Encyclopedia of Signaling Molecules

2018 Edition
| Editors: Sangdun Choi

Gamma-Interferon-Inducible Lysosomal Thiol Reductase (GILT)

Reference work entry
DOI: https://doi.org/10.1007/978-3-319-67199-4_101662

Synonyms

Historical Background

In 1988, the laboratory of Dr. Jeffrey Ravetch characterized a gene which is upregulated upon exposure to the cytokine, interferon-γ (IFN-γ). The protein is produced in a precursor form of 35 kDa and processed into a mature form of 30 kDa. The protein was originally named inducible protein 30 (IP30) and the gene named IFI30. IP30 is constitutively expressed in many antigen-presenting cells (APCs) and induced in other cell types including fibroblasts, endothelial cells, and keratinocytes (Maric et al. 2001; Lackman and Cresswell 2006; Phipps-Yonas et al. 2013a; Nguyen et al. 2016).

Homology to protein folding patterns of a cellular thiol reductase, thioredoxin, suggested that IP30 may be capable of breaking disulfide bonds. Indeed, a thioredoxin-like motif, CXXC, is found in the IP30 protein sequence and is conserved across species with IP30 homologs. IP30 reduces disulfide bonds, and the cysteines in the CXXC motif are required for this activity (Arunachalam et al. 2000; Phan et al. 2000). Therefore, IP30 was given a new descriptive name, gamma-interferon-inducible lysosomal thiol reductase (GILT). GILT is the only known thiol reductase in the endocytic pathway. GILT is found in late endosomes and lysosomes where it co-localizes with major histocompatibility complex (MHC) class II (Arunachalam et al. 2000).

The reductase function and localization to the endocytic pathway led Dr. Peter Cresswell’s group to investigate the role of GILT in antigen processing and presentation. MHC class II-restricted processing and presentation require that proteins in the endocytic pathway are broken down into antigenic peptides, or epitopes, which are then presented in the context of MHC class II to generate CD4 T cell responses. GILT’s thiol reductase function proved to be critical for MHC class II-restricted processing and presentation of certain epitopes from disulfide bond containing proteins important in the immune response to pathogens, cancer, and autoimmunity (Haque et al. 2002; Sealy et al. 2008; Rausch et al. 2010; Bergman et al. 2012). In addition, GILT’s reductase function facilitates MHC class I-restricted cross-presentation by dendritic cells by enabling the retrotranslocation of exogenous antigens from the phagosome to the cytosol (Singh and Cresswell 2010). Thus, GILT’s reductase activity helps to shape the peptide repertoire for eliciting immune responses.

Additionally, GILT can restrict infection, as in the case of human immunodeficiency virus (HIV) and dengue virus (Kubo et al. 2016), or enhance microbial pathogenesis and escape from immune recognition, as in Listeria (Singh et al. 2008). Less well-described roles for GILT include decreasing oxidative stress, autophagy, and proliferation (Maric 2009; Chiang and Maric 2011). GILT expression is also associated with improved survival in certain cancer types (Phipps-Yonas et al. 2013a; Xiang et al. 2014). Each of these roles for GILT will be discussed in greater detail throughout this chapter.

Regulation, Trafficking, and Enzymatic Activity

General Characteristics

GILT is synthesized from a 1045 base pair (bp) gene (IFI30) which includes an 85 bp 3′ untranslated region and a 13 bp poly-A tail for transcription termination. Upstream of the initiation codon, there are IFN-gamma-activated sites (GAS) for regulation by transcription factors (Srinivasan and Maric 2010). The full-length protein product is 261 amino acids (aa), including the 37 aa signal peptide for targeting to the endoplasmic reticulum (ER), where it is cleaved, resulting in the precursor form of GILT at 224 aa and 35 kDa (Fig. 1; Arunachalam et al. 2000). There are three potential N-linked glycosylation sites at residues 37, 69, and 82 (Arunachalam et al. 2000). The precursor form of human GILT contains 11 cysteine residues, each of which are required for either proper folding and maturation or enzymatic activity (Arunachalam et al. 2000). GILT exists in two primary forms: a 35 kDa dimeric secreted precursor and a mature 30 kDa monomer lacking the N- and C-terminal propeptides which is localized to the late endosomes and lysosomes.
Gamma-Interferon-Inducible Lysosomal Thiol Reductase (GILT), Fig. 1

