Vγ9Vδ2 T-cells Are Potent Inhibitors of SARS-CoV-2 Replication and Represent Effector Phenotypes in Patients With COVID-19.

Vγ9Vδ2 T cells play a key role in the innate immune response to viral infections through butyrophilin 3A (BTN3A). Here, we report blood Vγ9Vδ2 T cells decreased in clinically mild COVID-19 compared to healthy volunteers, and this was maintained up to 28 days and in the recovery period. Terminally differentiated Vγ9Vδ2 T cells tended to be enriched on the day of diagnosis, 28 days after, and during the recovery period. These cells showed cytotoxic and inflammatory activities following anti-BTN3A activation. BTN3A upregulation and Vγ9Vδ2 T-cell infiltration were observed in a lung biopsy from a fatal SARS-CoV-2 infection. In vitro, SARS-CoV-2 infection increased BTN3A expression in macrophages and lung cells that enhanced the anti-SARS-CoV-2 Vγ9Vδ2 T-cell cytotoxicity and interferon-γ and tumor necrosis factor-α. Increasing concentrations of anti-BTN3A lead to viral replication inhibition. Altogether, we report Vγ9Vδ2 T cells are important in the immune response against SARS-CoV-2 infection and activation by anti-BTN3A antibody may enhance their response. Clinical Trials Registration. NCT04816760.

Pan-microscopic examination of monkeypox virus in trophoblasts cells reveals new insights into virions release through filopodia-like projections.

Vertical transmission has been described following monkeypox virus (MPXV) infection in pregnant women. The presence of MPXV has been reported in the placenta from infected women, but whether pathogens colonize placenta remains unexplored. We identify trophoblasts as a target cell for MPXV replication. In a pan-microscopy approach, we decipher the specific infectious cycle of MPXV and inner cellular structures in trophoblasts. We identified the formation of a specialized region for viral morphogenesis and replication in placental cells. We also reported infection-induced cellular remodeling. We found that MPXV stimulates cytoskeleton reorganization with intercellular extensions for MPXV cell spreading specifically to trophoblastic cells. Altogether, the specific infectious cycle of MPXV in trophoblast cells and these protrusions that were structurally and morphologically similar to filopodia reveal new insights into the infection of MPXV.

Selected recent advances in understanding the role of human mast cells in health and disease.

Mast cells are highly granular tissue-resident cells and key drivers of inflammation, particularly in allergies as well as in other inflammatory diseases. Most mast cell research was initially conducted in rodents but has increasingly shifted to the human system, with the advancement of research technologies and methodologies. Today we can analyze primary human cells including rare subpopulations, we can produce and maintain mast cells isolated from human tissues, and there are several human mast cell lines. These tools have substantially facilitated our understanding of their role and function in different organs in both health and disease. We can now define more clearly where human mast cells originate from, how they develop, which mediators they store, produce de novo, and release, how they are activated and by which receptors, and which neighboring cells they interact with and by which mechanisms. Considerable progress has also been made regarding the potential contribution of mast cells to disease, which, in turn, has led to the development of novel approaches for preventing key pathogenic effects of mast cells, heralding the era of mast cell-targeted therapeutics. In this review, we present and discuss a selection of some of the most significant advancements and remaining gaps in our understanding of human mast cells during the last 25 years, with a focus on clinical relevance.

Disruption of the Expression of the Placental Clock and Melatonin Genes in Preeclampsia.

Circadian rhythms have been described in numerous tissues of living organisms and are necessary for homeostasis. The understanding of their role in normal and pathological pregnancy is only just emerging. It has been established that clock genes are expressed in the placenta of animals and humans, but the rhythmicity of placenta immune cells is not known. Macrophages from healthy placenta of women at term were isolated and the expression of clock genes BMAL1, CLOCK, PER2, CRY2, and NR1D1 was assessed by qRT-PCR every 4 h over 24 h. Raw data were treated with cosinor analysis to evaluate the significance of the oscillations. Placental macrophages exhibited significant circadian expression of clock genes but one third of placental macrophages lost clock gene rhythmicity; the clock gene oscillations were restored by co-culture with trophoblasts. We wondered if melatonin, a key hormone regulating circadian rhythm, was involved in the oscillations of placental cells. We showed that macrophages and trophoblasts produced melatonin and expressed MT2 receptor. In women who developed preeclampsia during pregnancy, circadian oscillations of placental macrophages were lost and could not be rescued by coculture with trophoblasts from healthy women. Moreover, production and oscillations of melatonin were altered in preeclamptic macrophages. For the first time to our knowledge, this study shows circadian rhythms and melatonin production by placental macrophages. It also shows that preeclampsia is associated with a disruption of the circadian rhythm of placental cells. These results represent a new scientific breakthrough that may contribute to the prevention and treatment of obstetrical pathologies.

