Role of immune microenvironment in gastrointestinal stromal tumours.

AIMS: The immune microenvironment is a prognostic factor for various malignancies. The significance of key players of this immune microenvironment, including tumour-infiltrating lymphocytes (TILs) and expression of programmed death-ligand 1 (PD-L1), indoleamine 2,3-dioxygenase (IDO) and tryptophanyl-tRNA synthetase (WARS) in gastrointestinal stromal tumours (GISTs) is largely unknown. METHODS AND RESULTS: Tissue microarrays were constructed from pathology files, 1996-2016. Immunohistochemistry for PD-L1, IDO and WARS was correlated with tumour size, mitoses and outcomes. TILs expressing CD3, CD4, CD8, FoxP3 and GBP5 were counted. A total of 129 GISTs were analysed. Mean patient age was 62.5 years; 52.0% were male. Tumour location included 89 stomach (69.0%), 33 small bowel (25.6%) and seven other (5.4%). Mean tumour size was 5.6 cm; mean mitoses were 7.2 per 50 high-power field. Nineteen patients (15.0%) developed disease progression, to abdominal wall (n = 8), liver (n = 6) and elsewhere (n = 5). Median progression-free survival was 56.6 months; five patients died of disease. PD-L1 was positive in 88 of 127 tumour samples (69.0%), 114 of 127 tumours were IDO-positive (89.8%) and 60 of 127 were positive for WARS (47.2%). PD-L1 was associated with increased size (P = 0.01), necrosis (P = 0.018) and mitoses (P = 0.006). Disease progression was not associated with PD-L1 (P = 0.44), IDO (P = 0.14) or WARS (P = 0.36) expression. PD-L1-positive GISTs with CD8+ or CD3+ TILs were significantly smaller than tumours with CD8+ or CD3+ TILs. CONCLUSIONS: PD-L1 expression was associated with increased size and mitoses. High CD8+ or CD3+ TIL counts were associated with decreased PD-L1/IDO+ GIST size. PD-L1 and IDO could be significant in GIST tumour biology, which invites consideration of immunotherapy as a potential treatment option.

Effect of Mini-Tyrosyl-tRNA Synthetase/Mini-Tryptophanyl-tRNA Synthetase on Angiogenesis in Rhesus Monkeys after Acute Myocardial Infarction.

AIMS: The purpose of this study was to clarify the effect of mini-tyrosyl-tRNA synthetase/mini-tryptophanyl-tRNA synthetase (mini-TyrRS/mini-TrpRS) in ischemic angiogenesis in rhesus monkeys with acute myocardial infarction (AMI). METHODS: A 27-gauge needle was incorporated percutaneously into the left ventricular myocardium of rhesus monkeys with AMI. All monkeys were randomized to receive adenoviral vector mini-TyrRS/mini-TrpRS, which was administered as five injections into the infarcted myocardium, or saline or ad-null (control groups). The injections were guided by EnSite NavX left ventricular electroanatomical mapping. Mini-TyrRS/mini-TrpRS proteins were detected by Western blot and immunoprecipitation analyses. Microvessel density (MVD) per section was measured using immunostaining with a CD34 monoclonal antibody. Proliferating cardiomyocytes were identified through histological and immunohistochemical analyses. Myocardial perfusion and cardiac function were estimated by G-SPECT. Infarction size was also measured. RESULTS: Western blot analyses showed that compared to the normal zone, the expression level of mini-TyrRS/mini-TrpRS was significantly different in the infarction zone. G-SPECT analysis indicated that the mini-TyrRS group had better cardiac function and myocardial perfusion after the injection of ad-mini-TyrRS than before, while mini-TrpRS injection had a totally opposite effect. After mini-TyrRS was administered, there was less of an infarction zone and more proliferating cardiomyocytes and capillaries in the mini-TyrRS group compared to both of the control groups, and the ad-mini-TrpRS group had a totally opposite effect. CONCLUSION: These results indicated that angiogenesis could be either stimulated by mini-TyrRS or inhibited by mini-TrpRS.

Overexpressed tryptophanyl-tRNA synthetase, an angiostatic protein, enhances oral cancer cell invasiveness.