GILT domains and conserved sites. The amino acid sequence of human GILT published by Arunachalam et al. (2000) is shown with amino acid numbering in precursor GILT. The signal peptide is shown in italics. The N-terminal propeptide (red) and the C-terminal propeptide (blue) are cleaved to generate the mature form of GILT. There are three putative N-linked glycosylation sites highlighted in gray. The 11 conserved cysteines are underlined. GILT’s reductase active site (CXXC) is highlighted in yellow at residues 46–49. An uncharacterized GILT signature motif (CQHGX2ECX2NX4C, green) is also conserved in GILT across multiple species

Conservation of GILT homologs across species is quite striking (Fig. 2). Species ranging from Paramecium to Arabidopsis (thale cress) to Drosophila (fruit fly) to Homo sapiens contain GILT homologs, with the cysteines from human GILT being largely conserved, including the CXXC reductase active site. An uncharacterized GILT signature motif (CQHGX2ECX2NX4C) is also conserved across GILT homologs. The vast array of species, many of which arose prior to the evolution of the adaptive immune system, with a GILT homolog containing the reductase active site, strongly suggests that GILT serves basic biochemical or metabolic functions in addition to facilitating antigen presentation.
Gamma-Interferon-Inducible Lysosomal Thiol Reductase (GILT), Fig. 2

Cladogram showing diversity of species with GILT homologs. Phylogenetic tree generated from amino acid sequences of known GILT proteins from a diverse array of species, using the neighbor-joining method in MEGA7 software. Evolutionary distance was calculated based on amino acid substitutions per site and is shown to scale

Expression and Regulation

GILT is expressed in a wide variety of cell types. GILT is constitutively expressed in most APCs, including monocytes, macrophages, B cells and dendritic cells (Arunachalam et al. 2000; Maric et al. 2001; Phipps-Yonas et al. 2013a). GILT is also constitutively expressed in thymocytes, mature T cells, and some fibroblasts (Maric et al. 2009). IFN-γ upregulates constitutive expression and induces GILT expression in other cell types, including immature monocytes and monocyte precursors, fibroblasts, endothelial cells, keratinocytes, fibrosarcoma, and melanoma cell lines (Haque et al. 2002; Lackman and Cresswell 2006). Heterogeneous GILT expression within the tumor and between patients is found in human cancer specimens and will be discussed later. Figure 3 demonstrates GILT expression in B cells and dendritic cells in human tonsil.
Gamma-Interferon-Inducible Lysosomal Thiol Reductase (GILT), Fig. 3

GILT expression in B cells and dendritic cells in human tonsil. GILT protein expression was assessed by immunohistochemistry with a brown chromagen. At low power (100x), the overall morphology of the tonsil can be seen: germinal center (light zone within follicle), mantle zone (ring of B cells around germinal center within follicle), and T cell zone (between follicles). At higher power (600x), there is vesicular staining of B cells and intense staining of follicular dendritic cells in the germinal center, vesicular staining within naïve B cells of the mantle zone, intense staining of interdigitating dendritic cells in the T cell zone, and intense staining of dendritic cells residing in the epithelium called Langerhans cells. GILT staining was not observed in epithelial cells or T cells. GILT expression in T cells may be below the limit of detection or not present in human T cells (Reused with permission from Phipps-Yonas et al. 2013a)

IFN-γ binds to a specific cell surface receptor which activates Janus kinases 1 and 2 (JAK1/JAK2) and the transcription factor signal transducer and activator of transcription 1 (STAT1). While IFN-γ-inducible expression of MHC class II and other genes in the class II antigen presentation pathway is regulated by IFN-γ-inducible isoforms of class II transactivator (CIITA), neither basal nor IFN-γ-inducible GILT expression is mediated by CIITA (O’Donnell et al. 2004). Instead, STAT1 regulates both constitutive and IFN-γ-inducible expressions of GILT (O’Donnell et al. 2004; Srinivasan and Maric 2010).

