MATRIN3 deficiency triggers autoinflammation via cGAS-STING activation.

Interferon-stimulated genes (ISGs) comprise a program of immune effectors important for host immune defense. When uncontrolled, ISGs play a central role in interferonopathies and other inflammatory diseases. The mechanisms responsible for turning on ISGs are not completely known. By investigating MATRIN3 (MATR3), a nuclear RNA-binding protein mutated in familial ALS, we found that perturbing MATR3 results in elevated expression of ISGs. Using an integrative approach, we elucidate a pathway that leads to activation of cGAS-STING. This outlines a plausible mechanism for pathogenesis in a subset of ALS, and suggests new diagnostic and therapeutic approaches for this fatal disease.

The neuroimmune CGRP-RAMP1 axis tunes cutaneous adaptive immunity to the microbiota.

The somatosensory nervous system surveils external stimuli at barrier tissues, regulating innate immune cells under infection and inflammation. The roles of sensory neurons in controlling the adaptive immune system, and more specifically immunity to the microbiota, however, remain elusive. Here, we identified a mechanism for direct neuroimmune communication between commensal-specific T lymphocytes and somatosensory neurons mediated by the neuropeptide calcitonin gene-related peptide (CGRP) in the skin. Intravital imaging revealed that commensal-specific T cells are in close proximity to cutaneous nerve fibers in vivo. Correspondingly, we observed upregulation of the receptor for the neuropeptide CGRP, RAMP1, in CD8+ T lymphocytes induced by skin commensal colonization. The neuroimmune CGRP-RAMP1 signaling axis functions in commensal-specific T cells to constrain Type 17 responses and moderate the activation status of microbiota-reactive lymphocytes at homeostasis. As such, modulation of neuroimmune CGRP-RAMP1 signaling in commensal-specific T cells shapes the overall activation status of the skin epithelium, thereby impacting the outcome of responses to insults such as wounding. The ability of somatosensory neurons to control adaptive immunity to the microbiota via the CGRP-RAMP1 axis underscores the various layers of regulation and multisystem coordination required for optimal microbiota-reactive T cell functions under steady state and pathology.

Illuminating T cell-dendritic cell interactions in vivo by FlAsHing antigens.

Delineating the complex network of interactions between antigen-specific T cells and antigen presenting cells (APCs) is crucial for effective precision therapies against cancer, chronic infections, and autoimmunity. However, the existing arsenal for examining antigen-specific T cell interactions is restricted to a select few antigen-T cell receptor pairs, with limited in situ utility. This lack of versatility is largely due to the disruptive effects of reagents on the immune synapse, which hinder real-time monitoring of antigen-specific interactions. To address this limitation, we have developed a novel and versatile immune monitoring strategy by adding a short cysteine-rich tag to antigenic peptides that emits fluorescence upon binding to thiol-reactive biarsenical hairpin compounds. Our findings demonstrate the specificity and durability of the novel antigen-targeting probes during dynamic immune monitoring in vitro and in vivo. This strategy opens new avenues for biological validation of T-cell receptors with newly identified epitopes by revealing the behavior of previously unrecognized antigen-receptor pairs, expanding our understanding of T cell responses.

Clinical and functional spectrum of RAC2-related immunodeficiency.

ABSTRACT: Mutations in the small Rho-family guanosine triphosphate hydrolase RAC2, critical for actin cytoskeleton remodeling and intracellular signal transduction, are associated with neonatal severe combined immunodeficiency (SCID), infantile neutrophilic disorder resembling leukocyte adhesion deficiency (LAD), and later-onset combined immune deficiency (CID). We investigated 54 patients (23 previously reported) from 37 families yielding 15 novel RAC2 missense mutations, including one present only in homozygosity. Data were collected from referring physicians and literature reports with updated clinical information. Patients were grouped by presentation: neonatal SCID (n = 5), infantile LAD-like disease (n = 5), or CID (n = 44). Disease correlated to RAC2 activity: constitutively active RAS-like mutations caused neonatal SCID, dominant-negative mutations caused LAD-like disease, whereas dominant-activating mutations caused CID. Significant T- and B-lymphopenia with low immunoglobulins were seen in most patients; myeloid abnormalities included neutropenia, altered oxidative burst, impaired neutrophil migration, and visible neutrophil macropinosomes. Among 42 patients with CID with clinical data, upper and lower respiratory infections and viral infections were common. Twenty-three distinct RAC2 mutations, including 15 novel variants, were identified. Using heterologous expression systems, we assessed downstream effector functions including superoxide production, p21-activated kinase 1 binding, AKT activation, and protein stability. Confocal microscopy showed altered actin assembly evidenced by membrane ruffling and macropinosomes. Altered protein localization and aggregation were observed. All tested RAC2 mutant proteins exhibited aberrant function; no single assay was sufficient to determine functional consequence. Most mutants produced elevated superoxide; mutations unable to support superoxide formation were associated with bacterial infections. RAC2 mutations cause a spectrum of immune dysfunction, ranging from early onset SCID to later-onset combined immunodeficiencies depending on RAC2 activity. This trial was registered at www.clinicaltrials.gov as #NCT00001355 and #NCT00001467.