Oral squamous cell carcinoma (OSCC) is one of the most common neoplasms worldwide. Previously, we identified the angiostatic agent tryptophanyl-tRNA synthetase (TrpRS) as a dysregulated protein in OSCC based on a proteomics approach. Herein, we show that TrpRS is overexpressed in OSCC tissues (139/146, 95.2%) compared with adjacent normal tissues and that TrpRS expression positively correlates with tumor stage, overall TNM stage, perineural invasion and tumor depth. Importantly, the TrpRS levels were significantly higher in tumor cells from metastatic lymph nodes than in corresponding primary tumor cells. TrpRS knockdown or treatment with conditioned media obtained from TrpRS-knockdown cells significantly reduced oral cancer cell viability and invasiveness. TrpRS overexpression promoted cell migration and invasion. In addition, the extracellular addition of TrpRS rescued the invasion ability of TrpRS-knockdown cells. Subcellular fractionation and immunofluorescence staining further revealed that TrpRS was distributed on the cell surface, suggesting that secreted TrpRS promotes OSCC progression via an extrinsic pathway. Collectively, our results demonstrated the clinical significance and a novel role of TrpRS in OSCC.

An expression system for the efficient incorporation of an expanded set of tryptophan analogues.

Biosynthetic incorporation of tryptophan (Trp) analogues in recombinant proteins using an E. coli Trp auxotroph expression host is limited to analogues modified with a small substituent like a fluoro atom or a hydroxyl or amine group. We report here the efficient incorporation (>89 %) of chloro- and bromo atoms containing Trp analogues in alloproteins at high expression levels using a Lactococcus lactis Trp auxotroph strain. This result was only obtained after coexpression of the enzyme tryptophanyl-tRNA synthetase (TrpRS) of L. lactis, an enzyme believed to show a more relaxed substrate specificity than TrpRS from E. coli. Chloro- and bromo-Trps are attractive intrinsic phosphorescence probes as these Trp analogues are much less sensitive for quenchers in the medium, like oxygen, than Trp. Coexpression of TrpRS was also essential for the biosynthetic incorporation (94 %) of the Trp analogue 5,6 difluoroTrp. This makes our expression system ideally suited to generate a set of methyl- and fluoro-substituted Trp analogue-containing alloproteins in high yield for investigating the involvement of the Trp residue in cation-pi or pi-pi interactions. Taken together, the presented Trp auxotroph expression system features the most relaxed specificity for Trp analogue structures reported to date and gives a high alloprotein yield.

Decreased IDO activity and increased TTS expression break immune tolerance in patients with immune thrombocytopenia.

INTRODUCTION: Indoleamine 2,3-dioxygenase (IDO) can promote peripheral immune tolerance and control autoimmune responses through tryptophan catabolism. Tryptophanyl-tRNA synthetase (TTS) can protect T cells from IDO-mediated cell injury. Impaired IDO-mediated tryptophan catabolism has been observed in some autoimmune diseases. MATERIALS AND METHODS: The concentrations of plasma kynurenine and tryptophan were detected by high-pressure liquid chromatography. The expressions of IDO and TTS were analyzed by real-time quantitative polymerase chain reaction and flow cytometry. RESULTS: Compared with healthy controls, the PBMCs of patients with immune thrombocytopenia (ITP) had significantly increased expressions of IDO and TTS, especially IDO. However, the plasma tryptophan concentration was significantly elevated, and kynurenine concentration was significantly reduced in ITP patients. In CD4(+) and CD8(+) T cells of the ITP patients, IDO expressions were significantly lower than those in healthy controls, but in CD19(+) and CD14(+) cells, IDO expression significantly increased. Conversely, TTS expressions in CD4(+) and CD8(+) T cells of the ITP patients were significantly higher than those in healthy controls, but there was no difference either in CD19(+) or CD14(+) cells. CONCLUSION: These results suggest that the activity of IDO enzyme is insufficient in ITP patients. Increased TTS expressions from CD4(+) and CD8(+) T cells might link to a pathogenic mechanism involved in increasing survival of autoreactive T cells in ITP patients.

Different angiogenesis effect of mini-TyrRS/mini-TrpRS by systemic administration of modified siRNAs in rats with acute myocardial infarction.

