Hypoxia Induced DC Tolerance as an Immune Evasion Mechanism in Adoptive Cellular Therapy

Preprints.org · 2026-04-24

Background/Objectives: Glioblastoma (GBM) remains a lethal primary CNS malignancy with limited response to immunotherapy. Adoptive cellular therapy (ACT) improves survival in preclinical models, yet tumors ultimately recur. While T cell exhaustion is a common mechanism of resistance, the contribution of dendritic cell (DC) dysfunction remains unclear. We aimed to define mechanisms of immune escape following ACT, focusing on DC function and the role of hypoxia. Methods: Using a murine glioma model (KR158B), mice were treated with ACT consisting of tumor RNA–pulsed DC vaccines and adoptively transferred T cells. Tumor-infiltrating immune populations were analyzed by flow cytometry. DC function was assessed using T cell activation assays. Bulk RNA sequencing and gene set enrichment analysis were performed on sorted DCs. Hypoxia was modeled in vitro, and HIF1α was perturbed using CRISPR-mediated knockout. Results: ACT significantly increased survival but did not prevent tumor recurrence. Escaped tumors contained abundant cytotoxic, non-exhausted T cells, indicating that T cell dysfunction was not the primary driver of resistance. Instead, tumor-associated DCs exhibited impaired T cell activation despite preserved antigen uptake. Transcriptomic analyses revealed reduced antigen presentation and co-stimulatory signaling, alongside increased expression of tolerogenic factors. ACT-treated tumors demonstrated heightened hypoxia pathway activation, with elevated HIF1α expression in DCs. Hypoxia induced DC tolerogenic programs and reduced their ability to activate T cells, an effect partially reversed by HIF1α disruption. Increased immune infiltration and inflammation following ACT further amplified hypoxia signaling. Conclusions: DC dysfunction is a key mechanism of immune escape following ACT in glioma. Hypoxia-driven tolerization of DCs impairs sustained anti-tumor immunity, highlighting the hypoxia–DC axis as a promising therapeutic target to enhance immunotherapy efficacy.

Basal IFNλ2/3 signaling is required for ISG expression and viral control in human intestinal epithelial cells

PLoS Pathogens · 2026-01-12

Interferon-lambdas (IFNλs) serve as critical mediators of antiviral defense at mucosal surfaces. Beyond their established role in regulating innate immune responses during infection, recent evidence demonstrates that IFNλs are constitutively expressed in pathogen-free environments, termed "basal" IFN expression. While intestinal epithelial cells constitutively express all basal IFNλ subtypes (IFNλ1, IFNλ2, and IFNλ3), their individual contributions to antiviral immunity remain poorly defined. Here, we systematically investigate the distinct roles of IFNλ1 and IFNλ2/3 in regulating intrinsic antiviral immunity using human intestinal epithelial T84 cells. Through genetic depletion of IFNλ1 or IFNλ2/3, we show that basal IFNλ2/3, but not IFNλ1, is essential for restricting replication and spread of diverse viruses, including vesicular stomatitis virus (VSV), mammalian orthoreovirus (MRV), rotavirus (RV), and vaccinia virus (VV). Transcriptomic profiling revealed that IFNλ2/3 selectively controls the basal expression of interferon-stimulated genes (ISGs), including key antiviral effectors and components of the IFN signaling machinery (e.g., STAT1, STAT2, IRF9). Loss of IFNλ2/3 reduced total STAT1 protein levels and blunted responsiveness to exogenous IFNλ, indicating compromised interferon signaling capacity. Furthermore, basal IFNλ2/3 was required for activating paracrine JAK/STAT signaling and ISG induction in neighboring bystander cells, thereby amplifying antiviral protection across the epithelial layer. These findings reveal a functional hierarchy among IFNλ subtypes and establish IFNλ2/3 as the dominant, non-redundant regulators of epithelial immune readiness. Our study provides the first comprehensive analysis of basal IFNλ subtype functions in the gut epithelium and underscores the central role of basal IFNλ2/3 in maintaining mucosal antiviral defense.

