CLASS-II-RESTRICTED PRESENTATION OF A HEN EGG LYSOZYME DETERMINANT DERIVED FROM ENDOGENOUS ANTIGEN SEQUESTERED IN THE CYTOPLASM OR ENDOPLASMIC-RETICULUM OF THE ANTIGEN PRESENTING CELLS

A cDNA encoding a form of hen egg lysozyme (HEL) lacking a leader sequ ence and predicted to be localized in the cytoplasm, was transfected i nto MHC class II-positive B lymphomacells. Cytoplasmically expressed H EL (cytHEL) had a half-life of less than 5 min and did not react with HEL specific mAb suggesting non-native conformation. Cells expressing cytoplasmic HEL, as well as cells previously reported to express a low level of HEL retained in the endoplasmic reticulum (ERHEL), constitut ively presented the HEL determinant encoded by residues 46-61 to a sen sitive class II-restricted T hybridoma (3A9). Constitutive presentatio n of HEL determinants was not detectable in cytHEL or ERHEL transfecta nts using T hybridomas with lower sensitivity to exogenous Ag. Constit utive presentation of HEL46-61 derived from cytoplasmic HEL was demons trable in multiple transfected clones and was most obvious when a CMV rather than SV40 promoter was used to express the cytHEL gene. The pre sentation of HEL46-61 by cytHEL transfectants was notdue to HEL reupta ke by bystander cells because there was no biochemical evidence of cyt HEL shedding and cytHEL supernatants added to indicator APC did not re sult in HEL46-61 presentation. Constitutive presentation of endogenous HEL46-61 by the cytHEL and ERHEL transfectants was inhibited by chlor oquine, and recovery of presentation of endogenous HEL was slower in c ytHEL compared with ERHEL transfectants. The findings indicate that cl ass II-restricted presentation of Ag retained in the cytoplasm or endo plasmic reticulum does take place but probably requires abundant level s of intracellular Ag and is easily disrupted by lysosomotropic agents . These pathways of presentation may be important when high levels of foreign endoplasmic reticulum-retained or cytoplasmic Ag are present ( e.g., viral infection), and during the acquisition of self-tolerance b y highly sensitive developing T cells.