Gamma-interferon-inducible Lysosomal Thiol Reductase Research Articles

Functional of tongue sole (Cynoglossus semilaevis) gamma-interferon-inducible lysosomal thiol reductase with implications in innate immune reponse depend on CXXC active site

The enzyme gamma-interferon-inducible lysosomal thiol reductase (GILT) plays an important role in promoting the processing and presentation of major histocompatibility complex (MHC) class II-restricted antigens. It is also involved in MHC I-restricted antigens catalyzing disulfide bond reduction in fishes’ adaptive immunity. The open reading frame of tongue sole (Cynoglossus semilaevis) GILT (tsGILT) gene is 771 bp long, encoding 257 amino acids, with a calculated molecular weight of 28.465 kDa and isoelectric point (pI) of 5.35. After induction with lipopolysaccharide, the expression of tsGILT mRNA was upregulated in spleen and kidney and recombinant tsGILT protein transferred to late endosomes and lysosomes in HeLa cells. The refolded tsGILT was capable of catalyzing the reduction of the interchain disulfide bonds against an IgG substrate depend on the active site CXXC motif at residues 75–78. The process of immune response to bacteria challenge needs GILT to catalyze the reduction of disulfide bond and unfolding native protein antigens, promoting their hydrolysis by proteases. Whether a single mutation or a double mutation of active site CXXC at residues75–78, the 3D structure of tsGILT protein has undergone major changes and lost its activity of catalyzing the reduction of the interchain disulfide bonds.

Role of GILT and MHC class II in melanoma cells on regulating the anti-tumor immune response

Abstract The MHC class I antigen presentation pathway in melanoma cells has a well-established role in immune-mediated destruction of melanoma. However, the role of the MHC class II pathway in melanoma cells is not fully understood. Gamma-interferon-inducible lysosomal thiol reductase (GILT) is critical for MHC class II-restricted presentation of multiple melanoma antigens by antigen presenting cells. While GILT and MHC class II expression is typically limited to antigen presenting cells, GILT and MHC class II can be expressed constitutively or induced by IFN-γ in melanoma cells. In human melanoma specimens, high GILT expression and an active and intact MHC class II pathway are associated with improved survival. The goal of this project is to investigate a causal role for GILT and MHC class II in melanoma cells, using Yale University Mouse Melanoma (YUMM) lines YUMM2.1, YUMMER1.7, and YUMMERG. These cell lines constitutively expressed GILT, and MHC class II expression was IFN-γ-inducible in a subset of cells under serum-free conditions. We have genetically engineered GILT−/− YUMM2.1 cells and MHC class II (H2Ab1)−/− YUMM2.1 cells via CRISPR/Cas9 and verified the deletion. We have generated lentiviral constructs expressing GILT alone, CIITA alone, and dual expression of H2Ab1 and CIITA, to allow for the generation of melanoma cell lines with and without GILT and MHC class II. Transduction of GILT−/− YUMM2.1 cells with GILT resulted in high transduction efficiency and overexpression. Thus, we have developed models to determine the effect of GILT and MHC class II expression in melanoma cells on regulating in vivo tumor growth and the anti-tumor immune response. This work was supported by a Basic Science Award from the Harry J. Lloyd Charitable Trust and a Merit Review Award I01-BX005336 from the US Department of Veterans Affairs (VA), Biomedical Laboratory Research and Development Service (K.T.H.). The contents do not represent the views of the VA or the US Government.

A Longitudinal Study of Humoral Immune Responses to SARS-CoV-2 Spike Proteins in Gamma-Interferon-inducible Lysosomal Thiol Reductase Deficient Mice

