Major histocompatibility complex class I assembly within endolysosomal pathways.

Major histocompatibility complex class I (MHC class I) molecules facilitate subcellular immune surveillance by presenting peptides on the cell surface. MHC class I assembly with peptides generally happens in the endoplasmic reticulum (ER). Peptides are processed in the cytosol, transported into the ER, and assembled with MHC class I heavy and light chains. However, as many pathogens reside within multiple subcellular organelles, peptide sampling across non-cytosolic compartments is also important. MHC class I molecules internalize from the cell surface into endosomes and constitutively traffic between endosomes and the cell surface. Within endosomes, MHC class I molecules assemble with both exogenous and endogenous antigens processed within these compartments. Human MHC classI polymorphisms, well known to affect ER assembly modes, also influence endosomal assembly outcomes, an area of current interest to the field.

Endo-lysosomal assembly variations among human leukocyte antigen class I (HLA class I) allotypes.

The extreme polymorphisms of human leukocyte antigen class I (HLA class I) proteins enable the presentation of diverse peptides to cytotoxic T lymphocytes. The canonical endoplasmic reticulum (ER) HLA class I assembly pathway enables presentation of cytosolic peptides, but effective intracellular surveillance requires multi-compartmental antigen sampling. Endo-lysosomes are generally sites of HLA class II assembly, but human monocytes and monocyte-derived dendritic cells (moDCs) also contain significant reserves of endo-lysosomal HLA class I molecules. We hypothesized variable influences of HLA class I polymorphisms upon outcomes of endo-lysosomal trafficking, as the stabilities and peptide occupancies of cell surface HLA class I molecules are variable. Consistent with this model, when the endo-lysosomal pH of moDCs is disrupted, HLA-B allotypes display varying propensities for reductions in surface expression, with HLA-B*08:01 or HLA-B*35:01 being among the most resistant or sensitive, respectively, among eight tested HLA-B allotypes. Perturbations of moDC endo-lysosomal pH result in accumulation of HLA-B*35:01 in LAMP1+ compartments and increase HLA-B*35:01 peptide receptivity. These findings reveal the intersection of the vacuolar cross-presentation pathway with a constitutive assembly pathway for some HLA-B allotypes. Notably, cross-presentation of epitopes derived from two soluble antigens was also more efficient for B*35:01 compared to B*08:01, even when matched for T cell response sensitivity, and more affected by cathepsin inhibition. Thus, HLA class I polymorphisms dictate the degree of endo-lysosomal assembly, which can supplement ER assembly for constitutive HLA class I expression and increase the efficiency of cross-presentation.

Concanamycin A counteracts HIV-1 Nef to enhance immune clearance of infected primary cells by cytotoxic T lymphocytes.

Nef is an HIV-encoded accessory protein that enhances pathogenicity by down-regulating major histocompatibility class I (MHC-I) expression to evade killing by cytotoxic T lymphocytes (CTLs). A potent Nef inhibitor that restores MHC-I is needed to promote immune-mediated clearance of HIV-infected cells. We discovered that the plecomacrolide family of natural products restored MHC-I to the surface of Nef-expressing primary cells with variable potency. Concanamycin A (CMA) counteracted Nef at subnanomolar concentrations that did not interfere with lysosomal acidification or degradation and were nontoxic in primary cell cultures. CMA specifically reversed Nef-mediated down-regulation of MHC-I, but not CD4, and cells treated with CMA showed reduced formation of the Nef:MHC-I:AP-1 complex required for MHC-I down-regulation. CMA restored expression of diverse allotypes of MHC-I in Nef-expressing cells and inhibited Nef alleles from divergent clades of HIV and simian immunodeficiency virus, including from primary patient isolates. Lastly, we found that restoration of MHC-I in HIV-infected cells was accompanied by enhanced CTL-mediated clearance of infected cells comparable to genetic deletion of Nef. Thus, we propose CMA as a lead compound for therapeutic inhibition of Nef to enhance immune-mediated clearance of HIV-infected cells.

Polymorphisms of HLA-B: influences on assembly and immunity.

The major histocompatibility class I (MHC-I) complex functions in innate and adaptive immunity, mediating surveillance of the subcellular environment. In humans, MHC-I heavy chains are encoded by three genes: the human leukocyte antigen (HLA)-A, HLA-B, and HLA-C. These genes are highly polymorphic, which results in the expression, typically, of six different HLA class I (HLA-I) proteins on the cell surface, and the presentation of diverse peptide antigens to CD8+ T cells for broad surveillance against many pathogenic conditions. Recent studies of HLA-B allotypes show that the polymorphisms, not surprisingly, also significantly impact protein folding and assembly pathways. The use of non-canonical assembly routes and the generation of non-canonical HLA-B conformers has consequences for immune receptor interactions and disease therapies.

Variations in HLA-B cell surface expression, half-life and extracellular antigen receptivity.

The highly polymorphic human leukocyte antigen (HLA) class I molecules present peptide antigens to CD8+ T cells, inducing immunity against infections and cancers. Quality control mediated by peptide loading complex (PLC) components is expected to ensure the cell surface expression of stable peptide-HLA class I complexes. This is exemplified by HLA-B*08:01 in primary human lymphocytes, with both expression level and half-life at the high end of the measured HLA-B expression and stability hierarchies. Conversely, low expression on lymphocytes is measured for three HLA-B allotypes that bind peptides with proline at position 2, which are disfavored by the transporter associated with antigen processing. Surprisingly, these lymphocyte-specific expression and stability differences become reversed or altered in monocytes, which display larger intracellular pools of HLA class I than lymphocytes. Together, the findings indicate that allele and cell-dependent variations in antigen acquisition pathways influence HLA-B surface expression levels, half-lives and receptivity to exogenous antigens.