Stage-specific regulation of KSHV infection by HIF-1α

Journal of Virology · 2026-03-10

ABSTRACT The impact of physiological stress conditions on Kaposi’s sarcoma-associated herpesvirus (KSHV) infection remains poorly understood. One such stressor, hypoxia, is regulated by the transcription factor HIF-1α. We recently reported that hypoxia, or HIF-1α expression alone, can promote lytic infection in cells that typically support latent infection under normoxia. Here, we show that hypoxia-induced lytic infection is reversible, leading to an abortive lytic cycle if the hypoxic condition ceases. Additionally, we found that HIF-1α induces lytic de novo infection only if expressed within the first 24 h post-infection (hpi). We show that HIF-1α can bind to viral promoters and induce lytic genes only during this early window of infection, before the KSHV genome undergoes heterochromatinization and establishes latency. In contrast, regardless of the timing of HIF-1α expression during KSHV infection, the induction of HIF-1α host target genes remains unaffected. These results indicate that the heterochromatinized KSHV DNA becomes resistant to HIF-1α-mediated activation after latency is established. These findings may explain why, despite the expression of HIF-1α in Kaposi’s sarcoma tumors, KSHV remains in latency, because HIF-1α cannot induce lytic genes once the viral DNA is heterochromatinized. Importantly, we also demonstrate that inhibition of the epigenetic repressor PRC2, which associates with lytic promoters after 24 hpi, restores HIF-1α’s ability to bind viral promoters and induce lytic gene expression post-latency. Collectively, our results indicate that not only the presence of HIF-1α, but also the timing and duration of its expression during KSHV infection, are critical determinants of its ability to drive lytic infection. IMPORTANCE The current view is that the default pathway of KSHV infection is the establishment of latency, however, how this is altered under physiological stress conditions remains largely unknown. We previously showed that hypoxia, or the expression of its transcription factor HIF-1α alone, promotes the establishment of lytic rather than latent KSHV infection. In this study, we show that the duration of hypoxia, as well as the timing and duration of HIF-1α expression, are crucial determinants in facilitating lytic de novo KSHV infection. Notably, we found that PRC2-mediated heterochromatin inhibits the HIF-1α-mediated upregulation of lytic genes as chromatinization of the KSHV genome progresses during infection. Our findings offer a deeper understanding of how epigenetic regulation intersects with host stress responses to influence viral pathogenesis.

Assessment of Epithelial Barrier Integrity by TEER and FITC-Dextran Permeability Assays

BIO-PROTOCOL · 2026-01-01

The integrity of epithelial barriers is essential for maintaining tissue homeostasis, particularly in the intestinal tract, where it separates the host from the complex luminal environment. Two complementary, standard methods for assessing this barrier are transepithelial electrical resistance (TEER), which provides a rapid, non-destructive measure of ionic conductance across tight junctions, and the fluorescein isothiocyanate (FITC)-dextran assay, which directly quantifies paracellular macromolecule flux. This protocol details a robust and reproducible method for performing both assays using intestinal epithelial cell monolayers (e.g., Caco-2, T84) cultured on permeable Transwell supports. We outline the procedure from cell culture and monolayer differentiation to TEER measurement with an Epithelial Volt/Ohm Meter 3 (EVOM3) and the subsequent FITC-dextran permeability assay. By combining these techniques, this protocol provides a comprehensive assessment of barrier function, making it ideal for studying tight junction dynamics and regulation under various experimental conditions, such as cytokine stimulation, drug screening, or microbial challenges. Key features • Combines electrical resistance (TEER) and macromolecular flux (FITC-dextran) assays for a comprehensive assessment of intestinal epithelial barrier integrity. • Applicable to various intestinal models, including Caco-2 and T84 cell lines; can be applied to other immortalized or primary epithelial and endothelial cells. • Utilizes the Epithelial Volt/Ohm Meter 3 (EVOM3) for accurate, non-destructive, and rapid TEER measurements in intestinal epithelial cell monolayers. • Provides clear guidelines for EVOM3 electrode handling and measurement to ensure reproducible results.