Abstract The disulfide (S-S) bonds located in the receptor binding domain (RBD) of Spike (S) proteins of SARS-CoV-2 are critical for structural folding and viral entry. Once entered cells, S proteins, like other exogenous Ag, can be transported into endo/lysosomes, where gamma-interferon-inducible lysosomal thiol reductase (GILT) is the only thiol reductase located, which can reduce S-S bonds. GILT mediated S-S bond reduction in APC is critical in unfolding of native S-S bonds containing Ag, subsequently processing/presenting Ag, and, finally, triggering CD4 T cell responses. Recently, we also find GILT plays a role in Ab responses. Whether GILT mediates anti-SARSCoV-2 host defense responses remains unknown. We hypothesized that GILT is important in eliciting host humoral immune responses against SARS-CoV-2 by reducing S-S bonds and exposing Ag epitopes. To address this, we used 3 different types of S protein derived Ag to immunize GILT KO or B6 control mice and measured the specific Ab responses in a longitudinal study. GILT KO or B6 wildtype mice were given RBD, S1 region and the full length of extracellular domain of S proteins with CFA initially, and boosted at week 3 with the same Ag in IFA. Sera were collected at weeks 3 and 5, months 3, 4 and 5 for quantification of specific anti-RBD, S1 and S Ab. At all timepoints examined, GILT KO mice generated lower specific Ab titers to the immunogens, as compared to B6. Furthermore, decay of anti-specific Ab in GILT KO was faster than that in controls after immunization. Our preliminary findings showed an important role of GILT in humoral immune responses against SARS-CoV-2, suggesting unfolding S-S bonds is critical in this process. Recombinant GILT as a therapeutic might benefit patients with COVID-19. Supported by Seed Grant, Univ. of Utah

High GILT Expression Is Associated with Improved Survival in Metastatic Melanoma Patients Treated with Immune Checkpoint Inhibition.

Simple SummarySkin cancer is the most common type of cancer, with melanoma being among the deadliest of skin cancers due to its propensity to metastasize. Immune checkpoint inhibitors (ICI) generate anti-tumor immune responses resulting in improved outcomes in patients with metastatic melanoma. However, only a subset of melanoma patients responds to these therapies, which are costly and come with a risk of adverse effects. Therefore, there is a need for biomarkers to predict which patients will respond to ICI. We found that ICI-treated metastatic melanoma patients with high GILT mRNA expression in bulk tumor samples had improved survival. Additionally, high GILT protein expression within metastatic melanoma cells was associated with improved survival in patients treated with ICI. This study suggests that GILT may serve as a biomarker to predict which patients will respond to ICI, which could improve patient care, reduce healthcare costs, and facilitate appropriate selection of therapies for patients with metastatic melanoma.Gamma-interferon-inducible lysosomal thiol reductase (GILT) is critical for MHC class II restricted presentation of multiple melanoma antigens. There is variable GILT protein expression in malignant melanocytes in melanoma specimens. High GILT mRNA expression in melanoma specimens is associated with improved overall survival, before the advent of immune checkpoint inhibitors (ICI). However, the association of GILT in metastatic melanoma with survival in patients treated with ICI and the cell type expressing GILT associated with survival have not been determined. Using RNA sequencing datasets, high GILT mRNA expression in metastatic melanoma specimens was associated with improved progression-free and overall survival in patients treated with ICI. A clinical dataset of metastatic melanoma specimens was generated and annotated with clinical information. Positive GILT immunohistochemical staining in antigen presenting cells and melanoma cells was observed in 100% and 65% of metastatic melanoma specimens, respectively. In the subset of patients treated with ICI in the clinical dataset, high GILT protein expression within melanoma cells was associated with improved overall survival. The association of GILT mRNA and protein expression with survival was independent of cancer stage. These studies support that high GILT mRNA expression in bulk tumor samples and high GILT protein expression in melanoma cells is associated with improved survival in ICI-treated patients. These findings support further investigation of GILT as a biomarker to predict the response to ICI.

Trichinella spiralis: Knockdown of gamma interferon inducible lysosomal thiol reductase (GILT) results in the reduction of worm burden.

