CD155 on Tumor Cells Drives Resistance to Immunotherapy by Inducing the Degradation of the Activating Receptor CD226 in CD8+ T Cells.

The activating receptor CD226 is expressed on lymphocytes, monocytes, and platelets and promotes anti-tumor immunity in pre-clinical models. Here, we examined the role of CD226 in the function of tumor-infiltrating lymphocytes (TILs) and resistance to immunotherapy. In murine tumors, a large proportion of CD8+ TILs had decreased surface expression of CD226 and exhibited features of dysfunction, whereas CD226hi TILs were highly functional. This correlation was seen also in TILs isolated from HNSCC patients. Mutation of CD226 at tyrosine 319 (Y319) led to increased CD226 surface expression, enhanced anti-tumor immunity and improved efficacy of immune checkpoint blockade (ICB). Mechanistically, tumor-derived CD155, the ligand for CD226, initiated phosphorylation of Y319 by Src kinases, thereby enabling ubiquitination of CD226 by CBL-B, internalization, and proteasomal degradation. In pre-treatment samples from melanoma patients, CD226+CD8+ T cells correlated with improved progression-free survival following ICB. Our findings argue for the development of therapies aimed at maintaining the expression of CD226.

Fibroblast-expressed LRRC15 is a receptor for SARS-CoV-2 spike and controls antiviral and antifibrotic transcriptional programs.

Although ACE2 is the primary receptor for Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection, a systematic assessment of host factors that regulate binding to SARS-CoV-2 spike protein has not been described. Here, we use whole-genome CRISPR activation to identify host factors controlling cellular interactions with SARS-CoV-2. Our top hit was a TLR-related cell surface receptor called leucine-rich repeat-containing protein 15 (LRRC15). LRRC15 expression was sufficient to promote SARS-CoV-2 spike binding where they form a cell surface complex. LRRC15 mRNA is expressed in human collagen-producing lung myofibroblasts and LRRC15 protein is induced in severe Coronavirus Disease 2019 (COVID-19) infection where it can be found lining the airways. Mechanistically, LRRC15 does not itself support SARS-CoV-2 infection, but fibroblasts expressing LRRC15 can suppress both pseudotyped and authentic SARS-CoV-2 infection in trans. Moreover, LRRC15 expression in fibroblasts suppresses collagen production and promotes expression of IFIT, OAS, and MX-family antiviral factors. Overall, LRRC15 is a novel SARS-CoV-2 spike-binding receptor that can help control viral load and regulate antiviral and antifibrotic transcriptional programs in the context of COVID-19 infection.

Autoimmunity Against Surfactant Protein B Is Associated with Pneumonitis During Checkpoint Blockade.

Rationale: Immune checkpoint inhibitor (ICI)-related pneumonitis is a serious autoimmune event affecting as many as 20% of patients with non-small-cell lung cancer (NSCLC), yet the factors underpinning its development in some patients and not others are poorly understood. Objectives: To investigate the role of autoantibodies and autoreactive T cells against surfactant-related proteins in the development of pneumonitis. Methods: The study cohort consisted of patients with NSCLC who provided blood samples before and during ICI treatment. Serum was used for proteomics analyses and to detect autoantibodies present during pneumonitis. T-cell stimulation assays and single-cell RNA sequencing were performed to investigate the specificity and functionality of peripheral autoreactive T cells. The findings were confirmed in a validation cohort comprising patients with NSCLC and patients with melanoma. Measurements and Main Results: Across both cohorts, patients in whom pneumonitis developed had higher pretreatment levels of immunoglobulin G autoantibodies targeting surfactant protein (SP)-B. At the onset of pneumonitis, these patients also exhibited higher frequencies of CD4+ IFN-γ-positive SP-B-specific T cells and expanding T-cell clonotypes recognizing this protein, accompanied by a proinflammatory serum proteomic profile. Conclusions: Our data suggest that the cooccurrence of SP-B-specific immunoglobulin G autoantibodies and CD4+ T cells is associated with the development of pneumonitis during ICI therapy. Pretreatment levels of these antibodies may represent a potential biomarker for an increased risk of developing pneumonitis, and on-treatment levels may provide a diagnostic aid.

Pulmonary Surfactant Proteins Are Inhibited by Immunoglobulin A Autoantibodies in Severe COVID-19.