GILT has a second regulatory pathway in primary and established human monocytes and macrophages that is IFN-independent. Bacterial stimuli, such as exposure to Escherichia coli or lipopolysaccharide (LPS), a component of gram-negative bacteria cell walls, result in the upregulation of GILT expression through Toll-like receptor 4 (TLR4) activation (Lackman and Cresswell 2006). This signaling pathway, though IFN-independent, is dependent upon the transcription factor nuclear factor kappa B (NFκB) and inflammatory cytokines tumor necrosis factor (TNF) and interleukin 1-beta (IL-1β), either of which is sufficient to induce GILT upregulation in the absence of TLR ligands (Lackman and Cresswell 2006). Activation of the IFN-independent pathway results in an increase in secreted precursor form over intracellular mature GILT due to the downregulation of the enzymes, γ subunit of N-acetylglucosamine (GlcNAc)-1-phosphotransferase (GNPT) and uncovering enzyme (UCE), involved in the generation of the mannose-6-phosphate tag responsible for lysosomal trafficking discussed below and shown in Fig. 4 (Lackman et al. 2007). The existence of two distinct regulatory pathways for GILT expression suggests the importance of GILT in immunological responses in both innate (IFN-independent) and adaptive (IFN-dependent) immunity.
Gamma-Interferon-Inducible Lysosomal Thiol Reductase (GILT), Fig. 4

GILT in MHC class II-restricted antigen processing and presentation. Class II molecules associate with invariant chain (Ii) in the endoplasmic reticulum (ER). Class II:Ii and GILT are trafficked through the Golgi and either follow the constitutive secretory pathway to the plasma membrane with possible endocytosis or traffic directly to endosomes by the cytoplasmic tail of Ii (Class II:Ii) or mannose-6-phoshate (M6P) tag (green circle, P on GILT). In the ER and early Golgi compartments, γ subunit of N-acetylglucosamine (GlcNAc)-1-phosphotransferase (GNPT) catalyzes the transfer of GlcNAc-1-phosphate from UDP-GlcNAc to certain C6 hydroxyl groups of mannose sugars on the α-1,6 branch. A subsequent phosphorylation may occur on the α-1,3 branch. In the Golgi, the uncovering enzyme (UCE) hydrolyzes the phosphodiester bond releasing GlcNAc and exposing the M6P, which is recognized by the mannose-6-phosphate receptor (M6PR). M6PRs dissociate from their ligand in the mildly acidic environment of the early endosomes and return to the Golgi to mediate additional rounds of transport. TLR4-mediated signals decrease the transcription of GNPT and UCE and, thus, decrease M6P tagging, resulting in a shift toward secretion of precursor GILT via the constitutive secretory pathway. In the endosome, Ii is cleaved to release the trimerization motif leaving the partial proteolytic product of Ii called leupeptin-induced protein (LIP) associated with class II (class II:LIP). Exogenous antigens are taken up by multiple mechanisms including phagocytosis, endocytosis, and pinocytosis. In the late endosomes and lysosomes, GILT catalyzes disulfide bond reduction to facilitate partial protein unfolding of antigens. Ii is further degraded leaving the class II-associated invariant chain peptide (CLIP) in the peptide-binding groove. Cathepsins (scissors) are responsible for Ii degradation and cleavage of antigenic proteins into peptides for loading onto class II. HLA-DM facilitates exchange of CLIP for locally generated peptides. HLA-DO acts as a competitive inhibitor of HLA-DM/class II interactions. MHC class II/peptide complexes are trafficked to the cell surface for presentation to CD4 T cells

Maturation and Trafficking

In the ER, precursor GILT either dimerizes (~20%) through interchain disulfide bond formation at Cys-222 and is either secreted through the Golgi apparatus or is posttranslationally modified with a mannose-6-phosphate at one of its three putative N-linked glycosylation sites (Fig. 4; Arunachalam et al. 2000). The M6PR recognizes this modification and results in trafficking of precursor GILT to the endocytic pathway, initially localizing to the early endosomes (Arunachalam et al. 2000). Here, precursor GILT is cleaved to form the mature 30 kDa form lacking N- and C-terminal propeptides. The N-terminus is cleaved at Asn-32 or Ala-33 by cathepsins D, L, and S, while the C-terminal propeptide is cleaved at Lys-206 by cathepsins B, L, and partially S in vitro (Phan et al. 2000). However, in vivo data suggests that there are other proteases besides cathepsins capable of processing precursor GILT to its mature form (Phan et al. 2000). Cathepsin S (CatS) is also responsible for turnover of mature GILT. The mature form of GILT primarily localizes in late endosomes and multilaminar lysosomes known as MIICs due to their association with MHC class II antigen presentation components (Arunachalam et al. 2000). Indeed, mature GILT co-localizes in MIICs with MHC class II (Arunachalam et al. 2000).