Trichinella spiralis is mammalian skeletal muscles parasite which may cause trichinellosis in animals and humans. Gamma interferon inducible lysosomal thiol reductase (GILT) is a widespread superfamily which plays key role in processing and presentation of MHC class II restricted antigen by catalyzing disulfide bond reduction. There are no reports about GILT in T. spiralis. In present study, GILT from T. spiralis (Tsp-GILT) was cloned, analyzed by multiple-sequence alignment, and predicted by 3D structure model. Recombinant Tsp-GILT (about 46 kDa) was efficiently expressed in Escherichia coli and thiol reductase activity suggested that in acidic environment the addition of a reducing agent is needed. Soaking method was used to knockdown expression of Tsp-GILT using small interference RNA (siRNA). Immunofluorescence assay confirmed the transformation of siRNA into muscle larva (ML) and new born larva (NBL). Quantitative real time-PCR (QRT-PCR) analysis revealed that transcription level of Tsp-GILT mRNA can be up-regulated by stimulation of mouse IFN-γ and down-regulated by siRNA2 in vitro. NBLs soaked with siRNA2 showed 32.3% reduction in the generation of MLs. MLs soaked with siRNA2 showed 26.2% reduction in the next generation of MLs, but no significant effect was observed on adult worms or NBLs. These findings concluded that GILT may play important roles in the development of T. spiralis parasite.

Antivirus activity, but not thiolreductase activity, is conserved in interferon-gamma-inducible GILT protein in arthropod

We have previously reported that gamma-interferon inducible lysosomal thiolreductase (GILT) functions as a host defense factor against retroviruses by digesting disulfide bonds on viral envelope proteins. GILT is widely conserved even in plants and fungi as well as animals. The thiolreductase active site of mammalian GILT is composed of a CXXC amino acid motif, whereas the C-terminal cysteine residue is changed to serine in arthropods including shrimps, crabs, and flies. GILT from Penaeus monodon (PmGILT) also has the CXXS motif instead of the CXXC active site. We demonstrate here that a human GILT mutant (GILT C75S) with the CXXS motif and PmGILT significantly inhibit amphotropic murine leukemia virus vector infection in human cells without alterning its expression level and lysosomal localization, showing that the C-terminal cysteine residue of the active site is not required for the antiviral activity. We have reported that human GILT suppresses HIV-1 particle production by digestion of disulfide bonds on CD63. However, GILT C75S mutant and PmGILT did not digest CD63 disulfide bonds, and had no effect on HIV-1 virion production, suggesting that they do not have thiolreductase activity. Taken together, this study found that antiviral activity, but not thiolreductase activity, is conserved in arthropod GILT proteins. This finding provides a new insight that the common function of GILT is antiviral activity in many animals.

Prognostic value of gamma-interferon-inducible lysosomal thiol reductase expression in female patients diagnosed with breast cancer.

Because of the high heterogeneity of breast cancer outcome, identification of novel prognostic biomarkers is critical to improve patient stratification and guide precise treatment. We examined the prognostic value of gamma-interferon-inducible lysosomal thiol reductase (GILT) expression in a training set of 416 breast cancer patients and a validation set of 210 patients, and performed functional studies to investigate the functions and underlying mechanisms of GILT on breast cancer prognosis. Our results indicated that high GILT expression in breast cancer cells was associated with improved disease-free survival (DFS; hazard ratio [HR]=0.189, 95% confidence interval [CI]: 0.099-0.361) and breast cancer-specific survival (BCSS; HR=0.187, 95% CI: 0.080-0.437) of breast cancer patients both in the training set and the external validation set (HR=0.453, 95% CI: 0.235-0.873 for DFS, HR=0.488, 95% CI: 0.245-0.970 for BCSS). In vitro and in vivo studies showed that GILT overexpression inhibited breast cancer cells proliferation, invasion, migration and tumor formation in nude mice and increased sensitivity of breast cancer cells to standard treatment. Proteomics analysis indicated that GILT inhibited reactive oxygen species (ROS) and autophagy activation in breast cancer cells, and GILT overexpression-mediated tumor growth was further enhanced in the presence of autophagy or ROS inhibitors. Our results demonstrate that GILT expression can be effectively used to predict the prognosis and guide treatment strategies of breast cancer patients.