The actin nucleation promoting factor WASH facilitates clathrin-independent endocytosis of human papillomaviruses

EMBO Reports · 2025-10-10 · 1 citations

Endocytosis is a fundamental cellular process facilitated by diverse mechanisms. Remarkably, several distinct clathrin-independent endocytic processes have been identified and characterized following virus uptake into cells. For some, however, mechanistic execution and biological function remain largely unclear. This includes an endocytic process exploited by human papillomavirus type 16 (HPV16). Using HPV16, we examine how vesicles are formed by combining systematic cellular perturbations with electron and video microscopy. Cargo uptake occurs by uncoated, inward-budding pits. Mechanistically, vesicle scission is facilitated by actin polymerization controlled through the actin nucleation-promoting factor WASH. While WASH typically functions in conjunction with the retromer complex on endosomes during retrograde trafficking, endocytic vesicle formation is largely independent of retromer itself and the heterodimeric membrane-bending SNX-BAR retromer adaptor, thereby uncovering a role of WASH in endocytosis in addition to its canonical role in intracellular membrane trafficking.

TMIC-38. Dendritic cell dysfunction driven by hypoxia pathway activated DC tolerance as an immune evasion mechanism in adoptive cellular therapy

Neuro-Oncology · 2025-11-01

Abstract BACKGROUND Our adoptive cellular therapy (ACT) enhances dendritic cell (DC) and T cell infiltration into tumors, improving survival in murine brain tumor models. However, tumors employ mechanisms to restrict immune surveillance and efficacy of immunotherapy. We hypothesize that tumor-induced DC dysfunction facilitates immune evasion during ACT. This study investigates the mechanisms of DC dysfunction in ACT-escaped gliomas. METHODS T cell phenotypes were characterized using flowcytometry, and DC function was assessed through T cell activation assays. Gene set enrichment analysis (GSEA) was performed on transcriptomic data to identify enriched pathways. The impact of hypoxia and factors secreted from tumor-T cell interactions on DC tolerance was evaluated using RT-qPCR. In vivo correlations between hypoxia and DC tolerance were analyzed using GeoMx spatial transcriptomics. RESULTS ACT-escaped tumors retained adoptively transferred cytotoxic but non-exhausted T cells that failed to recognize antigen-shifted tumors. DCs from both untreated primary and ACT-escaped tumors showed impaired T cell activation and reduced expression of antigen-presentation genes. DCs from ACT-escaped tumors exhibited increased expression of tolerance-associated genes, with significant enrichment in hypoxia pathway genes. Hypoxia induced the expression of DC tolerance genes in a HIF1α-dependent manner, impairing T cell activation. Spatial transcriptomics confirmed a strong correlation between HIF1α and ARG1 in tumor-bearing brains. Increased immune infiltration in ACT-treated gliomas exacerbated hypoxia, as shown by HIF1α and CD45 co-expression, further driving DC dysfunction. Inflammatory factors secreted during tumor-T cell interactions activated hypoxia pathways and induced DC tolerance genes. CONCLUSION Hypoxia-driven DC tolerance is a key driver of immune escape under ACT.

