia enterocolitica (YE) infection. Methods Mice were on a high iron diet for 4 weeks to generate iron overload followed by infection with YE. Control mice were fed control diet with normal iron. Results IL-33 was identified as a key mediator of iron-induced YE disease exacerbation, as IL-33 deficiency conferred marked protection, reducing neutrophilia, inflammatory cytokines, and multi-organ injury. Mechanistically, iron overload first upregulated IL-33 expression, and subsequent YE infection triggered excessive IL-33 release, driving lethal inflammation. Iron excess in also reprogrammed IL-33 receptor (ST2) expression by reducing ST2 expression on T regulatory cells (Treg) and their IL-13 secretion while enhancing ST2 expression on neutrophil and macrophages; thereby suppressing regulatory immunity and amplifying innate inflammation. Myeloid-specific ST2 deletion reduced cytokine storm and improved survival, whereas adoptive transfer of functional ST2+ Tregs or recombinant IL-13 restored immune regulation and protection. Conclusion The study reveal a previously unrecognized a nutritional—cytokine checkpoint in wherein excess iron dysregulates the IL-33/ST2 signaling, silencing Treg cell function and amplifying pathogenic IL-33 responses in myeloid cells. This dual reprogramming converts IL-33 from a tissue-protective alarmin into a proinflammatory amplifier that predisposes iron-overloaded hosts to lethal YE infection. Therapeutically, targeting the IL-33/ST2 axis or restoring Treg function may mitigate iron-driven inflammatory pathology. Funding Source NIH R01AI162670 Topic Categories Innate Immune Responses and Host Defense: Cellular Mechanisms (INC)
The role of an orphan scavenger receptor (CD163L1) in T cells 2310079
Abstract Introduction It is becoming increasingly clear that the developmental origins of T cells play a major role in their subsequent phenotypes. However, we lack markers to distinguish fetal- and adult-derived T cells. Using multiple RNA-seq datasets, we found CD163L1 to be selectively expressed by fetal-derived CD8+ T cells. CD163L1 is a member of the scavenger receptor cysteine-rich superfamily and has been shown to be expressed by γδ T cells. However, its role in the immune system is not known. Therefore, we developed several novel tools to determine the expression and function of CD163L1 across lymphocyte populations. Methods We hypothesized that CD163L1 would be preferentially expressed by innate-like lymphocytes made in early life. To test this, we generated a monoclonal antibody against CD163L1 and used it to characterize its expression across multiple lymphocyte subsets in tissues from mice of different ages. To understand its function, we generated a CD163L1 knockout mouse model using CRISPR—Cas9 system. Results Our results indicate that CD163L1 is expressed on many subsets of lymphocytes (e.g., NK cells, γδ T cells, αβ T cells) that are produced in early life and migrate to epithelial barriers, particularly the skin. Interestingly, lymphocyte subsets that express CD163L1 exhibit distinct effector molecules (FasL, IL13) and CD8+ T cells lacking CD163L1 are more efficient at forming memory. These data suggest that CD163L1+ lymphocytes may play a role in the removal of damaged cells and immune regulation. Conclusion CD163L1 expression defines a more innate-like and regulatory phenotype across distinct lymphocyte lineages. We are currently performing single-cell sequencing to gain deeper insight into the role of CD163L1+ lymphocytes during viral infection and autoimmune skin conditions. Funding Source R21AI167858 and CVG Scholars award 2023-2024 Topic Categories Immune Response Regulation: Molecular Mechanisms (IRM)
Inhibition of HIV-1 cell-to-cell transmission in-vitro using engineered CAR NK/T cells 2253162