Dermis resident macrophages orchestrate localized ILC2 eosinophil circuitries to promote non-healing cutaneous leishmaniasis.

Tissue-resident macrophages are critical for tissue homeostasis and repair. We previously showed that dermis-resident macrophages produce CCL24 which mediates their interaction with IL-4+ eosinophils, required to maintain their M2-like properties in the TH1 environment of the Leishmania major infected skin. Here, we show that thymic stromal lymphopoietin (TSLP) and IL-5+ type 2 innate lymphoid cells are also required to maintain dermis-resident macrophages and promote infection. Single cell RNA sequencing reveals the dermis-resident macrophages as the sole source of TSLP and CCL24. Generation of Ccl24-cre mice permits specific labeling of dermis-resident macrophages and interstitial macrophages from other organs. Selective ablation of TSLP in dermis-resident macrophages reduces the numbers of IL-5+ type 2 innate lymphoid cells, eosinophils and dermis-resident macrophages, and ameliorates infection. Our findings demonstrate that dermis-resident macrophages are self-maintained as a replicative niche for L. major by orchestrating localized type 2 circuitries with type 2 innate lymphoid cells and eosinophils.

TCR ligand potency differentially impacts PD-1 inhibitory effects on diverse signaling pathways.

Checkpoint blockade revolutionized cancer therapy, but we still lack a quantitative, mechanistic understanding of how inhibitory receptors affect diverse signaling pathways. To address this issue, we developed and applied a fluorescent intracellular live multiplex signal transduction activity reporter (FILMSTAR) system to analyze PD-1-induced suppressive effects. These studies identified pathways triggered solely by TCR or requiring both TCR and CD28 inputs. Using presenting cells differing in PD-L1 and CD80 expression while displaying TCR ligands of distinct potency, we found that PD-1-mediated inhibition primarily targets TCR-linked signals in a manner highly sensitive to peptide ligand quality. These findings help resolve discrepancies in existing data about the site(s) of PD-1 inhibition in T cells while emphasizing the importance of neoantigen potency in controlling the effects of checkpoint therapy.

The Influence of Wet Granulation Parameters on the Compaction Behavior and Tablet Strength of a Hydralazine Powder Mixture.

The aim of this paper was to describe the influence of high-shear wet granulation process parameters on tablet tensile strength and compaction behavior of a powder mixture and granules containing hydralazine. The hydralazine powder mixture and eight types of granules were compacted into tablets and evaluated using the Heckel, Kawakita and Adams analyses. The granules were created using two types of granulation liquid (distilled water and aqueous solution of polyvinylpyrrolidone), at different impeller speeds (500 and 700 rpm) and with different wet massing times (without wet massing and for 2 min). Granulation resulted in improved compressibility, reduced dustiness and narrower particle-size distribution. A significant influence of wet massing time on parameters from the Kawakita and Adams analysis was found. Wet massing time had an equally significant effect on tablet tensile strength, regardless of the granulation liquid used. Granules formed with the same wet massing time showed the same trends in tabletability graphs. Tablets created using a single-tablet press (batch compaction) and an eccentric tablet press showed opposite values of tensile strength. Tablets from granules with a higher bulk density showed lower strength during batch compaction and, conversely, higher strength during eccentric tableting.

Illuminating T cell-dendritic cell interactions in vivo by FlAsHing antigens.

Delineating the complex network of interactions between antigen-specific T cells and antigen presenting cells (APCs) is crucial for effective precision therapies against cancer, chronic infections, and autoimmunity. However, the existing arsenal for examining antigen-specific T cell interactions is restricted to a select few antigen-T cell receptor pairs, with limited in situ utility. This lack of versatility is largely due to the disruptive effects of reagents on the immune synapse, which hinder real-time monitoring of antigen-specific interactions. To address this limitation, we have developed a novel and versatile immune monitoring strategy by adding a short cysteine-rich tag to antigenic peptides that emits fluorescence upon binding to thiol-reactive biarsenical hairpin compounds. Our findings demonstrate the specificity and durability of the novel antigen-targeting probes during dynamic immune monitoring in vitro and in vivo. This strategy opens new avenues for biological validation of T-cell receptors with newly identified epitopes by revealing the behavior of previously unrecognized antigen-receptor pairs, expanding our understanding of T cell responses.