GILT’s 11 conserved cysteines play important roles in protein stability, folding, and maturation. Mutation of Cys-106, Cys-122, Cys-136, or Cys-152 results in undetectable expression of GILT, suggesting a role for these residues in proper folding or stability of GILT. GILT mutants of cysteines at positions 91, 98, 200, and 211 are expressed, but fail to mature, resulting in an increased amount of precursor GILT that is secreted. Interestingly, Cys-211 is in the C-terminal propeptide, and mutation of Cys-200 or Cys-211 resulted in decreased stability of the secreted precursor form, suggesting these two cysteines likely form a disulfide bond that contributes to the dimer’s stability. Active sites Cys-46 and Cys-49 are also involved in GILT maturation, although the mechanism by which these cysteines participate in GILT processing is unknown (Hastings et al. 2006).

Reductase Activity

The proposed mechanism of disulfide bond reduction by GILT resembles that of known thiol reductases (Fig. 5). Members of the thioredoxin family contain a conserved reductase motif, WCGH/PCK. GILT contains a nontraditional reductase active site motif 46-CXXC-49 in which Cys-46 is critical for disulfide bond reduction (Arunachalam et al. 2000), and it is strongly conserved across species. The N-terminal cysteine of the CXXC motif (Cys-46) initiates a nucleophilic attack on a disulfide bond in the substrate, resulting in a mixed GILT-substrate disulfide bond intermediate. This is followed by a second nucleophilic attack by the C-terminal cysteine (Cys-49), allowing the reduced substrate to escape and leaving an oxidized GILT which must be reduced before another reaction can occur (Phan et al. 2000). Mutation of Cys-49 results in “trapped” intermediates (Fig. 5, reaction 2) due to decreased enzymatic efficiency, which permits identification of GILT substrates (Phan et al. 2000; Singh et al. 2008; Singh and Cresswell 2010). The in vivo reducing agent required for GILT regeneration is unknown. In vitro, both cysteine and cysteinylglycine can serve as activators while glutathione cannot (Arunachalam et al. 2000; Phan et al. 2000). Both the mature form and precursor GILT exhibit optimal reducing activity at pH 4–5, the expected pH of lysosomal compartments, and maintain activity at pH = 7 (Arunachalam et al. 2000; Phan et al. 2000). Thus, GILT is the only known thiol reductase in the endocytic pathway and functions optimally at an acidic pH, which are key characteristics for facilitating MHC class II antigen processing and presentation.
Gamma-Interferon-Inducible Lysosomal Thiol Reductase (GILT), Fig. 5

Proposed mechanism of GILT-mediated protein disulfide bond reduction. The active site of GILT consists of a CXXC motif. The N-terminal Cys (C-46) initiates a nucleophilic attack on the substrate’s disulfide bond (1) resulting in a mixed disulfide intermediate (2). The intermediate is resolved when GILT’s C-terminal Cys (C-49) attacks the N-terminal Cys (C-46), allowing escape of the reduced substrate and leaving an oxidized GILT (3). GILT’s active site must be regenerated prior to the next substrate reduction. This can be achieved through cysteine or cysteinylglycine in vitro

Antigen Presentation

MHC Class II Presentation

The MHC class II-restricted antigen processing and presentation pathway is reviewed in Fig. 4. Newly synthesized MHC II α and β chains associate with invariant chain and form a trimer of trimers in the ER. Class II-invariant chain complexes can traffic to endosomes either via the cell surface or directly from the Golgi, directed by targeting motifs on the cytoplasmic tail of invariant chain. Cathepsin proteases are responsible for the sequential cleavage of invariant chain, proteolytic cleavage of antigenic proteins, and converting precursor GILT to its mature form. Proteolytic degradation of invariant chain ultimately leaves only the class II-associated invariant chain peptide (CLIP) in the peptide-binding groove of class II. CLIP prevents premature loading of peptides onto class II until the late endosome/lysosome (or MIIC) compartments. HLA-DM catalyzes the exchange of CLIP for a newly generated peptide and functions as a peptide editor. HLA-DO binds the same face of class II as HLA-DM and thus functions as a competitive inhibitor of class II interaction with HLA-DM. GILT-mediated reduction of protein disulfide bonds facilitates MHC class II-restricted presentation of certain epitopes from disulfide bond-containing proteins, presumably through exposing structurally constrained or buried regions of the protein for class II binding and protection from further proteolytic degradation (Hastings et al. 2006). The peptide/MHC class II complexes are directed to the plasma membrane for presentation to CD4 T cells.