Infection and Persistence of Coxiella burnetii Clinical Isolate in the Placental Environment.

Infection by Coxiella burnetii, the etiological agent of Q fever, poses the risk of causing severe obstetrical complications in pregnant women. C. burnetii is known for its placental tropism based on animal models of infection. The Nine Mile strain has been mostly used to study C. burnetii pathogenicity but the contribution of human isolates to C. burnetii pathogenicity is poorly understood. In this study, we compared five C. burnetii isolates from human placentas with C. burnetii strains including Nine Mile (NM) as reference. Comparative genomic analysis revealed that the Cb122 isolate was distinct from other placental isolates and the C. burnetii NM strain with a set of unique genes involved in energy generation and a type 1 secretion system. The infection of Balb/C mice with the Cb122 isolate showed higher virulence than that of NM or other placental isolates. We evaluated the pathogenicity of the Cb122 isolate by in vitro and ex vivo experiments. As C. burnetii is known to infect and survive within macrophages, we isolated monocytes and placental macrophages from healthy donors and infected them with the Cb122 isolate and the reference strain. We showed that bacteria from the Cb122 isolate were less internalized by monocyte-derived macrophages (MDM) than NM bacteria but the reference strain and the Cb122 isolate were similarly internalized by placental macrophages. The Cb122 isolate and the reference strain survived similarly in the two macrophage types. While the Cb122 isolate and the NM strain stimulated a poorly inflammatory program in MDM, they elicited an inflammatory program in placenta macrophages. We also reported that the Cb122 isolate and NM strain were internalized by trophoblastic cell lines and primary trophoblasts without specific replicative profiles. Placental explants were then infected with the Cb122 isolate and the NM strain. The bacteria from the Cb122 isolate were enriched in the chorionic villous foetal side. It is likely that the Cb122 isolate exhibited increased virulence in the multicellular environment provided by explants. Taken together, these results showed that the placental isolate of C. burnetii exhibits a specific infectious profile but its pathogenic role is not as high as the host immune response in pregnant women.

Congenital Infection of Severe Acute Respiratory Syndrome Coronavirus 2 With Intrauterine Fetal Death: A Clinicopathological Study With Molecular Analysis.

BACKGROUND: Observations of vertical transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection from mother to fetus have recently been described in the literature. However, the consequences of such transmission, whether fetal or neonatal, are poorly understood. METHODS: From a case of in utero fetal death at 24+2 weeks of gestation that occurred 7 days after the diagnosis of symptomatic SARS-CoV-2 infection in the mother, we isolated the incriminating virus by immunochemistry and molecular techniques in several fetal tissues, with a variant analysis of the SARS-CoV-2 genome. RESULTS: The fetal demise could be explained by the presence of placental histological lesions, such as histiocytic intervillositis and trophoblastic necrosis, in addition to fetal tissue damage. We observed mild fetal growth retardation and visceral damage to the liver, causing hepatocellular damage and hemosiderosis. To the best of our knowledge, this is the first report in the literature of fetal demise secondary to maternal-fetal transmission of SARSCoV- 2 with a congenital infection and a pathological description of placental and fetal tissue damage. CONCLUSIONS: SARS-CoV-2 was identified in both specimens using 3 independent techniques (immunochemistry, real-time quantitative polymerase chain reaction, and realtime digital polymerase chain reaction). Furthermore, the incriminating variant has been identified.

RadA, a Key Gene of the Circadian Rhythm of Escherichia coli.

Circadian rhythms are present in almost all living organisms, and their activity relies on molecular clocks. In prokaryotes, a functional molecular clock has been defined only in cyanobacteria. Here, we investigated the presence of circadian rhythms in non-cyanobacterial prokaryotes. The bioinformatic approach was used to identify a homologue of KaiC (circadian gene in cyanobacteria) in Escherichia coli. Then, strains of E. coli (wild type and mutants) were grown on blood agar, and sampling was made every 3 h for 24 h at constant conditions. Gene expression was determined by qRT-PCR, and the rhythmicity was analyzed using the Cosinor model. We identified RadA as a KaiC homologue in E. coli. Expression of radA showed a circadian rhythm persisting at least 3 days, with a peak in the morning. The circadian expression of other E. coli genes was also observed. Gene circadian oscillations were lost in radA mutants of E. coli. This study provides evidence of molecular clock gene expression in E. coli with a circadian rhythm. Such a finding paves the way for new perspectives in antibacterial treatment.