Specific regulation of mechanical nociception by Gß5 involves GABA-B receptors.

Mechanical, thermal, and chemical pain sensation is conveyed by primary nociceptors, a subset of sensory afferent neurons. The intracellular regulation of the primary nociceptive signal is an area of active study. We report here the discovery of a Gß5-dependent regulatory pathway within mechanical nociceptors that restrains antinociceptive input from metabotropic GABA-B receptors. In mice with conditional knockout (cKO) of the gene that encodes Gß5 (Gnb5) targeted to peripheral sensory neurons, we demonstrate the impairment of mechanical, thermal, and chemical nociception. We further report the specific loss of mechanical nociception in Rgs7-Cre+/- Gnb5fl/fl mice but not in Rgs9-Cre+/- Gnb5fl/fl mice, suggesting that Gß5 might specifically regulate mechanical pain in regulator of G protein signaling 7-positive (Rgs7+) cells. Additionally, Gß5-dependent and Rgs7-associated mechanical nociception is dependent upon GABA-B receptor signaling since both were abolished by treatment with a GABA-B receptor antagonist and since cKO of Gß5 from sensory cells or from Rgs7+ cells potentiated the analgesic effects of GABA-B agonists. Following activation by the G protein-coupled receptor Mrgprd agonist ß-alanine, enhanced sensitivity to inhibition by baclofen was observed in primary cultures of Rgs7+ sensory neurons harvested from Rgs7-Cre+/- Gnb5fl/fl mice. Taken together, these results suggest that the targeted inhibition of Gß5 function in Rgs7+ sensory neurons might provide specific relief for mechanical allodynia, including that contributing to chronic neuropathic pain, without reliance on exogenous opioids.

Dermis resident macrophages orchestrate localized ILC2-eosinophil circuitries to maintain their M2-like properties and promote non-healing cutaneous leishmaniasis.

Tissue-resident macrophages (TRMs) are critical for tissue homeostasis/repair. We previously showed that dermal TRMs produce CCL24 (eotaxin2) which mediates their interaction with IL-4 producing eosinophils, required to maintain their number and M2-like properties in the TH1 environment of the Leishmania major infected skin. Here, we unveil another layer of TRM self-maintenance involving their production of TSLP, an alarmin typically characterized as epithelial cell-derived. Both TSLP signaling and IL-5+ innate lymphoid cell 2 (ILC2s) were shown to maintain the number of dermal TRMs and promote infection. Single cell RNA sequencing identified the dermal TRMs as the sole source of TSLP and CCL24. Development of Ccl24-cre mice permitted specific labeling of dermal TRMs, as well as interstitial TRMs from other organs. Genetic ablation of TSLP from dermal TRMs reduced the number of dermal TRMs, and disease was ameliorated. Thus, by orchestrating localized type 2 circuitries with ILC2s and eosinophils, dermal TRMs are self-maintained as a replicative niche for L. major.

Nuclear translocation of spike mRNA and protein is a novel feature of SARS-CoV-2.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes severe pathophysiology in vulnerable older populations and appears to be highly pathogenic and more transmissible than other coronaviruses. The spike (S) protein appears to be a major pathogenic factor that contributes to the unique pathogenesis of SARS-CoV-2. Although the S protein is a surface transmembrane type 1 glycoprotein, it has been predicted to be translocated into the nucleus due to the novel nuclear localization signal (NLS) "PRRARSV," which is absent from the S protein of other coronaviruses. Indeed, S proteins translocate into the nucleus in SARS-CoV-2-infected cells. S mRNAs also translocate into the nucleus. S mRNA colocalizes with S protein, aiding the nuclear translocation of S mRNA. While nuclear translocation of nucleoprotein (N) has been shown in many coronaviruses, the nuclear translocation of both S mRNA and S protein reveals a novel feature of SARS-CoV-2.

Chemokine positioning determines mutually exclusive roles for their receptors in extravasation of pathogenic human T cells.

