Defective antigen processing in GILT-free mice.

Processing of proteins for major histocompatibility complex (MHC) class II-restricted presentation to CD4-positive T lymphocytes occurs after they are internalized by antigen-presenting cells (APCs). Antigenic proteins frequently contain disulfide bonds, and their reduction in the endocytic pathway facilitates processing. In humans, a gamma interferon-inducible lysosomal thiol reductase (GILT) is constitutively present in late endocytic compartments of APCs. Here, we identified the mouse homolog of GILT and generated a GILT knockout mouse. GILT facilitated the processing and presentation to antigen-specific T cells of protein antigens containing disulfide bonds. The response to hen egg lysozyme, a model antigen with a compact structure containing four disulfide bonds, was examined in detail.

Gamma-interferon-inducible lysosomal thiol reductase (GILT). Maturation, activity, and mechanism of action.

We recently identified a gamma-interferon-inducible lysosomal thiol reductase (GILT), constitutively expressed in antigen-presenting cells, that catalyzes disulfide bond reduction both in vitro and in vivo and is optimally active at acidic pH. GILT is synthesized as a 35-kDa precursor, and following delivery to major histocompatibility complex (MHC) class II-containing compartments (MIICs), is processed to the mature 30-kDa form via cleavage of N- and C-terminal propeptides. The generation of MHC class II epitopes requires both protein denaturation and reduction of intra- and inter-chain disulfide bonds prior to proteolysis. GILT may be important in disulfide bond reduction of proteins delivered to MIICs and consequently in antigen processing. In this report we show that, like its mature form, precursor GILT reduces disulfide bonds with an acidic pH optimum, suggesting that it may also be involved in disulfide bond reduction in the endocytic pathway. We also show that processing of precursor GILT can be mediated by multiple lysosomal proteases and provide evidence that the mechanism of action of GILT resembles that of other thiol oxidoreductases.

Enzymatic reduction of disulfide bonds in lysosomes: characterization of a gamma-interferon-inducible lysosomal thiol reductase (GILT).

Proteins internalized into the endocytic pathway are usually degraded. Efficient proteolysis requires denaturation, induced by acidic conditions within lysosomes, and reduction of inter- and intrachain disulfide bonds. Cytosolic reduction is mediated enzymatically by thioredoxin, but the mechanism of lysosomal reduction is unknown. We describe here a lysosomal thiol reductase optimally active at low pH and capable of catalyzing disulfide bond reduction both in vivo and in vitro. The active site, determined by mutagenesis, consists of a pair of cysteine residues separated by two amino acids, similar to other enzymes of the thioredoxin family. The enzyme is a soluble glycoprotein that is synthesized as a precursor. After delivery into the endosomal/lysosomal system by the mannose 6-phosphate receptor, N- and C-terminal prosequences are removed. The enzyme is expressed constitutively in antigen-presenting cells and induced by IFN-gamma in other cell types, suggesting a potentially important role in antigen processing.

Ribavirin polarizes human T cell responses towards a Type 1 cytokine profile.

BACKGROUND/AIMS: The therapeutic benefit of ribavirin, a nucleoside analog, in the treatment of chronic HCV infection is seen even in the absence of any apparent direct antiviral effect. We surmised that ribavirin may act by eliciting altered virus-specific immune responses. Because antiviral immunity is predominantly mediated by cytotoxic T cells and antiviral cytokines, we sought to determine whether ribavirin could promote antiviral (Type 1) cytokine expression in human T cells. METHODS: Isolated human T cells were activated in vitro with enterotoxin B or with phorbol ester plus ionomycin. Cytokine ELISAs were performed on culture supernatants, cytokine mRNA was detected following RT-polymerase chain reaction of T cell RNA, and T cell proliferation measured using MTT assay. RESULTS: Ribavirin enhanced a Type 1 (IL-2, IFNgamma, TNFalpha) while suppressing a Type 2 cytokine response (IL-4, IL-5 and IL-10), at the level of both protein and mRNA expression. Ribavirin mediated comparable effects on cytokine expression both following activation of specific T cell subpopulations with superantigen and following activation of a larger percentage of T cells via pharmacologic means. The in vitro effect on cytokine expression following ribavirin treatment was comparable in both CD4+ or CD8+ T cell subsets and was observed in a dose range that promoted T cell proliferation. CONCLUSIONS: These data support the view that ribavirin promotes a Type 1 cytokine-mediated immune response, a property which may account in part for its ability to enhance the antiviral activity of interferon-alpha in the treatment of chronic HCV infection.

Oligonucleotide-mediated inhibition of CD28 expression induces human T cell hyporesponsiveness and manifests impaired contact hypersensitivity in mice.

Ligation of CD28 provides a costimulatory signal essential for Ag-mediated T cell activation via the TCR. Blocking CD28 ligation can inhibit cytokine expression and elicits a state of T cell hyporesponsiveness. In this study, we examined the effect of inhibiting CD28 expression on in vitro and in vivo T cell responses. To address this, we have synthesized a series of G-rich phosphorothioate oligonucleotides that inhibited activation-induced transcription and cell surface expression of CD28 on human T cells. CD28 blockade was selective, as expression of other activation-induced receptors was unaffected by oligonucleotide treatment. Using strategic changes to base composition, we identified a minimal 12-mer sequence, containing two sets of four contiguous guanosines separated by 3 to 5 bases, which conferred activity in vitro. Furthermore, inhibition of CD28 expression mediated by one representative active oligonucleotide, GR1, resulted in a concomitant dose-dependent diminution of anti-CD3/PMA-induced cytokine (IL-2, IFN-gamma, IL-8) production. Inhibition of IL-2 synthesis was dependent on CD28 expression, as GR1 failed to abrogate activated IL-2 production in a CD28-deficient T cell line, HUT 78. The inhibitory activity of GR1 reduced T cell proliferative responses in MLR and induced Ag-specific T cell hyporesponsiveness to alloantigens. Finally, s.c. administration of GR1 impaired in vivo contact hypersensitivity responses in mice and was associated with substantially decreased CD28 and IFN-gamma mRNA expression in lymph node cells. Collectively, our studies show the tolerogenic potential of oligonucleotide-mediated CD28 inhibition on T cell activation, in vitro and in vivo.