Deficient Peptide Loading and MHC Class II Endosomal Sorting in a Human Genetic Immunodeficiency Disease: the Chediak-Higashi Syndrome

The Chediak-Higashi syndrome (CHS) is a human recessive autosomal disease caused by mutations in a single gene encoding a protein of unknown function, called lysosomal-trafficking regulator. All cells in CHS patients bear enlarged lysosomes. In addition, T- and natural killer cell cytotoxicity is defective in these patients, causing severe immunodeficiencies. We have analyzed major histocompatibility complex class II functions and intracellular transport in Epstein Barr Virus–transformed B cells from CHS patients. Peptide loading onto major histocompatibility complex class II molecules and antigen presentation are strongly delayed these cells. A detailed electron microscopy analysis of endocytic compartments revealed that only lysosomal multilaminar compartments are enlarged (reaching 1–2 μm), whereas late multivesicular endosomes have normal size and morphology. In contrast to giant multilaminar compartments that bear most of the usual lysosomal markers in these cells (HLA-DR, HLA-DM, Lamp-1, CD63, etc.), multivesicular late endosomes displayed reduced levels of all these molecules, suggesting a defect in transport from the trans-Golgi network and/or early endosomes into late multivesicular endosomes. Further insight into a possible mechanism of this transport defect came from immunolocalizing the lysosomal trafficking regulator protein, as antibodies directed to a peptide from its COOH terminal domain decorated punctated structures partially aligned along microtubules. These results suggest that the product of the Lyst gene is required for sorting endosomal resident proteins into late multivesicular endosomes by a mechanism involving microtubules.Major histocompatibility complex (MHC)¹ class II molecules are composed of an αβ dimer that associates in the ER with a third membrane molecule, the invariant chain (Ii; 33, 24). The αβ−Ii chain complexes are transported via the Golgi apparatus to the endocytic pathway, directed by a signal localized in the cytoplasmic tail of Ii chain (7, 41). Ii chain is then degraded (12), and upon complete removal of the remaining Ii fragments (60), antigenic peptides are loaded onto class II molecules under the control of HLA-DM (65, 22).Ii chain cleavage and antigen processing to fitting peptides occurs in endosomal and/or lysosomal compartments (24). Depending on the species origin of the cell, cell types, or even on the maturation status in the case of dendritic cells, accumulation of MHC class II molecules may occur in different endocytic compartments (43, 51). In human Epstein Barr virus–transformed B (EBV-B) cells, HLA-DR molecules accumulate in lysosomal compartments named MHC class II compartments (MIICs; 49). In murine splenic lipopolysaccharide-activated B cells (18) as well as in macrophages and human melanoma cells (30, 52), MHC class II is found all along the endocytic pathway, from early endosomes to lysosomes. In contrast, A20 murine B lymphoma cells accumulate MHC class II molecules in endosomal compartments, the class II vesicles (2, 4), whereas few class II molecules are found in conventional endosomes and lysosomes. However, upon inhibition of Ii chain degradation, class II molecules redistribute into lysosomal compartments (14).Recent results from the laboratory of H. Geuze (50, 35) showed that the distribution of MHC class II molecules in EBV-B cells is not as restricted as initially envisioned. Indeed, HLA-DR accumulates in two types of compartments: (a) in endosomes containing multiple internal vesicles that are reached by fluid phase markers after 20–30 min of internalization and contain some Ii chain (multivesicular late endosomes); and (b) in vesicles containing internal membranes organized in onion-like structures that accumulate fluid phase markers only after 60 min and contain no Ii chain (multilaminar lysosomal compartments). Both types of compartments also contain Lamp1/2, CD63, and HLA-DM.The functional relevance of this heterogeneity of endocytic MHC class II–containing compartments is still unclear, and the precise role of multivesicular and multilaminar endosomes in MHC class II transport and Ii chain degradation is not known. Moreover, it has recently been shown that the antigenic peptides generated in endosomal and lysosomal compartments might not be the same (30). In addition, we have recently shown that antigen internalization through different membrane receptors that may deliver antigens to particular endocytic compartments results in presentation of different antigenic peptides (3).To evaluate the role of this heterogeneity of endocytic compartments in MHC class II transport and function, we examined EBV-B cells of patients suffering from a rare genetic immunodeficiency disease, the Chediak-Higashi Syndrome (CHS), which affects the morphology and function of endocytic compartments. CHS results from mutations in a gene encoding a large cytosolic protein called lysosomal trafficking regulator (LYST), which displays limited sequence homology to a regulatory subunit of the yeast phosphatidyl-inositol-3 kinase (PI3K), VPS15 (9, 45). LYST also includes several WD40 and HEAT/ARM domains, a domain of limited homology to stathmin, as well as a unique domain that has been called BEACH (9, 8, 10, 45).Despite having identified several subdomains in the CHS protein, the precise function of the protein is not known. We do know, however, that mutations in this gene result in immunological disorders and susceptibility to multiple childhood infections. The lysosomal compartments in all cell types of CHS patients are enlarged, reaching over 1 μm/vesicle (70). In hematopoietic cells, including T lymphocytes, NK cells, and granulocytes, cytotoxicity is defective, most likely because of a defect in regulated secretion (61, 29, 6). In nonhematopoietic cells such as melanocytes and kidney cells, enlarged lysosomal morphology and defects in lysosomal enzyme secretion have been reported (15). It is yet unclear whether the defect in the secretory function of lysosomes in hematopoietic cells is a consequence or a cause of the abnormal lysosomal morphology. It is also possible that both phenotypes arise from a unique upstream defect in the endocytic pathway.Here we show that antigen presentation and MHC class II intracellular transport are affected in EBV-B cells from CHS patients. Surprisingly, only lysosomal multilaminar MHC class II–containing compartments are enlarged, while multivesicular late endosomes displayed normal size and morphology. However, a severe reduction in the staining of multivesicular endosomes for MHC class II, Lamp 1/2, CD63, CD82, and β-hexosaminidase was observed, suggesting that transport of these markers from the TGN and/or early endosomes into late endosomes is affected. Missorting of resident lysosomal proteins to the plasma membrane and early endosomes was also observed, as well as a striking redistribution of the cation-dependent mannose-6-phosphate receptor (CD-MPR) into giant multilaminar lysosomes. In addition, we showed that LYST partially colocalizes with microtubules, which have previously been shown to play a critical role in transport from early to late endosomes (19). Together, these results show severe missorting of membrane proteins along the endocytic pathway of CHS cells, and suggest that LYST may be directly involved in microtubule-dependent transport into late endocytic compartments.