APCs from GILT−/− mice have defects in MHC class II-restricted processing and presentation of particular epitopes (Maric et al. 2001). For instance, the model antigen hen egg lysozyme (HEL) contains four disulfide bonds. GILT is required for intracellular processing of HEL protein to generate the HEL peptide consisting of residues 74–88 (HEL74–88), which contains two cysteine residues that are each involved in disulfide bonds. Processing of HEL46–61, which does not contain cysteine residues, is partially diminished in the absence of GILT. The processing of two other HEL epitopes, HEL20–35 and HEL30–53, which share a cysteine that is involved a disulfide bond, is not affected by the absence of GILT. Thus, the mere presence of a cysteine involved in a disulfide bond in the epitope does not necessarily predict GILT dependence. A requirement for GILT may depend on the epitope’s location within the tertiary structure. A buried or disulfide bond constrained region is predicted to require GILT-mediated reduction for presentation, whereas a GILT-independent epitope may be surface exposed or, in a less structurally constrained site, accessible for class II binding without prior reduction. Despite not all HEL epitopes requiring GILT for efficient processing and presentation, the overall CD4 T cell response to HEL is decreased by 90% in GILT−/− mice compared to wild-type mice.

Subsequently, many other epitopes have been identified as GILT-dependent (presented more efficiently in the presence of GILT), GILT-independent (presented similarly in the presence or absence of GILT), or GILT-prevented (presented more efficiently in the absence of GILT; Table 1). Mass spectrometric analysis of the steady-state MHC class II-bound peptide repertoire from GILT−/− and wild-type mouse splenocytes revealed that 5.5% of self-peptides are more abundant in the presence of GILT compared with 94.5% of peptides which are more abundant in the absence of GILT (Bogunovic et al. 2010). Specifically, 2.0% of self-peptides are unique to GILT−/− APCs, and 3.5% of self-peptides are 10–60-fold more abundant in GILT−/− APCs at steady state. The GILT-prevented epitopes in this study were essentially all derived from the N- and C-termini of proteins, which typically lack structural constraints and are anticipated to be readily available for class II binding without reduction. Thus, epitopes from these regions may be overrepresented in the absence of GILT. Many GILT-influenced epitopes have been characterized for several pathogens, cancers, allergens, and autoimmune antigens (to be discussed further below).
Gamma-Interferon-Inducible Lysosomal Thiol Reductase (GILT), Table 1

Class I and II epitopes altered by GILT expression. Class I epitopes (red) and class II epitopes (blue) are shown as GILT-dependent, GILT-independent, or GILT-prevented. Dependent epitopes require GILT expression for processing and presentation, while independent epitopes are presented regardless of GILT expression. GILT-prevented epitopes are either more abundantly or uniquely presented in the absence of GILT. Epitopes were determined by (1) reactivity of T cell hybridomas specific for a given peptide/MHC, (2) recall responses to specific peptides following immunization or infection of mice, and (3) peptide elution from MHC class II followed by mass spectrometry

Cross-Presentation on MHC Class I

Antigen presentation on MHC class I for induction of CD8 T cell responses can occur through direct presentation by infected APCs or via cross-presentation of phagocytosed exogenous antigens by dendritic cells. A major mechanism of cross-presentation involves the transfer of antigens or antigen fragments from the phagosome to the cytosol (Fig. 6). In the cytosol the antigens are degraded by the proteasome, and resulting peptides are translocated into the ER via the transporter associated with antigen processing (TAP), and those with the appropriate sequence and length are loaded onto MHC class I in the ER.
Gamma-Interferon-Inducible Lysosomal Thiol Reductase (GILT), Fig. 6

GILT in MHC class I-restricted cross-presentation. Exogenous antigens are phagocytosed by dendritic cells. In the cytosolic pathway of cross-presentation, exogenous proteins or protein fragments are retrotranslocated from the phagosome to the cytosol. Large proteins in the phagosome must be unfolded and/or partially cleaved for retrotranslocation. GILT-mediated protein disulfide bond reduction in the phagosome facilitates retrotranslocation. In the cytosol, the antigen is further digested by the proteasome into small peptides, which are translocated into the endoplasmic reticulum (ER) by the transporter associated with antigen processing (TAP). The ER aminopeptidase associated with antigen processing (ERAP1 and/or ERAP2) trims the N-termini of peptides. Peptides of the appropriate sequence and length bind MHC class I molecules in the peptide-loading complex composed of class I (dark green), β2m (light green), tapasin (yellow), ERp57 (orange), and calreticulin (pink). Peptide binding triggers dissociation of the peptide-loading complex, and peptide/MHC class I complexes are directed to the cell surface for stimulation of CD8 T cells

