Jose Aramburu

Effector T lymphocytes are avid nutrient consumers, but can function in nutrient-poor tumor microenvironments. Availability of key nutrients such as glucose inside the tumor is not homogeneous, and how tumor-infiltrating T lymphocytes (TILs) differ between regions with better and poorer blood perfusion is not well known. Here we show that in vitro-stimulated TILs can induce substantial production of hallmark glucose-dependent cytokines under glucose concentrations 20 times lower than in blood. In vivo, effector TILs in tumor regions with poor access to blood show comparable capacity for inducing IFNγ and granzyme B to TILs with fuller accessibility; exhibit an enhanced type I IFN response supported by local myeloid cells; and unexpectedly, have reduced expression of immune checkpoint and Treg-associated markers. TILs with poor blood accessibility also have lower biosynthetic activity than highly blood-accessible TILs, yet both compartments depend fundamentally on glucose for ATP production. Thus, effector T lymphocytes in poorly perfused tumor regions can maintain specific glucose-dependent responses, and might be partially protected from inhibitory and exhausting pressure from the tumor microenvironment.

Immunotherapy has revolutionized cancer treatment, yet responses vary significantly based on tumour characteristics and microenvironment. Here, we developed and analysed subcutaneous and orthotopic immunocompetent mice models of mismatch repair-deficient (dMMR) lung adenocarcinoma (LUAD) by selectively ablating Mlh1. Subcutaneous tumours demonstrated partial sensitivity to anti-PD1 therapy, characterized by tumour volume reduction without significant changes in immune infiltration. In contrast, orthotopic tumours exhibited robust responses, with substantial reductions in tumour burden, enhanced immune infiltration, and increased CD4⁺memory T cells, highlighting the critical role of anatomical site and tumour microenvironment in shaping immunotherapy outcomes. Our findings emphasise the relevance of orthotopic models for preclinical evaluation and suggest that they more accurately reflect clinical responses to immune checkpoint blockade in dMMR LUAD.

Hematopoietic stem cells (HSCs) readily recover from acute stress, but persistent stress can reduce their viability and long-term potential. Here, we show that the nuclear factor of activated T cells 5 (NFAT5), a transcription modulator of inflammatory responses, protects the HSC pool under stress. NFAT5 restrains HSC differentiation to multipotent progenitors after bone marrow transplantation and bone marrow ablation with ionizing radiation or chemotherapy. Correspondingly, NFAT5-deficient HSCs fail to support long-term reconstitution of hematopoietic progenitors and mature blood cells after serial transplant. Evidence from competitive transplant assays shows that these defects are HSC intrinsic. NFAT5-deficient HSCs exhibit enhanced expression of type 1 interferon (IFN-1) response genes after transplant, and suppressing IFN-1 receptor prevents their exacerbated differentiation and cell death after reconstitution and improves long-term regeneration potential. Blockade of IFN-1 receptor also prevented the overdifferentiation of NFAT5-deficient HSCs after bone marrow ablation. These findings show that long-term IFN-1 responses to different hematopoietic stressors drive HSCs toward more differentiated progenitors, and that NFAT5 has an HSC-intrinsic role, limiting IFN-1 responses to preserve reconstitution potential. Our identification of cell-intrinsic mechanisms that strengthen the resistance of HSCs to stress could help to devise approaches to protect long-term stemness during the treatment of hematopoietic malignancies.

Effector T lymphocytes are avid glucose consumers, but can function in the nutrient-poor environments of tumors. However, availability of blood-delivered nutrients throughout the tumor is not homogeneous, and how this affects effector T cells is not well known. Here we have isolated tumor-infiltrating T lymphocytes (TILs) from mouse solid tumors by their capacity to capture blood-transported probes, and compared them with glucose-restricted T cells. Glucose restriction in vitro arrested cell proliferation but reduced only moderately the induction of hallmark glucose-dependent cytokines interferon gamma (IFNγ) and IL-17. In vivo , effector TILs with reduced access to blood had characteristics of glucose-restricted cells, such as reduced expression of IFNγ and genes associated with cell proliferation. However, they expressed more CXCR3, which identifies effective antitumor T lymphocytes, showed an enhanced IFN response signature, and had reduced expression of surface PD-1. We also identified genes regulated by the enzyme ACSS2, which allows TILs to sustain gene expression in glucose-poor environments. Thus, effector T lymphocytes infiltrating tumors express different gene signatures in regions with different accessibility to blood, and can maintain specific glucose-dependent responses even in poorly perfused tumor regions. Our results can help better understand nutrient-dependent TIL heterogeneity in changing tumor microenvironments. Abstract Figure:

TSS-Seq analysis of the kidney from renal tubular cell-specific NFAT5 KO mice uncovered novel genes that are possibly regulated by NFAT5 in the kidney under physiological conditions. The study further implied disorders of innate and adaptive immune systems in NFAT5 KO mice, thereby exacerbating renal fibrosis at pathological states. Our results may implicate the involvement of renal tubular NFAT5 in the progression of renal fibrosis. Further studies would be worthwhile for the development of novel therapy to treat chronic kidney disease.