Rationale: Coronavirus disease 2019 (COVID-19) can lead to acute respiratory distress syndrome with fatal outcomes. Evidence suggests that dysregulated immune responses, including autoimmunity, are key pathogenic factors. Objectives: To assess whether IgA autoantibodies target lung-specific proteins and contribute to disease severity. Methods: We collected 147 blood, 9 lung tissue, and 36 BAL fluid samples from three tertiary hospitals in Switzerland and one in Germany. Severe COVID-19 was defined by the need to administer oxygen. We investigated the presence of IgA autoantibodies and their effects on pulmonary surfactant in COVID-19 using the following methods: immunofluorescence on tissue samples, immunoprecipitations followed by mass spectrometry on BAL fluid samples, enzyme-linked immunosorbent assays on blood samples, and surface tension measurements with medical surfactant. Measurements and Main Results: IgA autoantibodies targeting pulmonary surfactant proteins B and C were elevated in patients with severe COVID-19 but not in patients with influenza or bacterial pneumonia. Notably, pulmonary surfactant failed to reduce surface tension after incubation with either plasma or purified IgA from patients with severe COVID-19. Conclusions: Our data suggest that patients with severe COVID-19 harbor IgA autoantibodies against pulmonary surfactant proteins B and C and that these autoantibodies block the function of lung surfactant, potentially contributing to alveolar collapse and poor oxygenation.

Autoreactive T cells targeting type II pneumocyte antigens in COVID-19 convalescent patients.

BACKGROUND: The role of autoreactive T cells on the course of Coronavirus disease-19 (COVID-19) remains elusive. Type II pneumocytes represent the main target cells of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Autoimmune responses against antigens highly expressed in type II pneumocytes may influence the severity of COVID-19 disease. OBJECTIVE: The aim of this study was to investigate autoreactive T cell responses against self-antigens highly expressed in type II pneumocytes in the blood of COVID-19 patients with severe and non-severe disease. METHODS: We collected blood samples of COVID-19 patients with varying degrees of disease severity and of pre-pandemic controls. T cell stimulation assays with peptide pools of type II pneumocyte antigens were performed in two independent cohorts to analyze the autoimmune T cell responses in patients with non-severe and severe COVID-19 disease. Target cell lysis assays were performed with lung cancer cell lines to determine the extent of cell killing by type II PAA-specific T cells. RESULTS: We identified autoreactive T cell responses against four recently described self-antigens highly expressed in type II pneumocytes, known as surfactant protein A, surfactant protein B, surfactant protein C and napsin A, in the blood of COVID-19 patients. These antigens were termed type II pneumocyte-associated antigens (type II PAAs). We found that patients with non-severe COVID-19 disease showed a significantly higher frequency of type II PAA-specific autoreactive T cells in the blood when compared to severely ill patients. The presence of high frequencies of type II PAA-specific T cells in the blood of non-severe COVID-19 patients was independent of their age. We also found that napsin A-specific T cells from convalescent COVID-19 patients could kill lung cancer cells, demonstrating the functional and cytotoxic role of these T cells. CONCLUSIONS: Our data suggest that autoreactive type II PAA-specific T cells have a protective role in SARS-CoV-2 infections and the presence of high frequencies of these autoreactive T cells indicates effective viral control in COVID-19 patients. Type II-PAA-specific T cells may therefore promote the killing of infected type II pneumocytes and viral clearance.

Fingolimod and tumor-infiltrating lymphocytes in checkpoint-inhibitor treated cancer patients.

Immune checkpoint inhibitors (ICIs) are emerging as the new standard of care for treating various metastatic cancers. It is known that effective anti-tumor immune responses are associated with a stronger presence of tumor-infiltrating lymphocytes (TILs) in solid tumor tissue. Cancer patients with relapsing-remitting multiple sclerosis (RRMS) are often under continuous treatment with fingolimod, an immune-modulating drug that inhibits lymphocyte egress from secondary lymphatic organs. Little is known about the effect of fingolimod on ICI cancer therapy, as fingolimod may limit the number of TILs. Here we present three patients with RRMS, who developed various cancers during fingolimod treatment. Histology of all tumors consistently showed low numbers of TILs. A second biopsy taken from one of the tumors, a melanoma, revealed a significant increase of TILs after stopping fingolimod and starting pembrolizumab, indicating a surge in the number and re-invigoration of T cells. Our study suggests that fingolimod limits the number of TILs in solid tumors and may, thus, inhibit anti-cancer immune responses.