Large protein antigens must be unfolded or undergo partial proteolysis for translocation from the phagosome to the cytosol. Thus, proteins constrained by disulfide bonds may require reduction for translocation. For herpes simplex virus 1 (HSV-1), cross-presentation of an immunodominant epitope from an envelope protein glycoprotein B (gB498–505), which contains five disulfide bonds, requires GILT’s reductase active site (Singh and Cresswell 2010). In contrast, direct presentation of gB498–505 in infected dendritic cells and cross-presentation of an epitope from an HSV-1 protein (ICP6822–829), which does not contain disulfide bonds, are GILT-independent. GILT is also required for cross-priming gB498–505-specific CD8 T cells in vivo. This role is not limited to HSV-1, as GILT is essential for cross-priming CD8 T cell responses following infection with influenza virus. GILT is required for efficient cross-priming of multiple epitopes from hemagglutinin (HA) and neuraminidase (NA) proteins, which contain disulfide bonds, while cross-priming of epitopes from polymerase (PA) and nucleoprotein (NP) proteins is GILT-independent (Table 1). Thus, GILT is not only involved in MHC class II-restricted processing in the endocytic pathway, but is also critical for facilitating cross-presentation on MHC class I.

T Cell Development and Activation

The effect of GILT in MHC class II-restricted antigen processing and presentation influences T cell development and activation. GILT is required for efficient MHC class II-restricted presentation of a self- and melanoma antigen, tyrosinase-related protein 1 (TRP1109–230; Rausch et al. 2010). Presentation of TRP1109–230 is not detected in GILT-deficient B cells, and presentation is ~100-fold more efficient in dendritic cells expressing GILT. Transfer of naïve TRP1-specific CD4 cells into recipient mice with and without GILT demonstrates that GILT accelerates the onset and severity of TRP1-specific T cell-mediated autoimmune vitiligo, which is associated with an increased frequency of TRP1-specific T cells with an effector memory phenotype. In addition to enhancing the T cell activation and function of mature T cells recognizing a GILT-dependent epitope, GILT also influences T cell development in the thymus. TRP1 is a self-antigen expressed in the thymus. In GILT-expressing mice, there is increased deletion of TRP1-specific T cells in the thymus (Rausch and Hastings 2012), demonstrating that expression of GILT enhances the thymic deletion of T cells recognizing a GILT-dependent epitope from an endogenously expressed self-protein. Thus, GILT’s role in antigen presentation modulates T cell development and peripheral activation.

Lysosomal/Phagosomal Proteolysis

GILT is the only known enzyme in the lysosomes and phagosomes to catalyze disulfide bond reduction with the potential to influence antigen processing and proteolysis within these compartments. The cysteine protease CatS is required for the cleavage of invariant chain and proteolysis of exogenous and endogenous proteins in these compartments for antigen presentation. However, excess proteolysis results in loss of epitopes and diminished antigen presentation. Thus, control of CatS activity can modulate proteolysis and antigen presentation. The proteolytic activity of CatS requires the active site cysteine to be in the reduced thiol state. An increasingly oxidizing environment, as in the case following respiratory burst, renders CatS less active. During respiratory burst, the phagocyte NADPH oxidase (NOX2) generates reactive oxygen species (ROS), namely, superoxide, which is then converted to H2O2. Macrophages alternately activated in response to IL-4 have decreased NOX2 expression and increased GILT expression and, thus, maintain CatS in the active state and display efficient proteolysis in phagosomes. In alternatively activated macrophages or in classically activated macrophages with NOX2 inhibition, GILT enhances bulk proteolysis, and in an entirely in vitro system, recombinant GILT enhances CatS activity, suggesting that GILT is responsible for maintaining the CatS cysteine active site in the reduced/active state in macrophages where proteolysis is favored (Balce et al. 2014).

In contrast, GILT expression in B cells inhibits proteolysis by CatS and may fine-tune the degree of proteolysis to favor antigen processing. In B cells, GILT’s reductase activity decreases the steady-state protein expression of CatS, and GILT expression decreases the half-life of CatS, suggesting that GILT-mediated reduction of protein disulfide bonds enhances CatS degradation (Phipps-Yonas et al. 2013b). Lysates from GILT-expressing B cells exhibit decreased proteolysis of a CatS selective substrate. Thus, in B cells, GILT inhibits proteolysis by CatS thereby regulating the degree of proteolysis to favor MHC class II-restricted antigen processing. Therefore, GILT may serve different functions regarding the regulation of cathepsins in different cell types and activation states and influence the bias toward proteolysis or antigen presentation.