Gamma-interferon inducible lysosomal thiolreductase (GILT) effect on breast tumor microenvironment through regulating CXCR6/CXCL16 axis.

e15081 Background: Gamma-interferon Inducible Lysosomal Thiolreductase (GILT) is constitutively expressed in most antigens endocytosed by antigen presenting cells (APCs), and its function is to catalyze the reduction of disulfide (S-S) bonds in protein substrates. The cytokine CXCL16 is one of the only two known plasma membrane chemokines which induces chemotaxis of activated T cells and bone marrow plasma cells in tumor microenvironment. It contains a free end folded by two sulfur bonds and therefore could also be a zymolyte of GILT. Previous studies found that specific receptor of CXCL16, CXCR6, was significantly overexpressed in breast cancer tissues and metastatic axillary lymph nodes. We suppose whether CXCR6/CXCL16 axis is regulated by GILT and affect tumor microenvironment, thereby eliciting specific anti-tumor immune responses in breast cancer (BC). Methods: GILT expression in BC was evaluated using publicly available data from The Cancer Genome Atlas (TCGA). GILT gene was analyzed in UALCAN ( http://ualcan.path.uab.edu/analysis-prot.html ) . In vitro, Immunohistochemistry (IHC) was conducted to examine the location and relation of GILT and CXCR6. Gene Set Enrichment Analysis (GSEA) was performed to mine the biological pathways involved in BC related GILT regulatory network. The expression of GILT at protein and RNA levels were detected by Western Blot and RT-PCR assay. Overexpression and knockdown of GILT in BC cell lines was carried out to further analyzed the correlation between GILT and CXCL16/CXCR6. Results: TCGA database showed that GILT expression was increased in the stroma of BC compared with normal, and was correlated to shorter BC overall survival. GSEA suggested that the expression of GILT was associated with chemotactic factors. Pearson analysis and IHC showed GILT had a strong correlation with CXCL16/CXCR6 axis in the aspect of angiogenesis and immunity. qRT-PCR and Western Blot assay also revealed that GILT had high expression in BC. Besides, patients with high expression of GILT in IHC simultaneously showed high immunoreactive to macrophage markers, which was related to neovascularization and anti-tumor immune responses. Compared with the normal breast cell line MCF-10A, GILT protein had high expression in Hs578T and low expression in MDA-MB-231 cell line. GILT was overexpressed in MDA-MB-231 and knockdown in Hs578T. Result showed that high level GILT promoted the production of CXCL16/CXCR6,while GILT siRNA knockdown inhibited the production of CXCL16/CXCR6. Conclusions: GILT could catalyze CXCL16 in BC, function as a key mechanism to affect tumor microenvironment through CXCR6/CXCL16 pathway. GILT-activated CXCL16 levels showed a strong connection with unfavorable outcomes in BC, which could be a potential biomarker of prognosis and a novel therapeutic target.

Variable GILT protein expression in melanoma cells of metastatic tumor specimens

Melanoma is the sixth most common cancer in the US. Immunotherapies, such as anti-CTLA-4 and anti-PD-1, have transformed the treatment of advanced melanoma; however, a subset of patients fail to respond to immunotherapy. Thus, improved predictors of the anti-melanoma immune response and response to immunotherapy are needed. We have previously shown that 1) gamma-interferon-inducible lysosomal thiol reductase (GILT) is critical for MHC class II-restricted presentation of a melanoma antigen, 2) there is variable GILT protein expression in melanoma cells from primary and cutaneous metastatic melanoma specimens, and 3) high GILT mRNA expression in melanoma specimens is associated with improved overall survival. Here, we created a database of n = 43 metastatic melanoma patients (stage III or IV) annotated with clinical outcome. Formalin-fixed, paraffin-embedded sections of pre-treatment metastatic melanoma specimens were tested for GILT protein expression using immunohistochemistry and scored by a dermatopathologist and dermatologist. GILT expression in melanoma cells was detected in 65% of lymph node and distant organ metastases, consistent with our prior finding of GILT expression in 58% of cutaneous metastases. The frequency of GILT-staining melanoma cells was determined by the ratio of the area of tumor cells with positive GILT staining to the total area of tumor cells. The frequency of GILT-staining melanoma cells among specimens with positive GILT staining was 15.2% ± 24.7% (mean ± SD). The median frequency of GILT staining was used to divide the patients into no/low and high GILT expression groups. The median overall survival was 52.8 months in the no/low GILT expression group, while the median overall survival was not reached in the high GILT expression group (log-rank, p = 0.67). For the n = 34 patients who were treated with immunotherapy targeting CTLA-4 and/or PD-1, the median overall survival was 38.2 months in the no/low GILT expression group, while the median overall survival was not reached in the high GILT expression group (log-rank, p = 0.83). These data suggest that high GILT expression in melanoma cells may be associated with improved overall survival, although a statistically significant association of GILT with survival was not observed in the whole group or the subset of patients treated with immunotherapy. A limitation of the current analysis is that 65% of patients have incomplete clinical outcome data. This study demonstrates variable GILT protein expression in tumor cells in metastatic melanoma specimens. Updated clinical outcome data will allow us to better evaluate the association of GILT protein expression in melanoma cells with overall survival and response to immunotherapy in metastatic melanoma patients.