Monocytes and Macrophages, Targets of Severe Acute Respiratory Syndrome Coronavirus 2: The Clue for Coronavirus Disease 2019 Immunoparalysis.

BACKGROUND: Coronavirus disease 2019 (COVID-19) clinical expression is pleiomorphic, severity is related to age and comorbidities such as diabetes and hypertension, and pathophysiology involves aberrant immune activation and lymphopenia. We wondered if the myeloid compartment was affected during COVID-19 and if monocytes and macrophages could be infected by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). METHODS: Monocytes and monocyte-derived macrophages (MDMs) from COVID-19 patients and controls were infected with SARS-CoV-2 and extensively investigated with immunofluorescence, viral RNA extraction and quantification, and total RNA extraction followed by reverse-transcription quantitative polymerase chain reaction using specific primers, supernatant cytokines (interleukins 6, 10, and 1ß; interferon-ß; transforming growth factor-ß1, and tumor necrosis factor-α), and flow cytometry. The effect of M1- vs M2-type or no polarization prior to infection was assessed. RESULTS: SARS-CoV-2 efficiently infected monocytes and MDMs, but their infection is abortive. Infection was associated with immunoregulatory cytokines secretion and the induction of a macrophagic specific transcriptional program characterized by the upregulation of M2-type molecules. In vitro polarization did not account for permissivity to SARS-CoV-2, since M1- and M2-type MDMs were similarly infected. In COVID-19 patients, monocytes exhibited lower counts affecting all subsets, decreased expression of HLA-DR, and increased expression of CD163, irrespective of severity. CONCLUSIONS: SARS-CoV-2 drives monocytes and macrophages to induce host immunoparalysis for the benefit of COVID-19 progression.SARS-CoV-2 infection of macrophages induces a specific M2 transcriptional program. In Covid-19 patients, monocyte subsets were decreased associated with up-expression of the immunoregulatory molecule CD163 suggesting that SARS-CoV-2 drives immune system for the benefit of Covid-19 disease progression.

Daytime variation in SARS-CoV-2 infection and cytokine production.

S. Ray and A. Reddy recently anticipated the implication of circadian rhythm in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which is the causative agent of the coronavirus disease (Covid-19). In addition to its key role in the regulation of biological functions, the circadian rhythm has been suggested as a regulator of viral infections. Specifically, the time of day of infection was found critical for illness progression, as has been reported for influenza, respiratory syncytial and parainfluenza type 3 viruses. We analyzed circadian rhythm implication in SARS-CoV-2 virus infection of isolated human monocytes, key actor cells in Covid-19 disease, from healthy subjects. The circadian gene expression of BMAL1 and CLOCK genes was investigated with q-RTPCR. Monocytes were infected with SARS-CoV-2 virus strain and viral infection was investigated by One-Step qRT-PCR and immunofluorescence. Interleukin (IL)-6, IL-1ß and IL-10 levels were also measured in supernatants of infected monocytes. Using Cosinor analysis, we showed that BMAL1 and CLOCK transcripts exhibited circadian rhythm in monocytes with an acrophase and a bathyphase at Circadian Time (CT)6 and CT17. After 48 h, the amount of SARS-CoV-2 virus increased in the monocyte infected at CT6 compared to CT17. The high virus amount at CT6 was associated with significant increased release in IL-6, IL-1ß and IL-10 compared to CT17. Our results suggest that time day of SARS-CoV-2 infection affects viral infection and host immune response. They support consideration of circadian rhythm in SARS-CoV-2 disease progression and we propose circadian rhythm as a novel target for managing viral progression.

A Granulocytic Signature Identifies COVID-19 and Its Severity.