Pro-inflammatory T cells co-express multiple chemokine receptors, but the distinct functions of individual receptors on these cells are largely unknown. Human Th17 cells uniformly express the chemokine receptor CCR6, and we discovered that the subgroup of CD4+CCR6+ cells that co-express CCR2 possess a pathogenic Th17 signature, can produce inflammatory cytokines independent of TCR activation, and are unusually efficient at transendothelial migration (TEM). The ligand for CCR6, CCL20, was capable of binding to activated endothelial cells (ECs) and inducing firm arrest of CCR6+CCR2+ cells under conditions of flow - but CCR6 could not mediate TEM. By contrast, CCL2 and other ligands for CCR2, despite being secreted from both luminal and basal sides of ECs, failed to bind to the EC surfaces - and CCR2 could not mediate arrest. Nonetheless, CCR2 was required for TEM. To understand if CCR2's inability to mediate arrest was due solely to an absence of EC-bound ligands, we generated a CCL2-CXCL9 chimeric chemokine that could bind to the EC surface. Although display of CCL2 on the ECs did indeed lead to CCR2-mediated arrest of CCR6+CCR2+ cells, activating CCR2 with surface-bound CCL2 blocked TEM. We conclude that mediating arrest and TEM are mutually exclusive activities of chemokine receptors and/or their ligands that depend, respectively, on chemokines that bind to the EC luminal surfaces versus non-binding chemokines that form transendothelial gradients under conditions of flow. Our findings provide fundamental insights into mechanisms of lymphocyte extravasation and may lead to novel strategies to block or enhance their migration into tissue.

Immunity to the microbiota promotes sensory neuron regeneration.

Tissue immunity and responses to injury depend on the coordinated action and communication among physiological systems. Here, we show that, upon injury, adaptive responses to the microbiota directly promote sensory neuron regeneration. At homeostasis, tissue-resident commensal-specific T cells colocalize with sensory nerve fibers within the dermis, express a transcriptional program associated with neuronal interaction and repair, and promote axon growth and local nerve regeneration following injury. Mechanistically, our data reveal that the cytokine interleukin-17A (IL-17A) released by commensal-specific Th17 cells upon injury directly signals to sensory neurons via IL-17 receptor A, the transcription of which is specifically upregulated in injured neurons. Collectively, our work reveals that in the context of tissue damage, preemptive immunity to the microbiota can rapidly bridge biological systems by directly promoting neuronal repair, while also identifying IL-17A as a major determinant of this fundamental process.

Epithelial-intrinsic defects in TGFßR signaling drive local allergic inflammation manifesting as eosinophilic esophagitis.

Allergic diseases are a global health challenge. Individuals harboring loss-of-function variants in transforming growth factor-ß receptor (TGFßR) genes have an increased prevalence of allergic disorders, including eosinophilic esophagitis. Allergic diseases typically localize to mucosal barriers, implicating epithelial dysfunction as a cardinal feature of allergic disease. Here, we describe an essential role for TGFß in the control of tissue-specific immune homeostasis that provides mechanistic insight into these clinical associations. Mice expressing a TGFßR1 loss-of-function variant identified in atopic patients spontaneously develop disease that clinically, immunologically, histologically, and transcriptionally recapitulates eosinophilic esophagitis. In vivo and in vitro, TGFßR1 variant-expressing epithelial cells are hyperproliferative, fail to differentiate properly, and overexpress innate proinflammatory mediators, which persist in the absence of lymphocytes or external allergens. Together, our results support the concept that TGFß plays a fundamental, nonredundant, epithelial cell-intrinsic role in controlling tissue-specific allergic inflammation that is independent of its role in adaptive immunity.

Adaptive immune responses to SARS-CoV-2 persist in the pharyngeal lymphoid tissue of children.

Most studies of adaptive immunity to SARS-CoV-2 infection focus on peripheral blood, which may not fully reflect immune responses at the site of infection. Using samples from 110 children undergoing tonsillectomy and adenoidectomy during the COVID-19 pandemic, we identified 24 samples with evidence of previous SARS-CoV-2 infection, including neutralizing antibodies in serum and SARS-CoV-2-specific germinal center and memory B cells in the tonsils and adenoids. Single-cell B cell receptor (BCR) sequencing indicated virus-specific BCRs were class-switched and somatically hypermutated, with overlapping clones in the two tissues. Expanded T cell clonotypes were found in tonsils, adenoids and blood post-COVID-19, some with CDR3 sequences identical to previously reported SARS-CoV-2-reactive T cell receptors (TCRs). Pharyngeal tissues from COVID-19-convalescent children showed persistent expansion of germinal center and antiviral lymphocyte populations associated with interferon (IFN)-γ-type responses, particularly in the adenoids, and viral RNA in both tissues. Our results provide evidence for persistent tissue-specific immunity to SARS-CoV-2 in the upper respiratory tract of children after infection.