Expression of gilt acts as a positive regulator of mouse hematopoietic progenitor cells

Gamma interferon inducible lysosomal thiol reductase (GILT), is known to be involved in immunity, but its role in hematopoiesis has not been previously reported. Herein, we demonstrate using gilt knockout (−/−) mice that loss of gilt associates with decreased numbers and cycling status of femoral hematopoietic progenitor cells (CFU-GM, BFU-E, and CFU-GEMM) with more modest effects on splenic progenitor cells. Thus, GILT is associated with positive regulation of hematopoietic progenitor cells in mice, mainly in bone marrow.

Transcriptome in combination with experimental validation unveils hub immune-related genes in oriental river prawn Macrobrachium nipponense against Spiroplasma eriocheiris challenge

Spiroplasma eriocheiris (S. eriocheiris) infection has caused heavy economic losses in Macrobrachium nipponense (M. nipponense) aquaculture industry. However, the interplay between immune responses of M. nipponense and S. eriocheiris infection has not yet been fully elucidated. Herein, full-length transcriptome profiling of M. nipponense hemocytes was performed with or without S. eriocheiris challenge. In total, 20,441 high quality unigenes were obtained, among which 733 differentially expressed genes (DEGs) were found, including 309 up-regulated and 424 down-regulated genes. Protein-protein interaction network (PPI) analysis of DEGs identified 4 hub immune-related genes, including MAP kinase-interacting serine/threonine-protein kinase 1 (MNK1), C-type lectin 4 (CTL4), gamma-interferon-inducible lysosomal thiol reductase (GILT), and anti-lipopolysaccharide factor (ALF). Tissue distribution and expression analysis of the key immune-associated genes demonstrated that they were widely expressed in 6 tissues, especially in gills, and were up-regulated by S. eriocheiris challenge. M. nipponense challenged by S. eriocheiris exhibited a significant reduction of bacterial clearance ability when the hub genes were silenced via RNA interference. Overall, our results indicated that the 4 hub immune-related genes play a vital role in anti-S. eriocheiris infection, thus providing insights into the immune defensive response mechanisms of M. nipponense against S. eriocheiris infection.

Planarian gamma-interferon-inducible lysosomal thiol reductase (GILT) is required for gram-negative bacterial clearance

The powerful regenerative ability of planarians has long been a concern of scientists, but recently, their efficient immune system has attracted more and more attention from researchers. Gamma-interferon-inducible lysosomal thiol reductase (GILT) is related not only to antigen presentation but also to bacteria invasions. But the systematic studies are not yet to be conducted on the relationship between bacterial infection. Our study reveals for the first time that GILT of planarian (DjGILT) plays an essential role in the clearance of Gram-negative bacteria by conducting H2O2 concentration in planarians. In animals that DjGILT was silenced, it persisted for up to 9 days before all bacteria were cleared, compared with 6 days of the control group. When infected with E. coli and V. anguillarum, the level of H2O2 was significantly increased in DjGILT-silenced planarians, and concomitantly, mRNA level of C-type lectin DjCTL, which modulates agglutination and clearance efficiency of invading bacteria, was decreased. Further study showed that the decrease of H2O2 level led to a significant increase in DjCTL transcripts. Collectively, we proposed a mechanism model for the involvement of GILT gene in bacterial elimination. We have for the first time revealed the specific mechanism of GILT in innate immune response against bacterial infection.