We aimed to clarify the different angiogenesis effects of mini-tyrosyl-tRNA synthetase (TyrRS)/minitryptophanyl-tRNA synthetase (TrpRS) in rodent primates with acute myocardial infarction, by delivering small interfering RNAs (siRNAs) systemically in a liposomal formulation. Left coronary artery ligation was used to establish the model of acute myocardial infarction in rats; mini-TyrRS/mini-TrpRS-specific siRNAs were encapsulated in stable nucleic acid lipid particles (SNALP), and administered by intravenous injection to rats. Rats were divided into four experiment groups: sham operated group (no left anterior descending artery [LAD] occlusion); negative control group (LAD occlusion + saline injection); mock transfection group (LAD occlusion + mock transfected injection); experiment group (LAD occlusion + mini-TyrRS/mini-TrpRS-specific siRNAs injection). Silencing efficiency was assayed by Western blotting. To determine whether mini-TyrRS/mini-TrpRS affected the angiogenesis activity of rats with myocardial infarction, we measured the myocardial infarction size by TTC staining, and the capillary density using immunohistochemistry staining, to investigate the expression of factor VIII. The myocardial infarction size and the capillary density of mini-TyrRS-siRNA group were respectively 18.89% and 8.64/0.1 mm(2) 1 month after ligation, while in the mini-TrpRS-siRNA group these values were 7.33% and 17.32/0.1 mm(2), significantly different compared with the mock transfection group (14.19%; 13.56/0.1 mm(2)) and negative control group (14.28%; 13.89/0.1 mm(2)), P < 0.05. There were no significant changes between the mock transfection group and the negative control group, P > 0.05. These results indicated that angiogenesis is either stimulated by mini-TyrRS or inhibited by mini-TrpRS in rat models with acute myocardial infarction.

Regulation of indoleamine 2,3-dioxygenase and tryptophanyl-tRNA-synthetase by CTLA-4-Fc in human CD4+ T cells.

Indoleamine-2,3-dioxygenase (IDO) and tryptophanyl-tRNA-synthetase (TTS) are interferon-gamma (IFN-gamma)-inducible enzymes that are responsible for tryptophan degradation and for its use in protein synthesis, respectively. IFN-gamma-induced IDO has immunomodulatory properties in murine and human models. A concomitant increase of TTS has been postulated to protect the IDO-expressing cells from tryptophan catabolism. IDO can be induced in dendritic cells (DCs) by recombinant soluble cytotoxic T lymphocyte antigen-4 (CTLA-4-Fc). We investigated the effects of CTLA-4-Fc on IDO and TTS mRNA expression in human peripheral blood mononuclear cells (PBMCs) and isolated leukocyte subsets. CTLA-4-Fc exposure induced increased IDO and TTS expression in unseparated PBMCs, as well as in monocyte-derived mature DCs. CD4(+) T cells isolated from CTLA-4-Fc-treated PBMCs showed increased IDO and TTS compared with untreated cells. CD8(+) T cells from CTLA-4-Fc-treated PBMCs expressed increased levels of TTS but not IDO. Pretreatment of PBMCs with CTLA-4-Fc inhibited the activation of CD4(+) T cells induced by influenza A virus (Flu) or phytohemagglutinin A (PHA), but had no effect on CD8(+) T cells. This is the first report of IDO and TTS regulation by the CTLA-4-B7 system in human CD4(+) and CD8(+) T cells, and raises the possibility that these 2 tryptophan-modulating enzymes provide an important mechanism for regulating immune responses.

Escherichia coli tryptophanyl-tRNA synthetase mutants selected for tryptophan auxotrophy implicate the dimer interface in optimizing amino acid binding.