Normothermic Machine Perfusion (NMP) Inhibits Proinflammatory Responses in the Liver and Promotes Regeneration.

Liver transplantation (LT) is a successful treatment for patients with liver failure. However, organ shortage results in over 11% of patients losing their chance of a transplant attributed to liver decompensation (LD) and death. Ischemia/reperfusion injury (IRI) following conventional cold storage (CS) is a major cause of injury leading to graft loss after LT. Normothermic machine perfusion (NMP), a method of organ preservation, provides oxygen and nutrition during preservation and allows aerobic metabolism. NMP has recently been shown to enable improved organ utilization and posttransplant outcomes following a phase I and a phase III randomized trial. The aim of the present study is to assess the impact of NMP on reducing IRI and to define the underlying mechanisms. We transplanted and compared 12 NMP with 27 CS-preserved livers by performing gene microarray, immunoprofiling of hepatic lymphocytes, and immunochemistry staining of liver tissues for assessing necrosis, platelet deposition, and neutrophil infiltration, and the status of steatosis after NMP or CS prereperfusion and postreperfusion. Recipients receiving NMP grafts showed significantly lower peak aspartate aminotransferase (AST) levels than those receiving CS grafts. NMP altered gene-expression profiles of liver tissue from proinflammation to prohealing and regeneration. NMP also reduced the number of interferon gamma (IFN-γ) and interleukin (IL)-17-producing T cells and enlarged the CD4pos CD25high CD127neg FOXP3pos regulatory T cell (Treg) pool. NMP liver tissues showed less necrosis and apoptosis in the parenchyma and fewer neutrophil infiltration compared to CS liver tissues. Conclusion: Reduced IRI in NMP recipients was the consequence of the combination of inhibiting inflammation and promoting graft regeneration.

CD14+ CD15- HLA-DR- myeloid-derived suppressor cells impair antimicrobial responses in patients with acute-on-chronic liver failure.

OBJECTIVE: Immune paresis in patients with acute-on-chronic liver failure (ACLF) accounts for infection susceptibility and increased mortality. Immunosuppressive mononuclear CD14+HLA-DR- myeloid-derived suppressor cells (M-MDSCs) have recently been identified to quell antimicrobial responses in immune-mediated diseases. We sought to delineate the function and derivation of M-MDSC in patients with ACLF, and explore potential targets to augment antimicrobial responses. DESIGN: Patients with ACLF (n=41) were compared with healthy subjects (n=25) and patients with cirrhosis (n=22) or acute liver failure (n=30). CD14+CD15-CD11b+HLA-DR- cells were identified as per definition of M-MDSC and detailed immunophenotypic analyses were performed. Suppression of T cell activation was assessed by mixed lymphocyte reaction. Assessment of innate immune function included cytokine expression in response to Toll-like receptor (TLR-2, TLR-4 and TLR-9) stimulation and phagocytosis assays using flow cytometry and live cell imaging-based techniques. RESULTS: Circulating CD14+CD15-CD11b+HLA-DR- M-MDSCs were markedly expanded in patients with ACLF (55% of CD14+ cells). M-MDSC displayed immunosuppressive properties, significantly decreasing T cell proliferation (p=0.01), producing less tumour necrosis factor-alpha/interleukin-6 in response to TLR stimulation (all p<0.01), and reduced bacterial uptake of Escherichia coli (p<0.001). Persistently low expression of HLA-DR during disease evolution was linked to secondary infection and 28-day mortality. Recurrent TLR-2 and TLR-4 stimulation expanded M-MDSC in vitro. By contrast, TLR-3 agonism reconstituted HLA-DR expression and innate immune function ex vivo. CONCLUSION: Immunosuppressive CD14+HLA-DR- M-MDSCs are expanded in patients with ACLF. They were depicted by suppressing T cell function, attenuated antimicrobial innate immune responses, linked to secondary infection, disease severity and prognosis. TLR-3 agonism reversed M-MDSC expansion and innate immune function and merits further evaluation as potential immunotherapeutic agent.

MerTK expressing hepatic macrophages promote the resolution of inflammation in acute liver failure.