GILT in thymic epithelial cells facilitates central CD4 T cell tolerance to a tissue-restricted, melanoma-associated self antigen

Abstract Central tolerance prevents autoimmunity, but also limits T cell responses to potentially immunodominant tumor epitopes with limited expression in healthy tissues. In peripheral antigen presenting cells (APCs), gamma-interferon-inducible lysosomal thiol reductase (GILT) is critical for MHC class II-restricted presentation of disulfide bond-containing proteins, including the self and melanoma antigen tyrosinase-related protein 1 (TRP1). The role of GILT in thymic antigen processing and generation of central tolerance has not been investigated. We found that GILT enhanced the negative selection of TRP1-specific thymocytes. GILT expression was enriched in thymic APCs capable of mediating deletion, namely medullary thymic epithelial cells (mTECs) and dendritic cells, while TRP1 expression was restricted solely to mTECs. GILT facilitated MHC class II-restricted presentation of endogenous TRP1 by pooled thymic APCs. Using bone marrow chimeras, GILT expression in thymic epithelial cells (TECs), but not hematopoietic cells, was sufficient for complete deletion of TRP1-specific thymocytes. An increased frequency of TRP1-specific regulatory T (Treg) cells was present in chimeras with increased deletion of TRP1-specific thymocytes. Only chimeras that lacked GILT in both TECs and hematopoietic cells had a high conventional T:Treg cell ratio and were protected from melanoma challenge. Thus, GILT expression in thymic APCs, and mTECs in particular, preferentially facilitates MHC class II-restricted presentation, negative selection and increased Treg cells, resulting in a diminished anti-tumor response to a tissue-restricted, melanoma-associated self antigen.

Penaeus monodon GILT enzyme restricts WSSV infectivity by reducing disulfide bonds in WSSV proteins.

Gamma-interferon-inducible lysosomal thiol reductase (GILT) is involved in the adaptive immune response via its effects on major histocompatibility complex (MHC)-restricted antigen presentation. In addition to antigen presentation, GILT exerts its antiviral activity by reducing disulfide bonds in proteins involved in viral infection and assembly, thereby inhibiting viral envelope-mediated infection and viral progeny production. In black tiger shrimp, Penaeus monodon GILT (PmGILT) was cloned and characterized, and found to be involved in the shrimp innate immune response and to exert neutralizing activity against white spot syndrome virus (WSSV) infection. However, the anti-WSSV mechanism of PmGILT in the shrimp innate immune response has not been defined. To explore the anti-WSSV activity of PmGILT, a yeast 2-hybrid (Y2H) assay was performed to identify WSSV proteins targeted by PmGILT. The assay revealed 4 potential PmGILT-interacting WSSV proteins: WSSV002, WSSV164, WSSV189, and WSSV471. Three of these 4 WSSV proteins (WSSV002, WSSV164 and WSSV189) were successfully produced and confirmed to interact with PmGILT in in vitro pull-down assays. WSSV189 and WSSV471 were previously identified as structural proteins, whereas WSSV164 is an immediate-early protein which has anti-melanization activity, and WSSV002 is an unknown. Because of the thiol reductase activity of PmGILT, WSSV164 and WSSV189, both of which are cysteine-containing WSSV proteins, were chosen for disulfide bond reduction assays. PmGILT reduced intrachain disulfide bonds in both WSSV proteins, suggesting that PmGILT exerts its anti-WSSV activity via its thiol reductase activity to disrupt the WSSV protein complex and restore the melanization activity of PmproPO1 and PmproPO2.

Involvement of gamma interferon inducible lysosomal thiol reductase in the innate immune responses of red swamp crayfish, Procambarus clarkii