Tryptophan auxotrophs of Escherichia coli in which mutations were mapped to the trpS locus (encoding tryptophanyl-tRNA synthetase) have been previously isolated. We have investigated the tryptophanyl-tRNA synthetase (TrpRS) purified from six auxotrophic strains for changes in amino acid activation and aminoacylation. Steady-state kinetic analyses show that these mutant TrpRS proteins have increases in the apparent KM for tryptophan, decreases in turnover number, or both, without significant changes in the apparent KM for ATP or tRNA(Trp). The crystal structure of a highly homologous tryptophanyl-tRNA synthetase from Bacillus stearothermophilus in a complex with the cognate aminoacyl adenylate allowed us to place the mutations in a structural context. The mutations in the enzymes are located in the KMSKS loop (M196I), in or near the active site (D112E, P129S, A133E) or far from the active site. The last three mutants (T60R, L91F, G329S) could not be predicted by examination of the protein structure as they line an interface between the C-terminal alpha-helix of one subunit and the Rossmann folds of both subunits, thus affecting a specific region of the dimer interface. These results support a role for dimerization in properly configuring the two active sites of the dimeric enzymes in the Trp/Tyr subclass of class I aminoacyl-tRNA synthetases in order to achieve optimal catalysis.

Differential regulation of the human, interferon inducible tryptophanyl-tRNA synthetase by various cytokines in cell lines.

Cytokines regulate the expression of specific sets of proteins which mediate their biological effects. We have comprehensively delineated the regulation of the human tryptophanyl-tRNA synthetase (hWRS) by eight different cytokines (including IFNs) and poly(I).poly(C) in several cell lines. Six non-lymphoid cell lines were tested, and all of these produced human, IFN inducible hWRS (gamma 2) mRNA upon stimulation with IFN-gamma. In all these cell lines the level of gamma 2 mRNA increased 2-4 h after induction reaching a stable plateau after 8-12 h. The IFN-gamma induction of gamma 2 mRNA could be blocked by cycloheximide in human amniotic (AMA) cells, epithelial HeLa cells and HT1080 fibroblasts, but not in T98G glioblastoma cells. IFN-alpha and poly(I).poly(C) elicited small, transient gamma 2 responses in a few of the non-lymphoid cell lines, whereas none of the other six cytokines tested elicited a response. The six lymphoid cell lines tested did not show the same induction pattern. In the monocytic cells, THP-1, gamma 2 mRNA was highly induced by IFN-gamma, whereas in the B-cell line, Daudi, gamma 2 mRNA was transiently induced by IFN-alpha and poly(I).poly(C), and not by IFN-gamma. Altered mRNA turnover rate as a consequence of IFN-gamma treatment did not appear to play a significant role in the accumulation of gamma 2 transcript, since the stability essentially was the same in induced versus non-induced cells. We conclude that the hWRS gene is induced preferentially by IFN-gamma, and that the induction pattern resembles the one reported for the IFN induced enzyme, indoleamine 2,3-dioxygenase (IDO).

The role of indoleamine 2,3-dioxygenase in the anti-tumour activity of human interferon-gamma in vivo.

We have studied the relationship between L-tryptophan metabolism and the response to human IFN-gamma in 3 human ovarian cancer xenografts growing in nude mice. During IFN-gamma therapy all 3 tumours showed a profound depletion in L-tryptophan and a corresponding rise in L-kynurenine. The microenvironment surrounding the tumours was also depleted of L-tryptophan. The IFN-gamma-inducible enzyme indoleamine dioxygenase, IDO, was induced in treated tumours. While there was a variability in IDO mRNA expression in the different xenografts tested, in situ hybridization showed that the gene was induced at all levels of the tumour, and not just the periphery. These results show that induction of IDO by IFN-gamma in vivo can metabolize L-tryptophan rapidly enough for it to become depleted, despite a continued supply of L-tryptophan from the host. The IDO mRNA and protein remained induced after the L-tryptophan levels had returned to normal, suggesting that the gene may be post-transcriptionally regulated and/or the IDO co-factor supply may be limited. Another IFN-gamma-inducible gene, tryptophanyl tRNA synthetase, was also induced in the tumour. It is possible that this enzyme, which is responsible for synthesizing tryptophanyl tRNA, acts in a compensatory manner by allowing protein synthesis to continue despite low free L-tryptophan concentrations. There was no correlation of the above parameters with the anti-tumour response to IFN-gamma, suggesting that other mechanisms must play a role. L-tryptophan depletion may be a contributor to a multifactorial growth inhibition of tumour cells following IFN-gamma treatment, but cannot on its own explain their growth inhibition.

Transcriptional regulation of the interferon-gamma-inducible tryptophanyl-tRNA synthetase includes alternative splicing.