OBJECTIVE: Acute liver failure (ALF) is characterised by overwhelming hepatocyte death and liver inflammation with massive infiltration of myeloid cells in necrotic areas. The mechanisms underlying resolution of acute hepatic inflammation are largely unknown. Here, we aimed to investigate the impact of Mer tyrosine kinase (MerTK) during ALF and also examine how the microenvironmental mediator, secretory leucocyte protease inhibitor (SLPI), governs this response. DESIGN: Flow cytometry, immunohistochemistry, confocal imaging and gene expression analyses determined the phenotype, functional/transcriptomic profile and tissue topography of MerTK+ monocytes/macrophages in ALF, healthy and disease controls. The temporal evolution of macrophage MerTK expression and its impact on resolution was examined in APAP-induced acute liver injury using wild-type (WT) and Mer-deficient (Mer-/-) mice. SLPI effects on hepatic myeloid cells were determined in vitro and in vivo using APAP-treated WT mice. RESULTS: We demonstrate a significant expansion of resolution-like MerTK+HLA-DRhigh cells in circulatory and tissue compartments of patients with ALF. Compared with WT mice which show an increase of MerTK+MHCIIhigh macrophages during the resolution phase in ALF, APAP-treated Mer-/- mice exhibit persistent liver injury and inflammation, characterised by a decreased proportion of resident Kupffer cells and increased number of neutrophils. Both in vitro and in APAP-treated mice, SLPI reprogrammes myeloid cells towards resolution responses through induction of a MerTK+HLA-DRhigh phenotype which promotes neutrophil apoptosis and their subsequent clearance. CONCLUSIONS: We identify a hepatoprotective, MerTK+, macrophage phenotype that evolves during the resolution phase following ALF and represents a novel immunotherapeutic target to promote resolution responses following acute liver injury.

HBV DNA Integration and Clonal Hepatocyte Expansion in Chronic Hepatitis B Patients Considered Immune Tolerant.

BACKGROUND & AIMS: Chronic infection with hepatitis B virus (HBV) progresses through different phases. The first, called the immune-tolerant phase, has been associated with a lack of disease activity. We examined HBV-DNA integration, clonal hepatocyte expansion, HBV antigen expression, and HBV-specific immune responses in patients in the immune-tolerant phase to assess whether this designation is appropriate or if there is evidence of disease activity. METHODS: We studied HBV-DNA integration, clonal hepatocyte expansion, and expression of hepatitis B surface antigen and core antigen in liver tissues from 26 patients with chronic HBV infection (ages, 14-39 y); 9 patients were positive for hepatitis B e antigen (HBeAg) in the immune-tolerant phase and were matched for age with 10 HBeAg-positive patients with active disease and 7 HBeAg-negative patients with active disease. Peripheral blood samples were collected and HBV-specific T cells were quantified for each group. RESULTS: Detection of HBV antigens differed among groups. However, unexpectedly high numbers of HBV-DNA integrations, randomly distributed among chromosomes, were detected in all groups. Clonal hepatocyte expansion in patients considered immune tolerant also was greater than expected, potentially in response to hepatocyte turnover mediated by HBV-specific T cells, which were detected in peripheral blood cells from patients in all phases of infection. CONCLUSIONS: We measured HBV-specific T cells, HBV-DNA integration, and clonal hepatocyte expansion in different disease phases of young patients with chronic hepatitis B, with emphasis on the so-called immune-tolerant phase. A high level of HBV-DNA integration and clonal hepatocyte expansion in patients considered immune tolerant indicated that hepatocarcinogenesis could be underway-even in patients with early stage chronic HBV infection. Our findings do not support the concepts that this phase is devoid of markers of disease progression or that an immune response has not been initiated. We propose that this early phase be called a high-replication, low-inflammation stage. The timing of therapeutic interventions to minimize further genetic damage to the hepatocyte population should be reconsidered.

Multivariate metabotyping of plasma predicts survival in patients with decompensated cirrhosis.