Abstract Introduction Despite the success of antiretroviral therapy in controlling HIV-1 replication, achieving a cure remains a significant challenge. Blocking viral replication has proven particularly challenging due to the highly efficient nature of cell-to-cell transmission (C-CT), as antibodies often fail to access the virological synapse. Here, we studied the effects of immune-based strategies, including ADCC and CAR NK/T cells, on HIV-1 transmission between cells. Methods We developed a highly sensitive assay for precise quantification of cell lysis using luciferase activity and built CAR NK/T based on the VRC01 and PGT121 bnAbs. We then utilized the assay to assess cellular cytotoxicity mediated by antibodies and CAR NK/T against target cells expressing HIV-1 envelope glycoproteins (Envs). We have previously developed an ultrasensitive HIV-1 C-CT assay, using a replication-competent HIV-1 vector and an activated nanoluciferase reporter system to quantify viral transmission in primary T cells. Using our nanoluciferase reporter assay, we studied the effects of a) PBMCs and soluble IgGs, and b) CAR NK/T on HIV-1 C-CT. Results We detected cytotoxic activity of T and NK cells engineered to express VRC01 and PGT121 CARs against CEM.NKR cells that express luciferase and HIV-1 Envs as targets. HIV-1 C-CT of the NL43(AD8) molecular clone, which contains all HIV-1 genes, exhibits significant resistance to bnAbs but could be efficiently blocked by ADCC. Moreover, CAR PBMCs exhibited increased specific killing across a range of envelope-expressing target cells at various E: T cell ratios. Among the CARs tested, the PGT121 bnAb-based CAR consistently outperformed others in mediating cellular cytotoxicity. Conclusion These results provide proof of concept that targeting HIV-1 C-CT through ADCC and CAR NK/T cell therapies can overcome the limitations of bnAbs. This study offers the first evidence to test these approaches to effectively target HIV-1 C-CT, highlighting their potential in studying HIV-1 treatment strategies. Funding Source NIH U01 Award, 2022 - 2027 Topic Categories Vaccines and Immunotherapy (VAC)
3D Printing of Magnetic Soft Materials for Functional Structures and Devices
ABSTRACT Magnetic soft materials (MSMs) have garnered widespread attention due to their advantages in wireless control, rapid response, and programmable anisotropy. Magnetically programmed MSM structures and devices have found extensive applications in fields such as miniature robots, biomedical engineering, and flexible electronics. However, their further development faces significant challenges in manufacturing, especially the need to create complex three‐dimensional (3D) structures with full degrees of freedom (DoFs) magnetic programming. Traditional manufacturing approaches and 3D printing followed by post‐magnetic programming face multistep workflows, limited programming freedom, and restricted filler selection. Magnetic field‐assisted 3D printing (MF3DP) addresses these limitations by coupling fabrication with in situ magnetic programming, enabling the direct manufacturing of MSM structures with programmable magnetic fillers orientations. In this paper, we first introduce magnetic materials, matrix materials, and their composites, focusing on their fundamental properties and response mechanisms. Then, we review traditional manufacturing approaches and 3D printing, followed by post‐magnetic programming. Next, we systematically review MF3DP technologies, providing a comprehensive discussion of magnetic field sources, field integration strategies, magnetic programming mechanisms, programming capabilities, and overall manufacturing performance. Finally, we discuss the current challenges and prospects of 3D printing of MSMs.