BACKGROUND: An unbiased approach to SARS-CoV-2-induced immune dysregulation has not been undertaken so far. We aimed to identify previously unreported immune markers able to discriminate COVID-19 patients from healthy controls and to predict mild and severe disease. METHODS: An observational, prospective, multicentric study was conducted in patients with confirmed mild/moderate (n = 7) and severe (n = 19) COVID-19. Immunophenotyping of whole-blood leukocytes was performed in patients upon hospital ward or intensive care unit admission and in healthy controls (n = 25). Clinically relevant associations were identified through unsupervised analysis. RESULTS: Granulocytic (neutrophil, eosinophil, and basophil) markers were enriched during COVID-19 and discriminated between patients with mild and severe disease. Increased counts of CD15+CD16+ neutrophils, decreased granulocytic expression of integrin CD11b, and Th2-related CRTH2 downregulation in eosinophils and basophils established a COVID-19 signature. Severity was associated with emergence of PD-L1 checkpoint expression in basophils and eosinophils. This granulocytic signature was accompanied by monocyte and lymphocyte immunoparalysis. Correlation with validated clinical scores supported pathophysiological relevance. CONCLUSIONS: Phenotypic markers of circulating granulocytes are strong discriminators between infected and uninfected individuals as well as between severity stages. COVID-19 alters the frequency and functional phenotypes of granulocyte subsets with emergence of CRTH2 as a disease biomarker.

Bacterial infection and non-Hodgkin's lymphoma.

One quarter of all cancers are linked to infectious diseases. The link between viral infection and cancer has been widely studied, but few reports have focused on the carcinogenic role of bacterial infection. Nonetheless, Helicobacter pylori, Chlamydia psittaci, Coxiella burnetii, Borrelia burgdorferi and Campylobacter jejuni are bacteria that can be associated with non-Hodgkin's lymphoma (NHL), the most common haematologic malignancy. Here, we review the evidence in favour of a link between these bacterial infections and NHL. Sero-epidemiological observation makes it possible to identify a link between H. pylori, C. burnetii, B. burgdorferi infection and NHL. Helicobacter pylori, Chlamydia psittaci, Coxiella burnetii, Borrelia burgdorferi and Campylobacter jejuni could be identified in NHL tissue samples at the site of chronic inflammation, where B and T lymphocytes are attracted to participate in follicle formation. Lymphoma remissions have been observed under antimicrobial therapies supporting the carcinogenic contribution of bacteria. If the theory of causality is characterized by the lack of universal criteria for establishing a causal link between two diseases, infection and lymphoma, epidemiological, clinical, and histological evidences reported here, should lead clinicians to pay attention to these infectious agents, to detect early lymphoma transformation.

Tropheryma whipplei Increases Expression of Human Leukocyte Antigen-G on Monocytes to Reduce Tumor Necrosis Factor and Promote Bacterial Replication.

BACKGROUND & AIMS: Infection with Tropheryma whipplei has a range of effects-some patients can be chronic carriers without developing any symptoms, whereas others can develop systemic Whipple disease, characterized by a lack a protective inflammatory immune response. Alterations in HLA-G function have been associated with several diseases. We investigated the role of HLA-G during T whipplei infection. METHODS: Sera, total RNA, and genomic DNA were collected from peripheral blood from 22 patients with classic Whipple's disease, 19 patients with localized T whipplei infections, and 21 asymptomatic carriers. Levels of soluble HLA-G in sera were measured by enzyme-linked immuosorbent assay, and expressions of HLA-G and its isoforms were monitored by real-time polymerase chain reaction. HLA-G alleles were identified and compared with a population of voluntary bone marrow donors. Additionally, monocytes from healthy subjects were stimulated with T whipplei, and HLA-G expression was monitored by real-time polymerase chain reaction and flow cytometry. Bacterial replication was assessed by polymerase chain reaction in the presence of HLA-G or inhibitor of tumor necrosis factor (TNF) (etanercept). RESULTS: HLA-G mRNAs and levels of soluble HLA-G were significantly increased in sera from patients with chronic T whipplei infection compared with sera from asymptomatic carriers and control individuals. No specific HLA-G haplotypes were associated with disease or T whipplei infection. However, T whipplei infection of monocytes induced expression of HLA-G, which was associated with reduced secretion of TNF compared with noninfected monocytes. A neutralizing antibody against HLA-G increased TNF secretion by monocytes in response to T whipplei, and a TNF inhibitor promoted bacteria replication. CONCLUSIONS: Levels of HLA-G are increased in sera from patients with T whipplei tissue infections, associated with reduced production of TNF by monocytes. This might promote bacteria colonization in patients.

Pru p 7 sensitization is a predominant cause of severe, cypress pollen-associated peach allergy.