BACKGROUND & AIMS: Predicting survival in decompensated cirrhosis (DC) is important in decision making for liver transplantation and resource allocation. We investigated whether high-resolution metabolic profiling can determine a metabolic phenotype associated with 90-day survival. METHODS: Two hundred and forty-eight subjects underwent plasma metabotyping by (1)H nuclear magnetic resonance (NMR) spectroscopy and reversed-phase ultra-performance liquid chromatography coupled to time-of-flight mass spectrometry (UPLC-TOF-MS; DC: 80-derivation set, 101-validation; stable cirrhosis (CLD) 20 and 47 healthy controls (HC)). RESULTS: (1)H NMR metabotyping accurately discriminated between surviving and non-surviving patients with DC. The NMR plasma profiles of non-survivors were attributed to reduced phosphatidylcholines and lipid resonances, with increased lactate, tyrosine, methionine and phenylalanine signal intensities. This was confirmed on external validation (area under the receiver operating curve [AUROC]=0.96 (95% CI 0.90-1.00, sensitivity 98%, specificity 89%). UPLC-TOF-MS confirmed that lysophosphatidylcholines and phosphatidylcholines [LPC/PC] were downregulated in non-survivors (UPLC-TOF-MS profiles AUROC of 0.94 (95% CI 0.89-0.98, sensitivity 100%, specificity 85% [positive ion detection])). LPC concentrations negatively correlated with circulating markers of cell death (M30 and M65) levels in DC. Histological examination of liver tissue from DC patients confirmed increased hepatocyte cell death compared to controls. Cross liver sampling at time of liver transplantation demonstrated that hepatic endothelial beds are a source of increased circulating total cytokeratin-18 in DC. CONCLUSION: Plasma metabotyping accurately predicts mortality in DC. LPC and amino acid dysregulation is associated with increased mortality and severity of disease reflecting hepatocyte cell death.

Patients with acute-on-chronic liver failure have increased numbers of regulatory immune cells expressing the receptor tyrosine kinase MERTK.

BACKGROUND & AIMS: Characteristics of decompensated cirrhosis and acute-on-chronic liver failure (ACLF) include susceptibility to infection, immuneparesis, and monocyte dysfunction. MER receptor tyrosine kinase (MERTK) is expressed by monocytes and macrophages and contributes to down-regulation of innate immune responses. We investigated whether MERTK expression is altered on monocytes from patients with liver failure. METHODS: We analyzed blood and liver samples collected from patients admitted to the liver intensive therapy unit at King's College Hospital in London from December 2012 through July 2014. Patients had either ACLF (n = 41), acute decompensation of cirrhosis without ACLF (n = 9), cirrhosis without decompensation (n = 17), or acute liver failure (n = 23). We also analyzed samples from healthy individuals (controls, n = 29). We used flow cytometry to determine the level of innate immune function, and associated the findings with disease severity. We developed an assay to measure recruitment and migration of immune cells from the tissue parenchyma. Immunohistochemistry and confocal microscopy were used to determine levels of MERTK in bone marrow, liver, and lymph node tissues. We performed immunophenotype analyses and measured the production of tumor necrosis factor and interleukin 6 and intracellular killing of Escherichia coli by monocytes and peritoneal macrophages incubated with lipopolysaccharide, with or without an inhibitor of MERTK (UNC569). RESULTS: The number of monocytes and macrophages that expressed MERTK was greatly increased in the circulation, livers, and lymph nodes of patients with ACLF, compared with patients with stable cirrhosis and controls. MERTK expression (mean fluorescence intensity) correlated with the severity of hepatic and extrahepatic disease and systemic inflammatory responses. Based on immunophenotype, migration, and functional analyses, MERTK-expressing monocytes migrate across the endothelia to localize into tissue sites and regional lymph nodes. Expression of MERTK reduced the response of cultured monocytes to lipopolysaccharide; the addition of UNC569 restored production of inflammatory cytokines in response to lipopolysaccharide. CONCLUSIONS: Patients with ACLF have increased numbers of immunoregulatory monocytes and macrophages that express MERTK and suppress the innate immune response to microbes. The number of these cells correlates with disease severity and the inflammatory response. MERTK inhibitors restore production of inflammatory cytokines by immune cells from patients with ACLF, and might be developed to increase the innate immune response in these patients.

Expression of the tumor antigens NY-ESO-1, tyrosinase, MAGE-A3, and TPTE in pediatric and adult melanoma: a retrospective case control study.