We have investigated the transcriptional control elements of the human interferon (IFN)-gamma-induced tryptophanyl-tRNA synthetase (hWRS) gene and characterized the transcripts. Transcription leads to a series of mRNAs with different combinations of the first exons. The full-length mRNA codes for a 55-kDa protein (hWRS), but a mRNA lacking exon II is present in almost as high amounts as the full-length transcript. This alternatively spliced mRNA is probably translated into a 48-kDa protein starting from Met48 in exon III. The predicted 48-kDa protein corresponds exactly to an IFN-gamma-inducible protein previously detected by two-dimensional gel electrophoresis. By isolation of genomic clones and construction of plasmids containing hWRS promoter fragments fused to the secreted alkaline phosphatase reporter gene we have mapped a promoter region essential for IFN-mediated gene activation. This region contains IFN-stimulated response elements (ISRE) as well as a Y-box and a gamma-activated sequence (GAS) element. IFN-gamma inducibility of hWRS depends on ongoing protein synthesis, suggesting that so far undescribed transcription factors apart from the latent GAS-binding protein p91 contribute to gene activation. This could be interferon-regulatory factor-1, which binds ISRE elements.

Effects of cytokines on the gamma interferon-induced tryptophanyl-tRNA synthetase expression by human keratinocytes.

Incubation of human keratinocytes with gamma interferon (gamma-IFN) has been shown to potently induce the synthesis of a 53 kDa protein which was recently identified as tryptophanyl-tRNA synthetase (TRS). However, in spite of the high sensitivity of cultured keratinocytes to TRS induction by gamma-IFN, the study of inflammatory skin lesions has allowed the detection of the protein only in a few cases, suggesting regulatory mechanisms from soluble endogenous mediators with antagonistic activity on the induction of TRS by gamma-IFN. Among these mediators, we wondered whether cytokines selected for possible anti-inflammatory activity and potentially derived from activated resident skin cells, such as IL-4, IL-10, TNF-alpha and TGF-beta, may be involved in the modulation of the keratinocyte TRS expression. To assess this possibility, we investigated the modulation of the synthesis of TRS by human cultured keratinocytes upon stimulation by various gamma-IFN/cytokine combinations. The effects were evaluated by immunoblotting assay revealed by enhanced chemiluminescence, with the aid of a specific antibody to the TRS protein. Results failed to demonstrate any effect of the tested cytokines, whether on the basal level of the TRS, or on the gamma-IFN-induced enzyme expression in keratinocytes. It is thus unlikely that such cytokines can account for the infrequency of the TRS detection in inflammatory skin processes. Further investigations of alternative working hypothesis should help elucidate the regulation of TRS in human keratinocytes.

A novel interferon-alpha-regulated, DNA-binding protein participates in the regulation of the IFP53/tryptophanyl-tRNA synthetase gene.

We have investigated the transcriptional response of the IFP53/tryptophanyl-tRNA synthetase gene to interferon-alpha (IFN-alpha). A single gamma-interferon activation site (GAS) in proximity to the transcription start sites was found to mediate the response of the IFP53 gene to IFN-alpha. This DNA element bound two distinct protein factors, alpha-interferon activation factor 1 (AAF1) and AAF2, which were rapidly activated in the cytoplasm of IFN-alpha-treated HeLa cells. AAF1, like the gamma-interferon activation factor, bound to the GAS from different IFN-responsive promoters and contained the 91-kDa ISGF3 protein (p91). However, in complexes with the IFP53 or Ly6A/E GAS, p91 was the only ISGF3 protein, whereas in the case of the GBP GAS, the 48-kDa protein (p48) was also present. AAF2 was found to preferentially bind to the IFP53 GAS, but not at all to the GBP GAS, and contained no ISGF3 protein. Therefore, GAS-binding regulatory factors in the IFN-alpha response can either consist of proteins found in ISGF3 or be formed by distinct proteins that are similarly linked to IFN-alpha-induced signal transduction.

Gamma interferon potently induces tryptophanyl-tRNA synthetase expression in human keratinocytes.

