Defective antigen processing correlates with a low level of intracellular glutathione.

Previously, we reported that Chinese hamster ovary (CHO) cells transfected with murine mouse major histocompatibility complex class II genes, exhibit a unique antigen (Ag) processing defect whereby these cells are impaired in processing only Ag with disulfide bonds. Here, we examined various aspects of the intracellular reducing environment in the CHO cells to understand the underlying mechanism causing the defect. A cell hybrid generated by the fusion of CHO cells and L cell fibroblasts was used for comparison due to their competency in processing Ag. The transport pathway of cysteine within the CHO cells appeared normal. However, these cells had a significantly lower level of glutathione, a major physiological reducing thiol, compared to the cell hybrid. Treatment of the CHO cells with N-acetyl-L-cysteine did not augment their glutathione content nor their ability to process Ag. When the cell hybrid was treated with L-buthionine-(S,R)-sulfoximine (BSO), which significantly decreased their glutathione level, the hybrid poorly processed hen egg lysozyme (HEL) and ovalbumin, which have disulfide bonds. In contrast, BSO treatment did not affect the capacity of the hybrid to process pigeon cytochrome c and carboxymethylated HEL, which lack disulfide bonds. Therefore, low intracellular glutathione levels in antigen-presenting cells correlated with defective processing of Ag with disulfide bonds, indicating that this thiol may be a critical factor in regulating productive Ag processing.

Characterization of fibroblasts with a unique defect in processing antigens with disulfide bonds.

A chinese hamster ovary (CHO) fibroblast, transfected with murine MHC class II genes, inefficiently stimulated CD4+ Th cells specific for OVA, hen egg lysozyme (HEL), and pork insulin which contain disulfide bonds. However, the fibroblasts elicited a T cell response to lambda repressor, which lacks disulfide bonds, and efficiently presented synthetic peptides. A somatic cell hybrid WALC, generated by fusing the hamster fibroblast with a murine L cell fibroblast, very efficiently processed OVA and HEL, suggesting that impaired processing was genetically complemented and was a recessive trait. The hamster fibroblasts were capable of processing two distinct denatured forms of OVA and carboxymethylated HEL, either as effectively or more efficiently than the B lymphoma cell. The CHO cells also displayed diminished disulfide reduction of an endocytosed [125I]tyramine linked to poly-(D-lysine) through a disulfide spacer compared with that of the cell hybrid, providing direct evidence for defective reductive cleavage by the CHO cells. Diminished aspartic acid-mediated proteolysis of Ag could not account for the phenotype, because cell lysates and separated organelles from the fibroblast possessed higher acidic aspartyl proteolytic activity than lysates and organelles from a B lymphoma cell. Thus, CHO cells exhibit a defect in processing Ag with disulfide bonds which is consistent with the impaired intracellular reduction of the disulfide bonds in endocytosed macromolecules.

Differences among various lineages of antigen-presenting cells in processing exogenous antigen internalized through transferrin receptors.

The Ag, pigeon cytochrome c, was coupled to human ferric transferrin by a heteroligation technique to target Ag into the endosomal transport pathway via transferrin receptors. The ability of various types of APC that do or do not express transferrin receptors to process exogenous Ag in their endosomes was investigated by the stimulation of Ag-specific CD4+ T cells with the transferrin-Ag conjugate in a serum-free assay. When two B lymphoma cells were the source of APC, the conjugate was significantly more potent than native Ag in activating the T cells, agreeing with our previous finding using a third B lymphoma cell. The conjugate and Ag were similarly presented by splenic B cells that lack transferrin receptors to the T cells. However, both a macrophage hybridoma and a MHC class II-L cell transfectant hardly elicited a T cell response to the conjugate, although a response to native Ag was readily observed. These findings could not be attributed to an absence of transferrin receptors or receptor-mediated internalization of the conjugate, nor to differential expression of MHC class II molecules or li chain by the APC. The poor presentation of the conjugate by the L cell transfectants was associated with diminished catabolism of the conjugate, however, the macrophage hybridoma rapidly degraded the conjugate, similar to the B lymphoma cell. Peritoneal macrophages, which lack transferrin receptors, and the macrophage hybridoma induced a response to the conjugate only at concentrations that allowed internalization by fluid phase pinocytosis. The lower potency of the conjugate compared with native Ag with non-B-presenting cells suggest that these cell types process the conjugate by a different mechanism than used by B cells. Differences in the mechanism of Ag processing used by APC of distinct cell lineages may possibly influence immune responsiveness.