Tumor-associated antigens (TAAs) are potential targets for T cell-based immunotherapy approaches in cutaneous melanoma. BNT111, an investigational lipoplex-formulated mRNA-based therapeutic cancer vaccine encoding melanoma TAAs NY-ESO-1, tyrosinase, MAGE-A3, and TPTE, is undergoing clinical testing in adults. Expression of these TAAs in pediatric melanoma is unclear but is a prerequisite for feasibility of this treatment approach in children with melanoma. Our main objective was to characterize expression of those TAAs in pediatric melanomas compared to control cohorts. In this retrospective case control study, protein and transcript expression of NY-ESO-1, tyrosinase, MAGE-A3, and TPTE were analyzed in a cohort of 25 pediatric melanomas, 31 melanomas of young adults, 29 adult melanomas, and 30 benign melanocytic nevi in children using immunohistochemical staining and digital pathology (QuPath) and reverse transcription quantitative PCR. Based on IHC analysis, pediatric melanomas expressed tyrosinase (100.0%), TPTE (44.0%), MAGE-A3 (12.0%), and NY-ESO-1 (8.0%). Young adult melanomas expressed tyrosinase (96.8%), NY-ESO-1 (19.4%), MAGE-A3 (19.4%), and TPTE (3.2%). Adult melanomas expressed tyrosinase (86.2%), MAGE-A3 (75.9%), NY-ESO-1 (48.3%), and TPTE (48.3%). Childhood melanocytic nevi only expressed tyrosinase (93.3%). Expression prevalence of individual TAAs did not differ between subtypes of pediatric melanoma, and no association with prognosis was found. All four TAAs were expressed in pediatric melanoma, albeit NY-ESO-1 and MAGE-A3 to a lesser extent than in adult melanoma. These data support the possibility of investigating vaccines targeting these TAAs for the treatment of pediatric melanoma.

Deep learning-based scoring of tumour-infiltrating lymphocytes is prognostic in primary melanoma and predictive to PD-1 checkpoint inhibition in melanoma metastases.

BACKGROUND: Recent advances in digital pathology have enabled accurate and standardised enumeration of tumour-infiltrating lymphocytes (TILs). Here, we aim to evaluate TILs as a percentage electronic TIL score (eTILs) and investigate its prognostic and predictive relevance in cutaneous melanoma. METHODS: We included stage I to IV cutaneous melanoma patients and used hematoxylin-eosin-stained slides for TIL analysis. We assessed eTILs as a continuous and categorical variable using the published cut-off of 16.6% and applied Cox regression models to evaluate associations of eTILs with relapse-free, distant metastasis-free, and overall survival. We compared eTILs of the primaries with matched metastasis. Moreover, we assessed the predictive relevance of eTILs in therapy-naïve metastases according to the first-line therapy. FINDINGS: We analysed 321 primary cutaneous melanomas and 191 metastatic samples. In simple Cox regression, tumour thickness (p < 0.0001), presence of ulceration (p = 0.0001) and eTILs ≤16.6% (p = 0.0012) were found to be significant unfavourable prognostic factors for RFS. In multiple Cox regression, eTILs ≤16.6% (p = 0.0161) remained significant and downgraded the current staging. Lower eTILs in the primary tissue was associated with unfavourable relapse-free (p = 0.0014) and distant metastasis-free survival (p = 0.0056). In multiple Cox regression adjusted for tumour thickness and ulceration, eTILs as continuous remained significant (p = 0.019). When comparing TILs in primary tissue and corresponding metastasis of the same patient, eTILs in metastases was lower than in primary melanomas (p < 0.0001). In therapy-naïve metastases, an eTILs >12.2% was associated with longer progression-free survival (p = 0.037) and melanoma-specific survival (p = 0.0038) in patients treated with anti-PD-1-based immunotherapy. In multiple Cox regression, lactate dehydrogenase (p < 0.0001) and eTILs ≤12.2% (p = 0.0130) were significantly associated with unfavourable melanoma-specific survival. INTERPRETATION: Assessment of TILs is prognostic in primary melanoma samples, and the eTILs complements staging. In therapy-naïve metastases, eTILs ≤12.2% is predictive of unfavourable survival outcomes in patients receiving anti-PD-1-based therapy. FUNDING: See a detailed list of funding bodies in the Acknowledgements section at the end of the manuscript.

AXL Expression on Homeostatic Resident Liver Macrophages Is Reduced in Cirrhosis Following GAS6 Production by Hepatic Stellate Cells.