Host—Microbiota Interactions Fuel Skin Cancer in the Context of TLR4 Deficiency and Epithelial IKKα Reduction 2254771
Abstract Introduction Pattern recognition receptors (PRRs) detect pathogen-associated molecular patterns (PAMPs) maintain epithelial immune balance. PRRs such as Toll-like receptor 4 (TLR4) are well studied in infection and inflammation, their roles in skin carcinogenesis and host—microbiota interactions are poorly defined. Analysis of TCGA revealed recurrent TLR4 mutations in human skin cancers, suggesting a tumor-suppressive function. We define how TLR4 deficiency and epithelial IKKα reduction cooperate with the microbiota to promote squamous cell carcinoma (SCC). Methods We used a mouse model carrying a keratin 5-driven hemizygous Ikka deletion (IkkaΔKC/+), which develops rare skin spontaneous SCCs. These were crossed with Tlr4—/— mice. Skin microbiotas were analyzed by 16S rRNA sequencing. 3D skin organoids derived from primary keratinocytes were exposed to tumor-associated microbial communities. DNA damage (γH2AX), cytokine expression, and lipid metabolism were assessed. Parallel experiments used IkkaΔKC/+ and IkkaΔKC/+; Il4R—/— mice derived organoids. Results IkkaΔKC/+; Tlr4—/— mice showed a marked increase in SCC incidence compared with controls. Tumors exhibited loss of the wild-type Ikka allele, consistent with a two-hit model. Microbial profiling revealed elevated total bacterial load with enrichment of Gram positive Firmicutes. Organoids exposed to tumor-associated microbiota displayed increased DNA damage, IL-1β induction, and lipid metabolic reprogramming, all significantly reduced in IkkaΔKC/+; Il4R—/— organoids. In vivo, IkkaΔKC/+; Il4R—/— mice were resistant to tumor formation. Antibiotic treatment suppressed tumor development, confirming a microbiota-dependent mechanism. Conclusion TLR4—microbiota—IL-4R signaling axis that fuels epithelial carcinogenesis in the context of reduced IKKα. Skin organoids provide a powerful ex vivo model to dissect host—microbiota—immune interactions. Targeting IL-4R signaling or microbial modulation may offer new therapeutic avenues for inflammation-driven skin cancers. Funding Source NIH Topic Categories Mucosal and Regional Immunology (MUC)
Stress-adapted Immunity: The Integrated Stress Response as a Driver of S. aureus Biofilm Persistence 2256775
Abstract Introduction Craniotomy, a neurosurgical procedure for treating brain tumors, epilepsy, and cranial bleeds, carries a 1-5% infection risk, often due to Staphylococcus aureus biofilm formation, rendering infections tolerant to antibiotics and immune clearance. Treatment often requires multiple surgeries, heightening patient morbidity. However, the mechanisms driving immune dysfunction and biofilm persistence remain poorly understood. The integrated stress response (ISR), marked by eIF2α phosphorylation (eIF2α-P) and ATF4 induction, coordinates cellular adaptation to stress and was markedly increased in numerous leukocyte infiltrates in our mouse model of S. aureus craniotomy infection. We propose that the ISR is central to infection persistence and immune dysfunction. Methods Evidence of ISR activation was assessed by scRNA-seq of mouse and human craniotomy infection tissues and validated by Western blot. Salubrinal (ISR inducer) and ISRIB (inhibitor) were administered during early craniotomy infection to evaluate effects on bacterial clearance and immune activation. Results scRNA-seq revealed marked enrichment of ISR-associated transcripts in neutrophils, macrophages, and G-MDSCs. Both eIF2α-P and ATF4 expression were elevated in the brain and galea at day 7 post-infection, coinciding with biofilm formation. During acute infection, salubrinal-mediated ISR activation significantly reduced S. aureus burdens, whereas ISR inhibition enhanced bacterial persistence, suggesting an early protective role for ISR signaling. Conclusion These findings identify the ISR as a pivotal regulator of immune dynamics and biofilm persistence. Understanding how stress-adaptive pathways shape leukocyte function may reveal novel immunomodulatory strategies to overcome treatment-refractory biofilm infections. Funding Source n/a Topic Categories Innate Immune Responses and Host Defense: Cellular Mechanisms (INC)
Rapid IFNg production by CD8+ TRM slows viral propagation but is insufficient to directly mediate clearance following respiratory virus transmission 2260225