The Gamma interferon inducible lysosomal thiol reductase (GILT) plays a key biological role in the immune responses and involves in the processing of class II MHC-restricted antigen by stimulating disulfide bond reduction in mammals. To determine the biological function of GILT in the innate immune system of crustaceans, we sequenced and cloned GILT gene from red swamp crayfish, Procambarus clarkii (Pc-GILT). The deduced amino acid sequence of Pc-GILT contained the putative conserved structures of the GILT family proteins: the GILT signature (CQHGX2ECX2NX4C) sequence and the active site (CXXS) motif. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis suggested that a recombinant Pc-GILT protein was successfully expressed in Escherichia coli (E. coli). Quantitative real-time PCR analysis showed that Pc-GILT transcript level was highest in the hepatopancreas followed by the gut, heart and muscles. Additionally, we analyzed the transcription level of Pc-GILT gene in hepatopancreas of red swamp crayfish under biotic stress conditions. The expression of Pc-GILT gene upregulated after viral (poly I:C) and bacterial (peptidoglycan, lipopolysaccharide) infection. The suppression of Pc-GILT by double stranded RNA influenced the transcript levels of various immune-related genes. These observations indicate that the Pc-GILT probably plays a key biological role in the innate immune responses of red swamp crayfish, since it modulates the expression of genes associated with immune pathways.

High GILT expression and an active and intact MHC class II antigen presentation pathway are associated with improved survival in melanoma

Abstract The MHC class I antigen presentation pathway in melanoma cells has a well-established role in immune-mediated destruction of tumors. However, the clinical significance of the MHC class II antigen presentation pathway in melanoma cells is less clear. In antigen presenting cells, gamma-interferon-inducible lysosomal thiol reductase (GILT) is critical for MHC class II-restricted presentation of multiple melanoma antigens. While not expressed in benign melanocytes of nevi, GILT and MHC class II expression is induced in malignant melanocytes in a portion of melanoma specimens. Analysis of The Cancer Genome Atlas (TCGA) cutaneous melanoma dataset showed that high GILT mRNA expression was associated with improved overall survival. Expression of IFN-γ, TNF-α, and IL-1β was positively associated with GILT expression in the TCGA melanoma specimens. These cytokines were capable of inducing GILT expression in melanoma cells in vitro. GILT protein expression in melanocytes was induced in halo nevi, which are nevi undergoing immune-mediated regression, and is consistent with the association of GILT expression with improved survival in melanoma. To explore potential mechanisms of GILT’s association with patient outcome, we analyzed the mRNA expression pattern in the MHC class II antigen presentation pathway. In contrast to healthy skin where the MHC class II pathway was nearly uniformly expressed and intact, there was substantial variation in the MHC class II pathway of the TCGA melanoma dataset. Both an active and intact MHC class II pathway were associated with improved overall survival in melanoma. These studies support a role for GILT and the MHC class II antigen presentation pathway in melanoma outcome.

GILT restricts the cellular entry mediated by the envelope glycoproteins of SARS-CoV, Ebola virus and Lassa fever virus

ABSTRACT Interferons (IFNs) control viral infections by inducing expression of IFN-stimulated genes (ISGs) that restrict distinct steps of viral replication. We report herein that gamma-interferon-inducible lysosomal thiol reductase (GILT), a lysosome-associated ISG, restricts the infectious entry of selected enveloped RNA viruses. Specifically, we demonstrated that GILT was constitutively expressed in lung epithelial cells and fibroblasts and its expression could be further induced by type II interferon. While overexpression of GILT inhibited the entry mediated by envelope glycoproteins of SARS coronavirus (SARS-CoV), Ebola virus (EBOV) and Lassa fever virus (LASV), depletion of GILT enhanced the entry mediated by these viral envelope glycoproteins. Furthermore, mutations that impaired the thiol reductase activity or disrupted the N-linked glycosylation, a posttranslational modification essential for its lysosomal localization, largely compromised GILT restriction of viral entry. We also found that the induction of GILT expression reduced the level and activity of cathepsin L, which is required for the entry of these RNA viruses in lysosomes. Our data indicate that GILT is a novel antiviral ISG that specifically inhibits the entry of selected enveloped RNA viruses in lysosomes via disruption of cathepsin L metabolism and function and may play a role in immune control and pathogenesis of these viruses.

Microglia express gamma-interferon-inducible lysosomal thiol reductase in the brains of Alzheimer's disease and Nasu-Hakola disease.