BACKGROUND: Peach is a common elicitor of food allergic reactions. Peach-induced immediate reactions may occur as benign pollen-food syndromes, usually due to birch pollen-related PR-10 cross-reactivity in temperate climates, and as potentially severe primary food allergies, predominantly related to nsLTP Pru p 3 in Mediterranean regions. The newly described peach allergen Pru p 7 has gained recent attention as a potential peach allergy severity marker. Sensitization to Pru p 7 and its allergenic homologues of the gibberellin-regulated protein family occurs in areas with high Cupressaceae tree pollen exposure. OBJECTIVE: We sought to investigate the distribution, clinical characteristics and molecular associations of Pru p 7 sensitization among subjects with suspected peach allergy in different regions of France. METHODS: Subjects with suspected peach allergy (n = 316) were included. Diagnostic work-up was performed according to current guidelines, including open food challenge when required. IgE antibody measurements and competition experiments were performed using the ImmunoCAP assay platform. RESULTS: Sensitization to Pru p 7 was present in 171 (54%) of all subjects in the study and in 123 of 198 (62%) diagnosed as peach allergic, more than half of whom were sensitized to no other peach allergen. Frequency and magnitude of Pru p 7 sensitization were associated with the presence of peach allergy, the clinical severity of peach-induced allergic reactions and the level of cypress pollen exposure. Cypress pollen extract completely outcompeted IgE binding to Pru p 7. Pru p 7 was extremely potent in basophil activation tests. CONCLUSION AND CLINICAL RELEVANCE: A subtype of Cupressaceae pollinosis, characterized by Pru p 7 sensitization, can be an underlying cause of severe peach allergy.

Paired acute-baseline serum tryptase levels in perioperative anaphylaxis: An observational study.

BACKGROUND: Anaphylaxis is recognized mainly through clinical criteria, which may lack specificity or relevance in the perioperative setting. The transient increase in serum tryptase has been proposed since 1989 as a diagnostic tool. Sampling for well-defined acute and baseline determinations has been recommended. We assessed the performance of four proposed algorithms with tightly controlled time frames for tryptase sampling, their robustness with inadequate sampling times, and the possible use of mature tryptase determination. METHODS: A retrospective study was performed on 102 adult patients from the Aix-Marseille University Hospitals who had experienced a perioperative hypersensitivity reaction clinically suggesting anaphylaxis. EAACI and ICON criteria were used to diagnose anaphylaxis. Mature and total serum tryptase levels were measured. RESULTS: Based on EAACI guidelines, clinical diagnostic criteria for anaphylaxis were found in 76 patients and lacking in 26. The most effective algorithm was the international consensus recommendation of 2012 that acute total tryptase levels should be greater than ([1.2×baseline tryptase] + 2] µg/L to be considered a clinically significant rise. In our cohort, this algorithm achieved 94% positive predictive value (PPV), 53% negative predictive value (NPV), 75% sensitivity, 86% specificity, and a Youden's index value of 0.61. A detectable acute mature tryptase level showed lower sensitivity, particularly in patients with acute total tryptase levels lower than 16 µg/L. Acute tryptase levels varied as a function of the clinical severity of anaphylaxis. CONCLUSION: Total tryptase levels in serum discriminated between nonanaphylactic and anaphylactic events in a perioperative setting when acute and baseline levels were collected and analyzed by the consensus algorithm.

P2X1 expressed on polymorphonuclear neutrophils and platelets is required for thrombosis in mice.

Adenosine triphosphate (ATP) and its metabolite, adenosine, are key regulators of polymorphonuclear neutrophil (PMN) functions. PMNs have recently been implicated in the initiation of thrombosis. We investigated the role of ATP and adenosine in PMN activation and recruitment at the site of endothelial injury. Following binding to the injured vessel wall, PMNs are activated and release elastase. The recruitment of PMNs and the subsequent fibrin generation and thrombus formation are strongly affected in mice deficient in the P2X1-ATP receptor and in wild-type (WT) mice treated with CGS 21680, an agonist of the A2A adenosine receptor or NF449, a P2X1 antagonist. Infusion of WT PMNs into P2X1-deficient mice increases fibrin generation but not thrombus formation. Restoration of thrombosis requires infusion of both platelets and PMNs from WT mice. In vitro, ATP activates PMNs, whereas CGS 21680 prevents their binding to activated endothelial cells. These data indicate that adenosine triphosphate (ATP) contributes to polymorphonuclear neutrophil (PMN) activation leading to their adhesion at the site of laser-induced endothelial injury, a necessary step leading to the generation of fibrin, and subsequent platelet-dependent thrombus formation. Altogether, our study identifies previously unknown mechanisms by which ATP and adenosine are key molecules involved in thrombosis by regulating the activation state of PMNs.