The neuroimmune CGRP-RAMP1 axis tunes cutaneous adaptive immunity to the microbiota.

The somatosensory nervous system surveils external stimuli at barrier tissues, regulating innate immune cells under infection and inflammation. The roles of sensory neurons in controlling the adaptive immune system, and more specifically immunity to the microbiota, however, remain elusive. Here, we identified a novel mechanism for direct neuroimmune communication between commensal-specific T lymphocytes and somatosensory neurons mediated by the neuropeptide Calcitonin Gene-Related Peptide (CGRP) in the skin. Intravital imaging revealed that commensal-specific T cells are in close proximity to cutaneous nerve fibers in vivo . Correspondingly, we observed upregulation of the receptor for the neuropeptide CGRP, RAMP1, in CD8 + T lymphocytes induced by skin commensal colonization. Neuroimmune CGRP-RAMP1 signaling axis functions in commensal-specific T cells to constrain Type 17 responses and moderate the activation status of microbiota-reactive lymphocytes at homeostasis. As such, modulation of neuroimmune CGRP-RAMP1 signaling in commensal-specific T cells shapes the overall activation status of the skin epithelium, thereby impacting the outcome of responses to insults such as wounding. The ability of somatosensory neurons to control adaptive immunity to the microbiota via the CGRP-RAMP1 axis underscores the various layers of regulation and multisystem coordination required for optimal microbiota-reactive T cell functions under steady state and pathology. Significance statement: Multisystem coordination at barrier surfaces is critical for optimal tissue functions and integrity, in response to microbial and environmental cues. In this study, we identified a novel neuroimmune crosstalk mechanism between the sensory nervous system and the adaptive immune response to the microbiota, mediated by the neuropeptide CGRP and its receptor RAMP1 on skin microbiota-induced T lymphocytes. The neuroimmune CGPR-RAMP1 axis constrains adaptive immunity to the microbiota and overall limits the activation status of the skin epithelium, impacting tissue responses to wounding. Our study opens the door to a new avenue to modulate adaptive immunity to the microbiota utilizing neuromodulators, allowing for a more integrative and tailored approach to harnessing microbiota-induced T cells to promote barrier tissue protection and repair.

Gata3 ZsG and Gata3 ZsG-fl: Novel murine Gata3 reporter alleles for identifying and studying Th2 cells and ILC2s in vivo.

T helper-2 (Th2) cells and type 2 innate lymphoid cells (ILC2s) play crucial roles during type 2 immune responses; the transcription factor GATA3 is essential for the differentiation and functions of these cell types. It has been demonstrated that GATA3 is critical for maintaining Th2 and ILC2 phenotype in vitro; GATA3 not only positively regulates type 2 lymphocyte-associated genes, it also negatively regulates many genes associated with other lineages. However, such functions cannot be easily verified in vivo because the expression of the markers for identifying Th2 and ILC2s depends on GATA3. Thus, whether Th2 cells and ILC2s disappear after Gata3 deletion or these Gata3-deleted "Th2 cells" or "ILC2s" acquire an alternative lineage fate is unknown. In this study, we generated novel GATA3 reporter mouse strains carrying the Gata3 ZsG or Gata3 ZsG-fl allele. This was achieved by inserting a ZsGreen-T2A cassette at the translation initiation site of either the wild type Gata3 allele or the modified Gata3 allele which carries two loxP sites flanking the exon 4. ZsGreen faithfully reflected the endogenous GATA3 protein expression in Th2 cells and ILC2s both in vitro and in vivo. These reporter mice also allowed us to visualize Th2 cells and ILC2s in vivo. An inducible Gata3 deletion system was created by crossing Gata3 ZsG-fl/fl mice with a tamoxifen-inducible Cre. Continuous expression of ZsGreen even after the Gata3 exon 4 deletion was noted, which allows us to isolate and monitor GATA3-deficient "Th2" cells and "ILC2s" during in vivo immune responses. Our results not only indicated that functional GATA3 is dispensable for regulating its own expression in mature type 2 lymphocytes, but also revealed that GATA3-deficient "ILC2s" might be much more stable in vivo than in vitro. Overall, the generation of these novel GATA3 reporters will provide valuable research tools to the scientific community in investigating type 2 immune responses in vivo.

