These studies show that a complete TOX knockout or silencing has no effect on exhaustion marker expression levels or the transcriptome in repeat-anti-CD3/anti-CD28-stimulated primary human T cells in vitro.
Abstract
T cells, which are central mediators of the adaptive immune response, can become dysfunctional when faced with persistent antigen stimulation, such as in chronic infections and cancer. This dysfunctional state, known as T cell exhaustion, limits pro-inflammatory T cell function, dampens cytotoxicity and proliferative capacity, and promotes expression of inhibitory receptors. Thymocyte Selection-Associated High Mobility Group Box (TOX) has been proposed as a master regulator of T cell exhaustion due to its necessity for survival of exhausted T cells as well as its role in shaping chromatin accessibility in murine models. Interestingly, partial Tox deficiency may improve control of murine tumors. In human tumor infiltrating lymphocytes, high TOX expression is associated with poor disease prognosis. However, the mechanisms by which TOX expression is regulated and its importance to human T cell exhaustion remain poorly understood. We report here a robust strategy for generating a genetic knockout of TOX via base editing or a knockout phenocopy via epigenome editing in primary human T cells ex vivo, with each approach resulting in near-complete elimination of TOX mRNA. Guided by enhancer prediction data, we use epigenome editing to identify several human cis-regulatory regions which function to silence TOX expression to varying levels when targeted with CRISPRoff. TOX deficiency had no measurable impact on survival or exhaustion marker levels in human CD8+ T cells in a model of anti-CD3/anti-CD28 stimulation in vitro. In agreement with these data, expression profiling revealed that TOX knockout effects on the transcriptome are limited to TOX itself, with no observable downstream effects. These studies show that a complete TOX knockout or silencing has no effect on exhaustion marker expression levels or the transcriptome in repeat-anti-CD3/anti-CD28-stimulated primary human T cells in vitro. Taken together, we developed a powerful toolkit of genome and epigenome editing strategies to modify expression of a gene of interest in primary human T cells and study its function. We propose that this framework can be applied to additional genes of interest both to gain mechanistic information about T cell function, as well as develop strategies for improvement of T cell immunotherapies.
Exhaustion of CD8 T cells during cancer or chronic infections remains a significant barrier to T cell immunotherapies. Recent studies showed that distinct epigenetic changes drive exhaustion by silencing effector and memory-related genes, thereby establishing the dysfunctional state of exhausted T cells (TEX). Thus, targeting epigenetic regulation of exhaustion is crucial for restoring TEX cell function. Short-chain fatty acids (SCFAs) are emerging as key mediators linking cellular metabolism to gene regulation. Notably, certain SCFAs naturally produced by human microbiota have been shown to modulate host immune responses through epigenetic mechanisms.
To investigate their effects on TEX cell epigenetic programming, we utilized innovative in vitro T cell exhaustion models that generate stable terminal dysfunction in both human and mouse CD8 T cells. By inducing a state of exhaustion that recapitulates key molecular and functional features observed in cancer and chronic infections, these models enabled us to assess how SCFA treatment affects T cell function and memory-associated stemness features.
We discovered that a specific microbial SCFA triggered a significant recovery of polyfunctionality and memory programs within both human and mouse dysfunctional T cells. SCFA-treated TEX cells exhibited renewed effector capabilities, such as enhanced cytokine production, degranulation, and tumor-killing activity. The enhanced effector functions persisted even following termination of SCFA treatment, suggesting stable reprogramming of TEX cells.
These findings identify a novel microbial SCFA as a potential metabolic-epigenetic regulator, capable of reactivating effector programs in TEX cells while blocking terminal exhaustion. These results provide insights into developing new therapeutic approaches to reprogram TEX cells and enhance the efficacy of T cell immunotherapy.
n/a
Tumor Immunology: Cellular Responses and Tumor Microevironment (TIME)
Asmaa M. Yousif, Amira Yousif, Ava Lowin et al.· Journal of Immunology· 0 citations
Adoptive T-cell therapies and immune checkpoint blockade have produced durable remissions in selected malignancies, yet most patients still fail to achieve lasting benefit. Two convergent obstacles underlie much of this failure: T-cell exhaustion and tumour immune evasion. T-cell exhaustion arises from chronic antigen stimulation in the tumour microenvironment (TME) and spans a hierarchy from reversible, stem-like progenitor-exhausted cells to terminally exhausted cells with limited functional recovery, which is a transition epigenetically enforced by transcription factors such as TOX and the NR4A family. In parallel, tumours evade recognition by silencing antigen-presentation pathways, including MHC class I. This review discusses a complementary therapeutic strategy that addresses both obstacles: engineering T cells for greater durability in the TME through knockout of exhaustion-associated transcription factors, and reprogramming tumour cells with DNA methyltransferase (DNMTi) and histone deacetylase (HDACi) inhibitors to restore immunogenicity. We also consider emerging evidence that metabolic and neuro-immune features of the TME, including nerve-to-tumour mitochondrial transfer, may contribute to immune resistance in some tumour contexts. Importantly, we emphasise that most supporting evidence derives from CAR-T and murine systems, and that direct validation in TCR-engineered T-cell (TCR-T) platforms is still required. We further outline a personalised, biomarker-guided framework that integrates T-cell signatures, the epigenetic landscape of the tumour, and tumour innervation density to match combination therapy to the individual patient. Integrating exhaustion-resistant T cells with a reprogrammed, immunologically visible tumour may help address mechanisms of immune resistance and improve therapeutic outcomes.