Gamma-interferon-inducible lysosomal thiol reductase (GILT), expressed in antigen-presenting cells (APCs), facilitates the reduction of disulfide bonds of endocytosed proteins in the endocytic pathway and they are further processed for presentation of immunogenic peptides loaded on major histocompatibility complex (MHC) class II. Although the constitutive and IFNγ-inducible expression of GILT was observed in various APCs, such as dendritic cells, monocytes/macrophages, and B cells, GILT-expressing cell types remain unknown in the human central nervous system (CNS). Nasu-Hakola disease (NHD) is a rare autosomal recessive disorder characterized by sclerosing leukoencephalopathy and multifocal bone cysts, caused by a loss-of-function mutation of either TYROBP (DAP12) or TREM2, both of which are expressed on microglia. A rare heterozygous variant of the TREM2 gene encoding p.Arg47His causes a 3-fold increase in the risk for late-onset Alzheimer's disease (LOAD), suggesting that both NHD and AD are induced by dysfunction of the microglial TREM2 signaling pathway in the brains. We studied by immunohistochemistry GILT expression in NHD and AD brains. GILT was expressed on amoeboid microglia with the highest levels of expression in AD brains, compared with those in non-neurological control (NC) brains and in NHD brains. In AD brains, the clusters of amoeboid microglia surrounding amyloid-beta (Aꞵ) deposition strongly expressed GILT. Furthermore, a human microglial cell line expressed GILT in response to IFNγ. These results indicate that microglia, expressing constitutively high levels of GILT, act as a principal cell type of APCs in AD brains, in contrast to baseline levels of GILT expression in NHD brains.

Molecular and biological characterization of gamma-interferon-inducible lysosomal thiol reductase in silver carp (Hypophthalmichthys molitrix)

Gamma-interferon-inducible lysosomal thiol reductase (GILT) plays an important role in the processing of major histocompatibility complex (MHC) class II-restricted antigens by catalyzing disulfide bonds reduction. Herein, a GILT homolog (ScGILT) was identified from silver carp. Its open reading frame covers 771 base pairs, encoding a protein of 256 amino acids that possesses GILT signature sequence CQHGX2ECX2NX4C, active-site CXXC motif, and two potential N-linked glycosylation sites. The predicted tertiary structures of ScGILT and other GILTs were quite similar in shape and positional arrangement of the key motifs. ScGILT mRNA was constitutively expressed in all detected tissues, with high-level expression in fish immune organs, spleen and head kidney. After stimulation with lipopolysaccharide, the expression of ScGILT mRNA significantly increased in spleen and head kidney cells, and ScGILT protein translocated to late endosomes and lysosomes in HeLa cells. Recombinant ScGILT fused with a His6 tag was expressed and purified, and could reduce the interchain disulfide bonds of IgG at pH 4.5. These results suggested that ScGILT was capable of catalyzing disulfide bonds reduction, and then might play an important role in the processing of MHC class II-restricted antigens in silver carp.

GILT in thymic epithelial cells preferentially facilitates MHC class II-restricted presentation in generating central T cell tolerance to a self and melanoma antigen

Abstract Central tolerance prevents autoimmunity, but limits T cell responses to tumor antigens that are self antigens. In peripheral APCs, gamma-interferon-inducible lysosomal thiol reductase (GILT) is critical for MHC class II-restricted presentation of disulfide bond-containing proteins, including the self and melanoma antigen tyrosinase-related protein 1 (TRP1). The role of GILT in thymic antigen processing and generation of central tolerance is unknown. We found that GILT enhanced the clonal deletion of TRP1-specific thymocytes. GILT expression was enriched in thymic APCs capable of mediating deletion, namely medullary thymic epithelial cells (mTECs) and dendritic cells. TRP1 expression was restricted to mTECs. GILT facilitated MHC class II-restricted presentation of endogenous TRP1 by pooled thymic APCs. Using bone marrow chimeras, GILT expression in thymic epithelial cells (TECs), but not hematopoietic cells, was sufficient for deletion of TRP1-specific thymocytes. Chimeras with GILT expression in TECs also had an increased frequency of TRP1-specific regulatory T (Treg) cells. These data demonstrate an advantage of TEC-derived GILT for antigen processing and subsequent T cell tolerance. Only chimeras that lacked GILT in both TECs and hematopoietic cells were protected from melanoma and had a high effector T cell:Treg cell ratio. Thus, GILT diminishes T cell-mediated protection from melanoma through promoting thymic deletion and Treg cells.