Inhibition of platelet activation prevents the P-selectin and integrin-dependent accumulation of cancer cell microparticles and reduces tumor growth and metastasis in vivo.

Venous thromboembolism constitutes one of the main causes of death during the progression of a cancer. We previously demonstrated that tissue factor (TF)-bearing cancer cell-derived microparticles accumulate at the site of injury in mice developing a pancreatic cancer. The presence of these microparticles at the site of thrombosis correlates with the size of the platelet-rich thrombus. The objective of this study was to determine the involvement of TF expressed by cancer cell-derived microparticles on thrombosis associated with cancer. We observed that pancreatic cancer cell derived microparticles expressed TF, its inhibitor tissue factor pathway inhibitor (TFPI) as well as the integrins αvß1 and αvß3. In mice bearing a tumor under-expressing TF, a significant decrease in circulating TF activity associated with an increase bleeding time and a 100-fold diminished fibrin generation and platelet accumulation at the site of injury were observed. This was mainly due to the interaction of circulating cancer cell-derived microparticles expressing TFPI with activated platelets and fibrinogen. In an ectopic model of cancer, treatment of mice with Clopidogrel, an anti-platelet drug, decreased the size of the tumors and restored hemostasis by preventing the accumulation of cancer cell-derived microparticles at the site of thrombosis. In a syngeneic orthotopic model of pancreatic cancer Clopidogrel also significantly inhibited the development of metastases. Together, these results indicate that an anti-platelet strategy may efficiently treat thrombosis associated with cancer and reduce the progression of pancreatic cancer in mice.

Tissue factor-positive neutrophils bind to injured endothelial wall and initiate thrombus formation.

For a long time, blood coagulation and innate immunity have been viewed as interrelated responses. Recently, the presence of leukocytes at the sites of vessel injury has been described. Here we analyzed interaction of neutrophils, monocytes, and platelets in thrombus formation after a laser-induced injury in vivo. Neutrophils immediately adhered to injured vessels, preceding platelets, by binding to the activated endothelium via leukocyte function antigen-1-ICAM-1 interactions. Monocytes rolled on a thrombus 3 to 5 minutes postinjury. The kinetics of thrombus formation and fibrin generation were drastically reduced in low tissue factor (TF) mice whereas the absence of factor XII had no effect. In vitro, TF was detected in neutrophils. In vivo, the inhibition of neutrophil binding to the vessel wall reduced the presence of TF and diminished the generation of fibrin and platelet accumulation. Injection of wild-type neutrophils into low TF mice partially restored the activation of the blood coagulation cascade and accumulation of platelets. Our results show that the interaction of neutrophils with endothelial cells is a critical step preceding platelet accumulation for initiating arterial thrombosis in injured vessels. Targeting neutrophils interacting with endothelial cells may constitute an efficient strategy to reduce thrombosis.

Ezurpimtrostat, A Palmitoyl-Protein Thioesterase-1 Inhibitor, Combined with PD-1 Inhibition Provides CD8+ Lymphocyte Repopulation in Hepatocellular Carcinoma.

BACKGROUND: Palmitoyl-protein thioesterase-1 (PPT1) is a clinical stage druggable target for inhibiting autophagy in cancer. OBJECTIVE: We aimed to determine the cellular and molecular activity of targeting PPT1 using ezurpimtrostat, in combination with an anti-PD-1 antibody. METHODS: In this study we used a transgenic immunocompetent mouse model of hepatocellular carcinoma. RESULTS: Herein, we revealed that inhibition of PPT1 using ezurpimtrostat decreased the liver tumor burden in a mouse model of hepatocellular carcinoma by inducing the penetration of lymphocytes into tumors when combined with anti-programmed death-1 (PD-1). Inhibition of PPT1 potentiates the effects of anti-PD-1 immunotherapy by increasing the expression of major histocompatibility complex (MHC)-I at the surface of liver cancer cells and modulates immunity through recolonization and activation of cytotoxic CD8+ lymphocytes. CONCLUSIONS: Ezurpimtrostat turns cold tumors into hot tumors and, thus, could improve T cell-mediated immunotherapies in liver cancer.