L. Saltis, Liew Jun Mun· Pathology, Research and Prac...· 0 citations
T cells differentiate into subtypes to maintain immune tolerance or mount inflammatory response upon antigen stimulation. This raises questions about whether and how T cell subtypes rely on fundamentally distinct epigenetic programs.
Using Wdr82, a component of the Set1/COMPASS histone H3K4 methyltransferase complex, as a model, we discover that this pathway is broadly required for the activation and function of both Te and Treg cells.
Counterintuitively, T cell-specific deletion of Wdr82 leads to Te activation and lethal spontaneous colitis. This dysregulation is nearly completely prevented by microbiome depletion or wild-type Treg transfer. Mechanistically, Set1/COMPASS complex interacts with Foxp3 in a TCR-signaling dependent manner. H3K4me3 pathway is preferentially required for Treg induction and for the expression of Treg functional genes such as Il10 and Rorc.
Thus, the generic H3K4me3 pathway plays a biased role in Treg-dependent immune homeostasis particularly in the presence of commensal microbiota. Our study depicts an epigenetic polarity governing Treg-mediated immune tolerance, highlighting a fundamental asymmetry at the epigenetic level that differentially regulates Treg and conventional T cells. Perturbation of this asymmetry by genetic and environmental factors would lead to autoimmune dysregulation.
National Institute of Allergy and Infectious Diseases
Immune Response Regulation: Molecular Mechanisms (IRM)
Wenjun Huang, Yongqiang Feng, Jun Li et al.· Journal of Immunology· 0 citations
Introduction Chronic inflammation has long been associated with cancer initiation, yet the mechanisms linking sustained immune activation to an immune-permissive tumor microenvironment remain incompletely defined. Prevailing explanations such as immune exhaustion (IEX) or free radical mediated tissue damage, fail to account for the active state of immune tolerance, a process driven by potent negative feedback loops that systematically suppress host effector responses. Methods To address this gap, we developed an in vitro model of macrophage tolerance driven by sustained Toll-like receptor 4 (TLR4) activation using microbial-associated molecular patterns (MAMPs). This system captures the full kinetic progression of the immune response, tracking macrophages from a resting baseline, through acute activation at 24 hours, to a chronic tolerant endpoint at 7–11 days. Methodologically, cells were maintained under a continuous media exchange (+/− E. coli O111:B4 LPS) featuring high glucose and an elevated volume-to-cell ratio. This setup effectively eliminates autocrine interference and toxic byproducts, successfully isolating the direct consequences of sustained TLR4 signaling across extended durations. Results Whole-transcriptome sequencing, validated by RT-PCR and select protein immunoblots, revealed that both “exhaustion” and “tolerance” are mischaracterized. Rather than a passive exhaustion state or a simple trajectory of diminishing returns, the resting-acute-chronic continuum drives a potent, active negative-feedback mechanism across an eight-phase bidirectional trajectory. By days 7–11, macrophages shifted to a TAM-like signature, overexpressing immune checkpoints (PD-L1/MSN, TIM-3, SPP1, CD73, CD44, LILRs) and regulatory suppressive networks (SOCS/JAK/STAT, IL-10, CCL2/7/12, CXCL2), while downregulating classical (H2-D1/K1) and non-classical (H2-Q/T) MHC-I antigen-presenting genes. These alterations coincided with the profound loss of interferon-stimulated genes (ISGs) including the IFIT family, Ly6e, Irf7, Rsad2/Viperin, and the Oas gene family, fundamentally crippling the machinery required for antiviral and antitumor immune surveillance. Moreover, this chronic stage drove the upregulation of degradative proteases (cathepsins, Adam8, S100a8, Klk9, carboxypeptidase D), integrins/adhesion molecules (Itga5, Marcks, Msr1/CD204, Alcam), iron-storage transcripts, lipid translocases (Cd36), and fatty acid-binding proteins. Concurrently, macrophages upregulated Nos2/Cox2 alongside the metabolic collapse of mitochondrial OXPHOS genes and Acod1 (itaconate). Uniquely, this negative feedback loop coincided with a sustained, massive surge in a cluster of poorly characterized small proline-rich proteins (SPRRs), specifically Sprr2b, 2e, 2d, 2f, 2g, 2h, 2i, 2j, and 2k. Discussion Overall, these results indicate that chronic inflammatory signaling can ultimately trigger a profound coordinated negative-feedback program consistent with a reduced immune recognition and defense pathway signatures. Ultimately, this study provides a reproducible in vitro macrophage model to investigate immune suppression. It offers deeper insights into how chronic inflammation impairs host defenses against viral and tumor cells.