Abstract Introduction Tissue-resident memory CD8+ T cells (TRM) in the respiratory tract can restrict viral spread and protect against transmission through secretion of IFN-gamma (IFNg), however, the precise mechanisms by which TRM-derived IFNg limits viral transmission remains poorly defined. We aimed to determine how IFNg alone, in the absence of other TRM-mediated effector mechanisms, contributes to protection against respiratory virus transmission Methods Using a natural mouse model of Sendai virus (SeV) transmission and bioluminescence imaging of viral dynamics, we developed an approach to activate WT or Ifng-/- influenza-specific TRM during SeV exposure to assess whether IFNg production from bystander, non-SeV-specific TRM in the upper respiratory tract (URT) could protect against developing a productive SeV infection. Results Activation of bystander TRM in the URT during SeV exposure significantly delayed viral propagation, and in some cases prevented productive SeV infection, in an IFNg-dependent manner. Using different immunization strategies, we found that the magnitude of this effect positively correlated by bystander TRM number. Bystander IFNg production enhanced early recruitment of SeV-specific T cells from circulation, rapidly induced antiviral programming in URT epithelial cells, and boosted local inflammatory cytokines. Notably, the ability of bystander TRM-derived IFNg to prevent infection was lost when the recruitment of pre-existing or de novo SeV-specific T cells from circulation was inhibited. Conclusion Overall, our data suggest that the primary role for rapid IFNg production during transmission is to reduce viral propagation in order to “buy time” for other effector mechanisms to mediate viral clearance. Understanding how different TRM effector mechanisms are coordinated to optimize viral clearance while limiting immunopathology will assist in the design of mucosal vaccines that provide broad, cross-protective immunity against respiratory viruses. Funding Source R01 AI190367 and R35 HL150803 Topic Categories Viral Immunology (VIR)
SLE risk variants shapes IRF5 alternative promoter isoform usage in B cells 2252005
Abstract Introduction Systemic lupus erythematosus (SLE) is a multifaceted autoimmune disease driven by a complex interplay of genetic and environmental factors. Emerging evidence highlights the contribution of alternative splicing and isoform dysregulation to SLE pathogenesis, although the underlying mechanisms require further elucidation. Methods We utilised a publicly available RNA-seq dataset to examine isoform usage in B cells from patients with SLE. Our analysis focused on five distinct B cell subsets to identify isoform alterations. We further investigated the role of Interferon Regulatory Factor 5 (IRF5) in double-negative 2 (DN2) B cells through CRISPR-Cas9 knockout and pharmacological inhibition experiments in primary B cells. Additionally, we integrated genome-wide association study fine-mapping, co-localisation analyses and CRISPR base-editing in the GM12878 cell line to identify a putative causal variant in the IRF5 locus. Results Our analysis identified extensive isoform alterations, notably within DN B cells, which displayed significant changes across hundreds of genes. IRF5, a known SLE risk locus, exhibited significant isoform dysregulation. By knocking out IRF5, we observed a substantial reduction in DN2 B cells, plasmablasts, and MKI67+ proliferating cells, accompanied by decreased production of IL-6 and IL-12. Furthermore, we identified a putative causal variant near the IRF5 transcription start site. Conversion of the non-risk allele to the risk allele induced significant upregulation of the E1B isoform. These findings were reproducible in primary B cells homozygous for the risk haplotype, which also displayed enhanced expansion of DN2-differentiating B cells on day 6 in vitro. Conclusion Our findings underscore a critical role for IRF5 in the differentiation of DN2 B cells and demonstrate how a genetic risk variant associated with lupus leads to isoform-specific dysregulation in B cells. Together, these provide new mechanistic insights into the molecular pathways involved in SLE pathogenesis. Funding Source Lupus Innovation Award (Gutierrez-Arcelus) Topic Categories Basic Autoimmunity (BA)
Antibody-Drug Conjugates (ADCs) as Adaptors for Universal CAR T cells 2259384