The kidney plays a crucial role in blood pressure (BP) regulation by controlling sodium reabsorption along the nephron. NFAT5 (nuclear factor of activated T-cells 5) is a transcription factor that is expressed in various tissues including the kidney and is activated at hypertonic conditions as observed in the renal medulla; the role for kidney NFAT5 in BP regulation, however, remains still obscure. In the present study, we generated inducible and renal tubular cell–specific NFAT5 knockout (KO) mice and characterized their phenotype. The NFAT5 KO mice exhibited high BP, hypernatremia, polyuria, and low urinary sodium excretion without significant alterations in the plasma renin activity or aldosterone concentration. The mice fed a high-salt diet further increased BP, revealing salt-sensitive hypertension. The KO mice ehibited the increased gene expression of the epithelial sodium channel. Protein expression of epithelial sodium channel in the membrane fraction was also significantly increased in KO mice than in wild-type mice. Treatment with amiloride, an epithelial sodium channel blocker, corrected high BP, hypernatremia, and decreased urinary sodium excretion in KO mice to the same levels of those in wild-type mice. Finally, the effects of high-salt diet and amiloride in KO mice were confirmed by the radiotelemetry method. In conclusion, these data indicate that renal tubular NFAT5 should play an important role in regulating sodium reabsorption through epithelial sodium channel under high-salt conditions, thereby preventing salt-dependent hypertension.

The ability of innate immune cells to respond to pathogen-associated molecular patterns across a wide range of intensities is fundamental to limit the spreading of infections. Studies on transcription responses to pathogen-activated TLRs have often used relatively high TLR ligand concentrations, and less is known about their regulation under mild stimulatory conditions. We had shown that the transcription factor NFAT5 facilitates expression of antipathogen genes under TLR stimulation conditions corresponding to low pathogen loads. In this study, we analyze how NFAT5 optimizes TLR-activated responses in mouse macrophages. We show that NFAT5 was required for effective recruitment of central effectors p65/NF-κB and c-Fos to specific proinflammatory target genes, such as Nos2, Il6, and Tnf in primary macrophages responding to low doses of the TLR4 ligand LPS. By contrast, NFAT5 was not required for p65/NF-κB recruitment in response to high LPS doses. Using the transposase-accessible chromatin with high-throughput sequencing assay, we show that NFAT5 facilitated chromatin accessibility mainly at promoter regions of multiple TLR4-responsive genes. Analysis of various histone marks that regulate gene expression in response to pathogens identified H3K27me3 demethylation as an early NFAT5-dependent mechanism that facilitates p65 recruitment to promoters of various TLR4-induced genes. Altogether, these results advance our understanding about specific mechanisms that optimize antipathogen responses to limit infections. Key Points NFAT5 amplifies TLR-induced antipathogen transcription responses. NFAT5 induces chromatin accessibility and H3K27me3 demethylation. NFAT5 facilitates recruitment of p65/NF-κB and c-Fos to promoters. Visual Abstract

Type I interferon (IFN-I) provides effective antiviral immunity but can exacerbate harmful inflammatory reactions and cause hematopoietic stem cell (HSC) exhaustion; therefore, IFN-I expression must be tightly controlled. While signaling mechanisms that limit IFN-I induction and function have been extensively studied, less is known about transcriptional repressors acting directly on IFN-I regulatory regions. We show that NFAT5, an activator of macrophage pro-inflammatory responses, represses Toll-like receptor 3 and virus-induced expression of IFN-I in macrophages and dendritic cells. Mice lacking NFAT5 exhibit increased IFN-I production and better control of viral burden upon LCMV infection but show exacerbated HSC activation under systemic poly(I:C)-induced inflammation. We identify IFNβ as a primary target repressed by NFAT5, which opposes the master IFN-I inducer IRF3 by binding to an evolutionarily conserved sequence in the IFNB1 enhanceosome that overlaps a key IRF site. These findings illustrate how IFN-I responses are balanced by simultaneously opposing transcription factors.