Incubation of human keratinocytes with gamma interferon (gamma-IFN) induces the synthesis of a 53-kDa protein of unknown nature and function. We report the identification of this protein through amino acid microsequencing. The NH2-terminal amino acid sequence of the 53-kDa antigen demonstrated that this protein was tryptophanyl-tRNA synthetase (Frolova et al, Gene 109:291-296, 1991, Genbank accession number 61715). This result was validated by the sequencing of tryptic peptides. Identification of the 53-kDa gamma-IFN-induced protein was confirmed by immunoblotting with an antiserum directed against beef pancreas tryptophanyl-tRNA synthetase. Northern blot analysis using a synthetic oligonucleotidic 32P-labeled probe evidenced a 3.1-kb transcript in gamma-IFN-treated cells indicating that the gene was regulated at the pre-translational level. These data show that gamma-IFN potently induces in keratinocytes the expression of an enzyme directly involved in protein biosynthesis. Elevated levels of tryptophanyl-tRNA synthetase in treated cultured keratinocytes might be involved in the cell-growth-inhibitory activity of gamma interferon.

An interferon-induced protein with release factor activity is a tryptophanyl-tRNA synthetase.

Interferon gamma induces expression of a protein termed IFP 53 according to its molecular weight of 53 kDa. IFP 53 shows significant sequence homology to rabbit peptide chain release factor as well as to bovine tryptophanyl-tRNA synthetase. IFP 53 has been shown to possess release factor activity for the UGA stop codon. We demonstrate here, by using a recombinant IFP 53 fusion protein, that IFP 53 tryptophanylates tRNA. These data indicate that IFP 53 is a protein with two activities: peptide chain termination and aminoacylation.

Interferon induces tryptophanyl-tRNA synthetase expression in human fibroblasts.

A cDNA clone complementary to an interferon (IFN)-induced mRNA was isolated and used to characterize the regulation of expression of its RNA by the IFNs and to identify the protein its RNA encodes. This cDNA hybridizes to IFN-induced 3.1- and 2.3-kilobase mRNAs that are synthesized in response to both IFN-alpha and IFN-gamma. IFN-gamma induces the sustained accumulation of these mRNAs while IFN-alpha induces their transient accumulation. Cycloheximide (50 micrograms/ml) failed to inhibit the induction of these mRNAs by either IFN-alpha or IFN-gamma, suggesting that their induction does not require de novo protein synthesis. DNA sequence analysis of this cDNA reveals that it encodes a protein of Mr 53,168 that has sequence homology with and the biological activity of a tryptophanyl-tRNA synthetase, an enzymatic activity that has been demonstrated to play a role in and be modulated by the growth of cells. Elevated levels of this enzyme may be involved in the cell growth inhibitory activity of the IFNs.

Regulation of tryptophanyl-tRNA synthetase formation.

A previously constructed trp-S-lacZ fusion encoding a hybrid protein with beta-galactosidase activity was subcloned from a multicopy plasmid onto a lambda vector. Single-copy lysogens of lambda trpS-lacZ were used to determine whether trpS was regulated in a manner similar to that of other aminoacyl-tRNA synthetases. trpS regulation was found to resemble that of the majority of synthetases, in that expression of the lysogen-encoded hybrid beta-galactosidase varied with growth rate; beta-galactosidase activity increased 2.5-fold as the generation time decreased from 150 to 37 min. This regulatory response was confirmed by DNA/RNA hybridization experiments, which also suggested that this form of metabolic regulation occurred at the transcriptional level. No alteration in the level of hybrid beta-galactosidase was observed, however, when cells were starved for tryptophan.

Assignment of a human gene for tryptophanyl-tRNA synthetase to chromosome 14 using human-mouse somatic cell hybrids.

Human tryptophanyl-tRNA SYNTHETASE (Trp-RS, EC 6.1.1.2) can be separated from its mouse counterpart by Cellogel electrophoresis. Analysis of the presence or absence of human Trp-RS and other human enzyme markers in eleven independently dervied cell lines of human-mouse somatic cell hybrids revealed that the expression of Trp-RS is correlated with the expression of human nucleoside phosphorylase (NP, EC 2.4.2.1). The syntenic relationship between Trp-RS and NP permits the assignment of the structural gene for Trp-RS to human chromosome 14. Karyotype and isozyme analysis of these hybrid clones rules out other linkage assignments.