BACKGROUND & AIMS: AXL and MERTK expression on circulating monocytes modulated immune responses in patients with cirrhosis (CD14+HLA-DR+AXL+) and acute-on-chronic liver failure (CD14+MERTK+). AXL expression involved enhanced efferocytosis, sustained phagocytosis, but reduced tumor necrosis factor-α/interleukin-6 production and T-cell activation, suggesting a homeostatic function. Axl was expressed on murine airway in tissues contacting the external environment, but not interstitial lung- and tissue-resident synovial lining macrophages. Here, we assessed AXL expression on tissue macrophages in patients with cirrhosis. METHODS: Using multiplexed immunofluorescence we compared AXL expression in liver biopsies in cirrhosis (n = 22), chronic liver disease (n = 8), non-cirrhotic portal hypertension (n = 4), and healthy controls (n = 4). Phenotype and function of isolated primary human liver macrophages were characterized by flow cytometry (cirrhosis, n = 11; control, n = 14) ex vivo. Also, AXL expression was assessed on peritoneal (n = 29) and gut macrophages (n = 16) from cirrhotic patients. Regulation of AXL expression was analyzed in vitro and ex vivo using primary hepatic stellate cells (HSCs), LX-2 cells, and GAS6 in co-culture experiments. RESULTS: AXL was expressed on resident (CD68+) but not tissue-infiltrating (MAC387+) liver macrophages, hepatocytes, HSCs, or sinusoidal endothelial cells. Prevalence of hepatic CD68+AXL+ cells significantly decreased with cirrhosis progression: (healthy, 90.2%; Child-Pugh A, 76.1%; Child-Pugh B, 64.5%; and Child-Pugh C, 18.7%; all P < .05) and negatively correlated with Model for End-Stage Liver Disease and C-reactive protein (all P < .05). AXL-expressing hepatic macrophages were CD68highHLA-DRhighCD16highCD206high. AXL expression also decreased on gut and peritoneal macrophages from cirrhotic patients but increased in regional lymph nodes. GAS6, enriched in the cirrhotic liver, appeared to be secreted by HSCs and down-regulate AXL in vitro. CONCLUSIONS: Decreased AXL expression on resident liver macrophages in advanced cirrhosis, potentially in response to activated HSC-secreted GAS6, suggests a role for AXL in the regulation of hepatic immune homeostasis.

Frequency and Genomic Aspects of Intrinsic Resistance to Vismodegib in Locally Advanced Basal Cell Carcinoma.

PURPOSE: Vismodegib is approved for the treatment of locally advanced basal cell carcinoma (laBCC), but some cases demonstrate intrinsic resistance (IR) to the drug. We sought to assess the frequency of IR to vismodegib in laBCC and its underlying genomic mechanisms. EXPERIMENTAL DESIGN: Response to vismodegib was evaluated in a cohort of 148 laBCC patients. Comprehensive genomic and transcriptomic profiling was performed in a subset of five intrinsically resistant BCC (IR-BCC). RESULTS: We identified that IR-BCC represents 6.1% of laBCC in the studied cohort. Prior treatment with chemotherapy was associated with IR. Genetic events that were previously associated with acquired resistance (AR) in BCC or medulloblastoma were observed in three out of five IR-BCC. However, IR-BCCs were distinct by highly rearranged polyploid genomes. Functional analyses identified hyperactivation of the HIPPO-YAP and WNT pathways at RNA and protein levels in IR-BCC. In vitro assay on the BCC cell line further confirmed that YAP1 overexpression increases the cell proliferation rate. CONCLUSIONS: IR to vismodegib is a rare event in laBCC. IR-BCCs frequently harbor resistance mutations in the Hh pathway, but also are characterized by hyperactivation of the HIPPO-YAP and WNT pathways.

Autoreactive napsin A-specific T cells are enriched in lung tumors and inflammatory lung lesions during immune checkpoint blockade.

