Cancer immunotherapy aims to boost T cell responses against tumor cells. However, a large fraction of patients with cancer exhibit intrinsic or acquired resistance to immunotherapy. To better understand resistance mechanisms in tumor cells, we used CRISPR-Cas9 whole-genome screening to uncover tumor-specific genes that influence sensitivity to T cell killing. Among the top hits, we identified the transcription factor zinc-finger protein X-linked (Zfx). Zfx knockout tumor cells are resistant to T cell killing both in vitro and in vivo. We demonstrate that Zfx regulates expression of the apoptotic machinery, with Caspase-3 being a central element in mediating T cell killing. Mechanistically, our ChIP-Seq analyses in multiple human cancer cell lines show ZFX directly binds to the promoters of key apoptosis genes, including Caspase-3, to control their expression. Notably, ZFX expression is decreased in several human cancer tissues compared to healthy tissues. Female patients with kidney renal clear cell carcinoma (KIRC) with high ZFX expression showed longer survival compared to patients with low ZFX expression. In addition, we find that higher ZFX expression in patients with melanoma correlates with a positive response to anti-PD-1 immunotherapy. Our results demonstrate a novel resistant mechanism in tumor cells, highlighting ZFX as a potential biomarker for immunotherapy response, and suggest that targeting tumor cell intrinsic resistance genes in combination with immune therapies could benefit patients with cancer.
© 2025 The Authors.

Strong T cell receptor (TCR) and interleukin (IL)-27 signaling influence type 1 regulatory (Tr1) T cell development, but whether other signals determine their differentiation is unclear. Utilizing Tg4 TCR transgenic mice, we established a model for rapid Tr1 cell induction. A single high dose of [4Y]-MBP peptide drove the differentiation of Il10 + T cells with Tr1 cell mRNA and protein signatures. Kinetic transcriptional and phenotypic analyses revealed that the Tr1 cell module was transient and preceded by Ifng transcription in other CD4+ T cells. Changes in Tr1 cell frequency correlated with altered macrophage activation, while neutralization of interferon (IFN)γ reduced Tr1 cell frequency and the TCR signal strength markers Nur77, inducible T cell costimulator (ICOS), and OX40. Antibody depletion experiments inferred that the relevant source of IFNγ was not natural killer (NK) cell derived. Additionally, blocking IL-27 in combination with IFNγ neutralization additively reduced Tr1 cell frequency in vivo. These findings reveal that IFNγ has a non-redundant role in augmenting Tr1 cell differentiation in vivo.
© 2025 The Author(s).

The RNA-binding protein human antigen R (HuR) has been shown to reduce cardiac remodeling following both myocardial infarction and cardiac pressure overload, but the full extent of the HuR-dependent mechanisms within cells of the myocardium has yet to be elucidated. Wild-type mice were subjected to 30 min of cardiac ischemia (via LAD occlusion) and treated with a novel small molecule inhibitor of HuR at the time of reperfusion, followed by direct in vivo assessment of cardiac structure and function. Direct assessment of HuR-dependent mechanisms was done in vitro using neonatal rat ventricular myocytes (NRVMs) and bone marrow-derived macrophages (BMDMs). HuR activity is increased within 2 h after ischemia/reperfusion (I/R) and is necessary for early post-I/R inflammatory gene expression in the myocardium. Despite an early reduction in inflammatory gene expression, HuR inhibition has no effect on initial infarct size at 24 h post-I/R. However, pathological remodeling is reduced with preserved cardiac function at 2 weeks post-I/R upon HuR inhibition. RNA sequencing analysis of gene expression in NRVMs treated with LPS to model damage-associated molecular pattern (DAMP)-mediated activation of toll-like receptors (TLRs) demonstrates a HuR-dependent regulation of pro-inflammatory chemokine and cytokine gene expression in cardiomyocytes. Importantly, we show that conditioned media transfer from NRVMs pre-treated with HuR inhibitor loses the ability to induce inflammatory gene expression and M1-like polarization in bone marrow-derived macrophages (BMDMs) compared to NRVMs treated with LPS alone. Functionally, HuR inhibition reduces macrophage infiltration to the post-ischemic myocardium in vivo. Additionally, we show that LPS-treated NRVMs induce the migration of peripheral blood monocytes in a HuR-dependent endocrine manner. These studies demonstrate that HuR is necessary for early pro-inflammatory gene expression in cardiomyocytes following I/R injury that subsequently mediates monocyte recruitment and macrophage activation in the post-ischemic myocardium.
© 2025 The Author(s). The FASEB Journal published by Wiley Periodicals LLC on behalf of Federation of American Societies for Experimental Biology.

The goal of therapeutic cancer vaccines and immune checkpoint therapy (ICT) is to promote T cells with anti-tumor capabilities. Here, we compared mutant neoantigen (neoAg) peptide-based vaccines with ICT in preclinical models. NeoAg vaccines induce the most robust expansion of proliferating and stem-like PD-1+TCF-1+ neoAg-specific CD8 T cells in tumors. Anti-CTLA-4 and/or anti-PD-1 ICT promotes intratumoral TCF-1- neoAg-specific CD8 T cells, although their phenotype depends in part on the specific ICT used. Anti-CTLA-4 also prompts substantial changes to CD4 T cells, including induction of ICOS+Bhlhe40+ T helper 1 (Th1)-like cells. Although neoAg vaccines or ICTs expand iNOS+ macrophages, neoAg vaccines maintain CX3CR1+CD206+ macrophages expressing the TREM2 receptor, unlike ICT, which suppresses them. TREM2 blockade enhances neoAg vaccine efficacy and is associated with fewer CX3CR1+CD206+ macrophages and induction of neoAg-specific CD8 T cells. Our findings highlight different mechanisms underlying neoAg vaccines and different forms of ICT and identify combinatorial therapies to enhance neoAg vaccine efficacy.
Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.

Clinical trials have identified ARID1A mutations as enriched among patients who respond favorably to immune checkpoint blockade (ICB) in several solid tumor types independent of microsatellite instability. We show that ARID1A loss in murine models is sufficient to induce anti-tumor immune phenotypes observed in ARID1A mutant human cancers, including increased CD8+ T cell infiltration and cytolytic activity. ARID1A-deficient cancers upregulated an interferon (IFN) gene expression signature, the ARID1A-IFN signature, associated with increased R-loops and cytosolic single-stranded DNA (ssDNA). Overexpression of the R-loop resolving enzyme, RNASEH2B, or cytosolic DNase, TREX1, in ARID1A-deficient cells prevented cytosolic ssDNA accumulation and ARID1A-IFN gene upregulation. Further, the ARID1A-IFN signature and anti-tumor immunity were driven by STING-dependent type I IFN signaling, which was required for improved responsiveness of ARID1A mutant tumors to ICB treatment. These findings define a molecular mechanism underlying anti-tumor immunity in ARID1A mutant cancers.
Copyright © 2024 Elsevier Inc. All rights reserved.