Basal IFN-λ2/3 expression mediates tight junction formation in human epithelial cells

The EMBO Journal · 2025-09-01 · 8 citations

Type-III interferons (or IFNλs) play important roles in antiviral defense and intestinal epithelial barrier integrity. While interferon expression has been primarily studied in response to pathogens, basal interferon expression also occurs in pathogen-free environments. However, the mechanisms regulating basal IFN-λ expression and their functions have not yet been elucidated. Here, we show that basal IFN-λ2/3 expression is linked to the development of an intact cellular epithelium characterized by formation of tight junctions and establishment of barrier function. Our findings indicate that basal IFN-λ2/3 expression depends on cGAS-STING-mediated mitochondrial DNA detection, while it is inhibited by the Hippo mechanotransduction pathway at low cellular densities. Cells lacking basal IFN-λ2/3 expression fail to develop proper tight junctions and establish normal barrier function. Mechanistically, IFN-λ2/3 suppresses Claudin-2 expression, thereby promoting barrier formation as cells become confluent. These results demonstrate a previously unknown function of basal IFNλ expression in regulating epithelial cell junction formation and highlight their importance not only during pathogen challenges but also in maintaining epithelial cell function under steady-state conditions.

Hypoxia increases susceptibility of human intestinal epithelial cells to rotavirus infection through repression of interferon induction

Gut Microbes · 2025-09-22 · 3 citations

Intestinal epithelial cells (IECs) serve as both a physical barrier and a source of robust antiviral interferon (IFN) response. As such, they constitute the primary barrier that enteric viruses, such as rotavirus, need to overcome to initiate infection. The gut is characterized by very low oxygen levels (hypoxia) within the lumen, resulting in a unique hypoxic physiological environment in which rotavirus infection occurs. Depending on the tissues or viruses, conflicting results have been described for the role of hypoxia in regulating viral infections, where hypoxia could have either a proviral or antiviral function. Since intestinal epithelial cells naturally exist in a hypoxic environment, it is essential to investigate how these conditions affect rotavirus infection. We found that hypoxia promotes rotavirus infection, resulting in increased virus replication and production of infectious virus particles. We showed that this increased production of rotavirus particles under hypoxia is due to a decreased induction of IFNs, leading to a decreased expression of IFN stimulated genes and antiviral protection. RNA sequencing showed a robust decrease in ISG production in hypoxia for both rotavirus infection and poly I:C transfection, suggesting a conserved inhibition of IECs' IFN response to viral pathogen challenges under hypoxic conditions. Functional analyses revealed that hypoxia impairs signal transduction leading to IFN expression by negatively regulating the activation of the master signaling molecule TBK1. Mechanistically, we determined that hypoxia induces the expression of the protein phosphatase PP2A which is responsible for the hypoxia-induced impairment of TBK1 activation. Importantly, we confirmed that this hypoxia-mediated dampening of immune response was not restricted to rotavirus infection but dampened the IFN induction of a broad range of viruses and immune stimuli. Together, we propose that hypoxia creates an immune-suppressive environment through downregulation of IFN, representing a novel proviral mechanism for hypoxia in the human gastro-intestinal tract.

361 The hypoxic twist: immunotherapy-enhanced pathway turns dendritic cell tolerogenic in tumor immune evasion

Regular and Young Investigator Award Abstracts · 2025-11-01

Rotavirus Spreads in a Spatially Controlled Manner

Cells · 2025-02-19

Rotavirus is an enteric virus that leads to 200,000 deaths worldwide every year. The live-cell imaging evaluating rotavirus infection of MA104 cells revealed that rotavirus replication and spread occurs in a spatially controlled manner. Specifically, following initial rotavirus infection, the infected cells die, and the second round of infection occurs in the restricted area surrounding the initially infected cell. Interestingly, we found that the time required to establish the secondary infection is shorter compared to the time required for the initial infection. To determine if this increase in the kinetic of secondary infection was due to the early release of viruses or priming of the cells that are infected during the secondary infection, we used a combination of live-cell microscopy, trypsin neutralization assays, and the pharmacological inhibition of calcium signaling. Together, our results show that the second round of infection required rotavirus to be released and accessible to extracellular proteases. In addition, we found that the calcium wave induced upon rotavirus infection was critical for initial infection but did not play a role in the establishment of a secondary infection. Finally, we uncovered that high viral titers released from the initial infection were sufficient to accelerate the rate of the secondary infection.