Cancer treatment with immune checkpoint blockade (ICB) often induces immune-related adverse events (irAEs). We hypothesized that proteins coexpressed in tumors and normal cells could be antigenic targets in irAEs and herein described DITAS (discovery of tumor-associated self-antigens) for their identification. DITAS computed transcriptional similarity between lung tumors and healthy lung tissue based on single-sample gene set enrichment analysis. This identified 10 lung tissue-specific genes highly expressed in the lung tumors. Computational analysis was combined with functional T cell assays and single-cell RNA sequencing of the antigen-specific T cells to validate the lung tumor self-antigens. In patients with non-small cell lung cancer (NSCLC) treated with ICB, napsin A was a self-antigen that elicited strong CD8+ T cell responses, with ICB responders harboring higher frequencies of these CD8+ T cells compared with nonresponders. Human leukocyte antigen (HLA) class I ligands derived from napsin A were present in human lung tumors and in nontumor lung tissues, and napsin A tetramers confirmed the presence of napsin A-specific CD8+ T cells in blood and tumors of patients with NSCLC. Napsin A-specific T cell clonotypes were enriched in lung tumors and ICB-induced inflammatory lung lesions and could kill immortalized HLA-matched NSCLC cells ex vivo. Single-cell RNA sequencing revealed that these T cell clonotypes expressed proinflammatory cytokines and cytotoxic markers. Thus, DITAS successfully identified self-antigens, including napsin A, that likely mediate effective antitumor T cell responses in NSCLC and may simultaneously underpin lung irAEs.

Early disappearance of tumor antigen-reactive T cells from peripheral blood correlates with superior clinical outcomes in melanoma under anti-PD-1 therapy.

BACKGROUND: Anti-programmed cell death protein 1 (PD-1) antibodies are now routinely administered for metastatic melanoma and for increasing numbers of other cancers, but still only a fraction of patients respond. Better understanding of the modes of action and predictive biomarkers for clinical outcome is urgently required. Cancer rejection is mostly T cell-mediated. We previously showed that the presence of NY-ESO-1-reactive and/or Melan-A-reactive T cells in the blood correlated with prolonged overall survival (OS) of patients with melanoma with a heterogeneous treatment background. Here, we investigated whether such reactive T cells can also be informative for clinical outcomes in metastatic melanoma under PD-1 immune-checkpoint blockade (ICB). METHODS: Peripheral blood T cell stimulation by NY-ESO-1 and Melan-A overlapping peptide libraries was assessed before and during ICB in two independent cohorts of a total of 111 patients with stage IV melanoma. In certain cases, tumor-infiltrating lymphocytes could also be assessed for such responses. These were characterized using intracellular cytokine staining for interferon gamma (IFN-γ), tumor negrosis factor (TNF) and CD107a. Digital pathology analysis was performed to quantify NY-ESO-1 and Melan-A expression by tumors. Endpoints were OS and progression-free survival (PFS). RESULTS: The initial presence in the circulation of NY-ESO-1- or Melan-A-reactive T cells which became no longer detectable during ICB correlated with validated, prolonged PFS (HR:0.1; p>0.0001) and OS (HR:0.2; p=0.021). An evaluation of melanoma tissue from selected cases suggested a correlation between tumor-resident NY-ESO-1- and Melan-A-reactive T cells and disease control, supporting the notion of a therapy-associated sequestration of cells from the periphery to the tumor predominantly in those patients benefitting from ICB. CONCLUSIONS: Our findings suggest a PD-1 blockade-dependent infiltration of melanoma-reactive T cells from the periphery into the tumor and imply that this seminally contributes to effective treatment.

Keratinocyte differentiation antigen-specific T cells in immune checkpoint inhibitor-treated NSCLC patients are associated with improved survival.

Immune checkpoint inhibitors (ICIs) have improved the survival of patients with non-small cell lung cancer (NSCLC) by reinvigorating tumor-specific T cell responses. However, the specificity of such T cells and the human leukocyte antigen (HLA)-associated epitopes recognized, remain elusive. In this study, we identified NSCLC T cell epitopes of recently described NSCLC-associated antigens, termed keratinocyte differentiation antigens. Epitopes of these antigens were presented by HLA-A 03:01 and HLA-C 04:01 and were associated with responses to ICI therapy. Patients with CD8+ T cell responses to these epitopes had improved overall and progression-free survival. T cells specific for such epitopes could eliminate HLA class I-matched NSCLC cells ex vivo and were enriched in patient lung tumors. The identification of novel lung cancer HLA-associated epitopes that correlate with improved ICI-dependent treatment outcomes suggests that keratinocyte-specific proteins are important tumor-associated antigens in NSCLC. These findings improve our understanding of the mechanisms of ICI therapy and may help support the development of vaccination strategies to improve ICI-based treatment of these tumors.