E. Mazzio, Andrew S. Barnes, R. Badisa et al.· Frontiers in Immunology· 0 citations
Rigorous research over the years has revealed that the cellular composition of the tumor microenvironment (TME) matters, and a major constituent that facilitates tumor progression is the regulatory T cell (Treg). Tregs are essential for maintaining immune homeostasis and preventing autoimmune conditions but can also be a detriment via their suppression of immune responses against cancer. The significance of Treg research is far-reaching as supported by the 2025 Nobel Prize in Physiology or Medicine awarded to Drs. Mary Brunkow, Fred Ramsdell and Shimon Sakaguchi, “for their discoveries concerning peripheral immune tolerance”. However, identifying differences in Treg subtypes to specifically disrupt the activities of tumor-promoting Tregs, without affecting Tregs that maintain immune homeostasis remain elusive. Here, we sought to discover how to blunt pro-tumorigenic Tregs without compromising the abilities of Tregs in mediating self-tolerance.
To this end, we used state-of-the-art methods in immunology, including high dimensional flow cytometry, lineage-specific loss-of-function studies and single-cell RNA sequencing, as well as multiple tumor models in animals to investigate these fundamental gaps in the field.
In doing so, we discovered that expression of the genome organizer special AT-rich sequence binding protein 1 (Satb1) could readily separate two Treg subtypes: pro-tumorigenic Tregs (termed Satb1+) and immune-regulatory Tregs (termed Satb1-). Deletion of Satb1 specifically in Tregs impaired the function of Satb1+ pro-tumorigenic Tregs, leading to enhanced CD8+ T cell antitumor immune responses, and complete tumor eradication without any systemic autoimmune conditions. Notably, Satb1- Tregs remained intact in knockout mice and were necessary for maintaining immune homeostasis and preventing autoimmunity.
Our results reveal a key mechanism to safely and potently enhance cancer immunity without causing systemic autoimmune diseases.
Pelotonia Institute for Immuno-Oncology
Tumor Immunology: Cellular Responses and Tumor Microevironment (TIME)
Ephraim A. Ansa-Addo, Parviz Azimnasab-sorkhabi, Musab Bouhajra et al.· Journal of Immunology· 0 citations
Post-transcriptional regulation (PTR) plays a crucial role in shaping T-cell gene expression programs that adapt to their microenvironment. Among RNA-binding proteins (RBPs) involved in PTR, Human antigen R (HuR; ELAVL1) binds to AU-rich elements within mRNAs encoding regulators of proliferation, inflammation, and immune responses. While HuR controls cytokine expression and T-cell development, its role in effector function and persistence remains unclear.
We utilized a T cell—specific HuR knockout (HuRfl/fl CD4-Cre) mouse model and employed flow cytometry, RIP-seq, and transcriptomic profiling to identify HuR-bound targets and their downstream effects. Functional analyses included chronic stimulation followed by characterization, ELISA, HuR and SerpinB9 overexpression, adoptive transfer into Rag ko mice (colitis), and the B16-F10 tumor model to assess T-cell function and persistence.
Using this approach, we found that HuR-deficient T cells exhibit increased effector cytokine secretion, decreased CD27 expression, elevated KLRG1 levels, and enhanced SA-β-Gal activity, characteristics of replicative senescence. Adoptive transfer of HuR-deficient T cells caused less severe colitis in Rag KO mice despite their effector phenotype, suggesting impaired persistence. Corroborating these findings, HuR expression was significantly reduced in tumor-infiltrating and aged T cells. Conversely, HuR overexpression decreased senescence and restored memory marker expression in both mouse and human aged T cells and TILs. Mechanistically, HuR bound transcripts involved in immune regulation, including SerpinB9, a granzyme B inhibitor vital for cytotoxic T-cell survival. Overexpressing SerpinB9 in wild-type and HuR-deficient T cells enhanced persistence under chronic stimulation and improved tumor control.
These findings highlight a HuR-SerpinB9 axis that regulates T-cell senescence and persistence, offering a potential therapeutic target in cancer and autoimmune diseases.
This work was supported by NIH grants R01CA300284, R01CA290201, R01CA282408, R42CA239952, R01DE030013, R01CA250458.
Tumor Immunology: Cellular Responses and Tumor Microevironment (TIME)
P. Chakraborty, Mrinmoyee Majumder, W. Wofford et al.· Journal of Immunology· 0 citations
A new method for surgically removing training examples from a model reveals that as datasets grow, the link between what a model learns and what it produces dissolves.
MIT News · Artificial Intelligence· news.mit.eduAug 17, 2026