Nuclear translocation of spike mRNA and protein is a novel pathogenic feature of SARS-CoV-2.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes severe pathophysiology in vulnerable older populations and appears to be highly pathogenic and more transmissible than SARS-CoV or MERS-CoV [1, 2]. The spike (S) protein appears to be a major pathogenic factor that contributes to the unique pathogenesis of SARS-CoV-2. Although the S protein is a surface transmembrane type 1 glycoprotein, it has been predicted to be translocated into the nucleus due to the novel nuclear localization signal (NLS) "PRRARSV", which is absent from the S protein of other coronaviruses. Indeed, S proteins translocate into the nucleus in SARS-CoV-2-infected cells. To our surprise, S mRNAs also translocate into the nucleus. S mRNA colocalizes with S protein, aiding the nuclear translocation of S mRNA. While nuclear translocation of nucleoprotein (N) has been shown in many coronaviruses, the nuclear translocation of both S mRNA and S protein reveals a novel pathogenic feature of SARS-CoV-2. Author summary: One of the novel sequence insertions resides at the S1/S2 boundary of Spike (S) protein and constitutes a functional nuclear localization signal (NLS) motif "PRRARSV", which may supersede the importance of previously proposed polybasic furin cleavage site "RRAR". Indeed, S protein's NLS-driven nuclear translocation and its possible role in S mRNA's nuclear translocation reveal a novel pathogenic feature of SARS-CoV-2.

CD4 T cells are rapidly depleted from tuberculosis granulomas following acute SIV co-infection.

HIV/Mycobacterium tuberculosis (Mtb) co-infected individuals have an increased risk of tuberculosis prior to loss of peripheral CD4 T cells, raising the possibility that HIV co-infection leads to CD4 T cell depletion in lung tissue before it is evident in blood. Here, we use rhesus macaques to study the early effects of simian immunodeficiency virus (SIV) co-infection on pulmonary granulomas. Two weeks after SIV inoculation of Mtb-infected macaques, Mtb-specific CD4 T cells are dramatically depleted from granulomas, before CD4 T cell loss in blood, airways, and lymph nodes, or increases in bacterial loads or radiographic evidence of disease. Spatially, CD4 T cells are preferentially depleted from the granuloma core and cuff relative to B cell-rich regions. Moreover, live imaging of granuloma explants show that intralesional CD4 T cell motility is reduced after SIV co-infection. Thus, granuloma CD4 T cells may be decimated before many co-infected individuals experience the first symptoms of acute HIV infection.

Metformin treatment rescues CD8+ T-cell response to immune checkpoint inhibitor therapy in mice with NAFLD.

BACKGROUND & AIMS: Non-alcoholic steatohepatitis (NASH) represents the fastest growing underlying cause of hepatocellular carcinoma (HCC) and has been shown to impact immune effector cell function. The standard of care for the treatment of advanced HCC is immune checkpoint inhibitor (ICI) therapy, yet NASH may negatively affect the efficacy of ICI therapy in HCC. The immunologic mechanisms underlying the impact of NASH on ICI therapy remain unclear. METHODS: Herein, using multiple murine NASH models, we analysed the influence of NASH on the CD8+ T-cell-dependent anti-PD-1 responses against liver cancer. We characterised CD8+ T cells' transcriptomic, functional, and motility changes in mice receiving a normal diet (ND) or a NASH diet. RESULTS: NASH blunted the effect of anti-PD-1 therapy against liver cancers in multiple murine models. NASH caused a proinflammatory phenotypic change of hepatic CD8+ T cells. Transcriptomic analysis revealed changes related to NASH-dependent impairment of hepatic CD8+ T-cell metabolism. In vivo imaging analysis showed reduced motility of intratumoural CD8+ T cells. Metformin treatment rescued the efficacy of anti-PD-1 therapy against liver tumours in NASH. CONCLUSIONS: We discovered that CD8+ T-cell metabolism is critically altered in the context of NASH-related liver cancer, impacting the effectiveness of ICI therapy - a finding which has therapeutic implications in patients with NASH-related liver cancer. LAY SUMMARY: Non-alcoholic steatohepatitis represents the fastest growing cause of hepatocellular carcinoma. It is also associated with reduced efficacy of immunotherapy, which is the standard of care for advanced hepatocellular carcinoma. Herein, we show that non-alcoholic steatohepatitis is associated with impaired motility, metabolic function, and response to anti-PD-1 treatment in hepatic CD8+ T cells, which can be rescued by metformin treatment.