Redox

Cellular Redox State

A series of studies have revealed that GILT regulates the cellular redox state. GILT expression in fibroblasts has been shown to decrease oxidative stress. Glutathione is the major low molecular weight redox buffer for controlling the cellular redox state, with an excess of the oxidized form of glutathione (GSH) over the reduced form of glutathione (GSSG) available to remove ROS, specifically H2O2. GILT expression diminishes oxidative stress by increasing GSH, thereby increasing the GSH/GSSG ratio, and altering the glutathione redox potential to maintain a more reduced state (Chiang and Maric 2011). Increased oxidative stress results in inactivation of multiple protein tyrosine phosphatases leading to increased phosphorylation of growth factor receptors and increased phosphorylation of mitogen-activated protein kinase cascades, including ERK1/ERK2. In the presence of GILT, there is decreased ERK1/ERK2 phosphorylation/activation and cell proliferation (Maric et al. 2009; Chiang and Maric 2011). GILT expression is also associated with increased levels of superoxide dismutase 2 (SOD2), a mitochondrial enzyme involved in removal of superoxide (Maric et al. 2009). This effect is likely indirect as GILT and SOD2 are localized to different cellular compartments (i.e., the late endosomes/lysosomes and the mitochondria, respectively). Oxidative stress and ERK1/ERK2 activation induce autophagy resulting in removal of dysfunctional mitochondria damaged by ROS. GILT-expressing fibroblasts exhibit decreased autophagy, which may in part explain increased SOD2 levels (Chiang and Maric 2011). Thus, GILT has been implicated in decreasing oxidative stress and impacting redox-sensitive cellular processes, proliferation, and autophagy.

T Cell Sensitivity

GILT expression in T cells results in a similar decrease in ROS, ERK1/ERK2 phosphorylation, and proliferation following T cell activating stimuli (Maric et al. 2009). These results suggest that GILT plays an inhibitory role in T cell activation by dampening the response to activating stimuli. In further support of this, GILT expression levels increase with T cell development from double-positive thymocytes to single-positive thymocytes or peripheral T cells and inversely correlates with induction of the early T cell activation marker CD69. Therefore, GILT expression may serve to control TCR sensitivity as T cells mature. In support of this, GILT−/− mice developed earlier onset and more severe hyperglycemia in streptozocin-induced diabetes, a CD8 T cell-mediated autoimmunity. Thus, GILT expression in T cells is critical for regulating T cell activation, which, when absent, can lead to aberrant responses and autoimmunity.

Clinical Impact

Autoimmunity

GILT’s role in autoimmunity has also been studied in experimental autoimmune encephalitis (EAE), a mouse model of multiple sclerosis. GILT expression alters the MHC class II epitopes recognized and the pathogenic mechanism in EAE (Bergman et al. 2012). Wild-type mice immunized with myelin oligodendrocyte glycoprotein (MOG) develop EAE that is mediated by CD4 T cells and depends on the presentation of the GILT-dependent epitope MOG35–55. MOG35–55 is the sole encephalitogenic epitope in MOG protein-induced EAE in wild-type mice. In contrast, T cells from GILT−/− mice immunized with MOG protein proliferate in response to several overlapping MOG peptides, which did not elicit responses in wild-type mice (Table 1). Consistent with the GILT dependence of the MOG35–55 epitope, GILT−/− mice do not generate T cell responses to MOG35–55 and are resistant to EAE induced by MOG35–55 peptide. Surprisingly, GILT−/− mice develop more severe EAE following immunization with MOG protein compared to wild-type mice. This unanticipated finding is due to a switch in the pathogenic mechanism from T cell-mediated autoimmunity in wild-type mice to antibody-mediated disease in GILT−/− mice.

Infection

Modulating antigen presentation is one way in which GILT may alter the host response to infection. As discussed above, several viruses have GILT-dependent MHC class I and II epitopes for generation of CD8 or CD4 T cell responses, respectively (Table 1). Some of these antigens include HSV gB and influenza HA and NA (Singh and Cresswell 2010). However, both viruses also have GILT-independent epitopes, including one in HA of influenza that is reduction-dependent, but GILT-independent. A GILT-dependent MHC class II epitope was identified for HIV in the envelope glycoprotein (Env/gp120; Sealy et al. 2008). This epitope lies at the base of the V1/V2 loop in an antiparallel beta sheet, with three disulfide bonds in the immediate vicinity. However, a second epitope in this region and overlapping with the GILT-dependent epitope is less affected by GILT.