Abstract Introduction Antibody—drug conjugates (ADCs) and chimeric antigen receptor (CAR) T cells are powerful cancer therapeutics, but each faces limitations. ADCs often fail to yield durable responses due to drug payload resistance or poor internalization. CAR T cells are often thwarted by challenges including immune suppression and poor persistence. To overcome these challenges, we developed a universal CAR T cell platform in which ADCs act as antigen-targeting “adaptors” that direct CAR T cell activity. In this system CAR T cells are co-administered with one or more ADCs. Instead of the CAR directly binding to a tumor antigen, ADCs bind to antigens, thus activating CAR signaling in addition to drug payload delivery. Methods We used our previously generated SNAP-CAR, a universal CAR that covalently binds to antibodies with a benzylguanine (BG) tag. We generated ADC adaptors by conjugating BG to several FDA-approved ADCs targeting HER2 and FOLR1. In parallel, we designed a novel universal anti-DM1/DM4 CAR that directly binds DM1 and DM4 drug payloads. Primary human CAR T cells were co-incubated with antigen-positive or -negative tumor cells and various adaptors, followed by flow cytometric assessment of T cell activation, target cell lysis, and bystander killing. Results SNAP-CAR T cells with ADC—BG adaptors showed strong, adaptor dose-dependent T cell activation and tumor lysis, outperforming unconjugated ADCs or BG-labeled antibodies. Additionally, these adaptors enabled bystander killing of antigen-negative tumor cells. Anti-DM1/DM4 CAR T cells were similarly effective showing dose-responsive activities induced by ADCs and in a HER2+ human tumor xenograft mouse models. Excitingly, both approaches were capable of potent killing of DM1/DM4-resistant cells, demonstrating their ability to overcome drug resistance limiting ADC therapies. Conclusion ADC-directed universal CAR T cells represent a highly modular approach that synergizes cytotoxic drug activity with immune function to overcome therapeutic resistance. Funding Source conditional R01 and the Gap Fund and Supported by the University of Pittsburgh Commercialization Gap Fund Topic Categories Tumor Immunology: Checkpoints, Prevention, and Treatment (TIPT)
Persistent uterine inflammation following murine chorioamnionitis predisposes to poor outcomes in a subsequent pregnancy 2310301
Abstract Introduction Chorioamnionitis, an infection and/or inflammation of the placenta and fetal membranes, is a leading cause of preterm birth (PTB) and an independent risk factor for neonatal morbidities. Women with a history of chorioamnionitis are at increased risk for complications (including implantation failure, PTBs, recurrent chorioamnionitis, and adverse neonatal outcomes) in future pregnancies; however, the mechanisms underlying these subsequent poor outcomes remain unclear. Pathological in-utero inflammation is a key driver of the morbidities seen with chorioamnionitis. Thus, we hypothesized that chorioamnionitis-associated inflammation persists postpartum in the uterus, predisposing to adverse outcomes in future pregnancies. Methods Chorioamnionitis was induced in pregnant C57BL/6 mice at embryonic day 14.5 via intravaginal inoculation with 100 CFU E. coli K1 (or PBS control). Four weeks postpartum, mice were either rebred or euthanized for uterine immune profiling by flow cytometry. Vaginal and uterine swabs were collected to assess persistent E. coli K1 infection. Results Postpartum, uteri from chorioamnionitis-exposed dams (CED) exhibited a significant increase in total immune cells, driven primarily by increased T cell numbers. Specifically, CED uteri showed elevated antigen-experienced CD4+ and CD8+ T cell numbers, increased Tregs with reduced Foxp3 expression, higher frequency and absolute number of inflammatory macrophages, and an accumulation of activated dendritic cells. Notably, uterine and vaginal E. coli K1 colonization persisted in only 12% of CED, indicating that sustained inflammation occurred largely in the absence of ongoing infection. These immune alterations correlated with poor outcomes in a second pregnancy, including dystocia, ∼50% perinatal mortality, and growth-restricted pups. Conclusion These findings suggest that chorioamnionitis drives chronic uterine inflammation postpartum, providing a potential immune-mediated mechanism for adverse outcomes in future pregnancies. Funding Source n/a Topic Categories Mucosal and Regional Immunology (MUC)