GILT also alters microbial pathogenesis independent of antigen processing. GILT increases the pathogenesis of certain bacteria strains which utilize hemolysins. For instance, during infection with Listeria monocytogenes, the bacterium is phagocytosed by macrophages and evades immune destruction using the pore-forming listeriolysin O to escape into the cytosol. GILT−/− mice are resistant to Listeria monocytogenes infection (Singh et al. 2008). GILT-mediated reduction activates listeriolysin O (LLO), thus permitting bacterial escape to the cytosol. In GILT−/− mice, bacterial escape from the phagosome is delayed, accounting for the diminished infectivity of Listeria in GILT−/− mice. Additionally, secreted precursor GILT can activate streptolysin O, a virulence factor of Streptococcus pyogenes, suggesting that secreted GILT may enhance hemolysin-mediated tissue damage.

GILT expression can also restrict virus infection at different stages of the viral life cycle. For example, GILT expression confers resistance to dengue virus by reducing translation and genome replication efficiency, strongly correlating with decreased autophagy. GILT can also limit virion production posttranslation. GILT appears to deter HIV virion production by reducing disulfide bonds in CD63, a member of the tetraspanin family localized to the endosome/lysosome, thus preventing proper Gag-CD63 complex formation required for assembly (Kubo et al. 2016). Enzymatically active, but not mutant, GILT also diminishes infectivity of viral vectors displaying envelope proteins from HIV, murine leukemia virus (MLV), and vesicular stomatitis virus (Kubo et al. 2016). Thus, GILT is presumably reducing disulfide bonds within the envelope proteins. Consistent with this, in the presence of GILT, MLV’s SU protein is specifically depleted from extracellular virions when SU is normally tethered to the virion through a disulfide bond with TM (Kubo et al. 2016). This is an intriguing role for GILT as a restriction factor for viral replication and infection by its apparent action on viral envelope proteins and host factors, although the proteins mentioned here have not been directly verified as GILT substrates.

Cancer Survival

GILT expression is associated with improved patient survival in multiple cancers. GILT expression in diffuse large B-cell lymphoma (DLBCL) is associated with improved overall survival, and the association of GILT expression with survival is independent of established prognostic factors (Phipps-Yonas et al. 2013a). Similarly, in breast cancer patients, GILT expression in tumors correlates with less adverse tumor characteristics and improved disease-free survival (Xiang et al. 2014). Consistent with findings that GILT expression in T cells and fibroblasts decreases proliferation, GILT expression in breast cancer inversely correlates with staining of the proliferation marker Ki67. Another cancer type with the potential for GILT expression to be associated with improved prognosis is melanoma. MHC class II-restricted epitopes from two melanoma antigens, tyrosinase and TRP1, are GILT-dependent (Table 1; Haque et al. 2002; Rausch et al. 2010). In patient specimens, GILT expression is upregulated in melanoma cells compared with benign melanocytes, and GILT expression in melanoma cells is heterogeneous between patients (Nguyen et al. 2016). Multiple known functions of GILT have the potential to improve cancer survival, including enhancing antigen presentation and immune recognition and diminishing oxidative stress, proliferation, and autophagy. However, whether GILT is directly responsible for improved cancer survival and the mechanism of GILT’s effect remains to be determined.

Summary

Overall, GILT has a diverse set of cellular functions. The most well-characterized function is GILT’s role in antigen presentation via disulfide bond reduction. GILT also alters the activity or stability of lysosomal proteases in different antigen-presenting cells. GILT influences the peptide repertoire presented and the subsequent T cell responses in autoimmunity, infection, and cancer. GILT can even alter the immune-mediated mechanism of autoimmune pathogenesis. A further understanding of the role of lysosomal reduction in proteolysis and antigen presentation in different cells types, such as antigen presentation in the thymus for T cell selection as well as different dendritic cells populations, is warranted. Outside of antigen presentation, GILT’s reductase activity is important in microbial pathogenesis as a host factor used by bacterial hemolysins for activity and restricting viral infection. GILT also is associated with decreased oxidative stress, proliferation, and autophagy and improved patient survival in cancer; however, the mechanisms of these effects remain to be defined.

See Also

References

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Copyright information

© Springer International Publishing AG 2018

Authors and Affiliations

  1. 1.Department of Basic Medical Sciences, College of MedicineUniversity of ArizonaPhoenixUSA