Point mutations in the α2 domain of HLA-A2.1 define a functionally relevant interaction with TAP

AbstractBackground: Glycoproteins encoded by the major histocompatibility complex class I region (MHC class I) present peptide antigens to cytotoxic T cells (CTLs). Peptides are delivered to the site of MHC class I assembly by the transporter associated with antigen processing (TAP), and cell lines that lack this transporter are unable to present endogenous antigens to CTLs. Although it has been shown that a fraction of newly synthesized class I molecules are in physical association with TAP, it is not known whether this interaction is functionally relevant, or where on the class I molecule the TAP binding site might be.Results C1R cells transfected with a mutant HLA-A2.1 heavy chain (HC), where threonine at position 134 in the α2 domain is changed to lysine (T134K), are unable to present endogenous antigens to CTLs. We have studied the biochemistry of this mutant in C1R cells, and found that a large pool of unstable empty class I HC–β2m (β-2 microglobulin) heterodimers exist that are rapidly transported to the cell surface. The T134K mutant seemed to bind peptide antigens and assemble with β2m as efficiently as wild-type HLA-A2.1. However, we show here that the inefficiency with which T134K presents intracellular antigen is associated with its inability to interact with the TAP heterodimer.Conclusion These experiments establish that the class I–TAP interaction is obligatory for the presentation of peptide epitopes delivered to the endoplasmic reticulum (ER) by TAP. Wild-type HLA-A2.1 molecules in TAP-deficient cells are retained in the ER, whereas T134K is rapidly released to the cell surface, but is unstable, suggesting a role for the TAP complex as an intracellular checkpoint that only affects the release of class I molecules with stably bound peptide ligands

A Single Residue Exchange Within a Viral CTL Epitope Alters Proteasome-Mediated Degradation Resulting in Lack of Antigen Presentation

AbstractCTL epitope (KSPWFTTL) encoded by AKV/MCF type of murine leukemia virus (MuLV) differs from the sequence in Friend/Moloney/Rauscher (FMR) type in one residue (RSPWFTTL). CTL experiments indicated defective processing of the FMR peptide in tumor cells. Proteasome-mediated digestion of AKV/MCF-type 26-mer peptides resulted in the early generation and higher levels of epitope-containing fragments than digestion of FMR-type peptides, explained by prominent cleavage next to R in the FMR sequence. The fragments were identified as 10- and 11-mer peptides and were efficiently translocated by TAP. The naturally presented AKV/MCF peptide is the 8-mer, indicating ER peptide trimming. In conclusion, a single residue exchange can cause CTL epitope destruction by specific proteasomal cleavage

Presentation of Cytosolic Glycosylated Peptides by Human Class I Major Histocompatibility Complex Molecules in Vivo

Antigens presented by class I major histocompatibility complex (MHC) molecules for recognition by cytotoxic T lymphocytes consist of 8–10-amino-acid-long cytosolic peptides. It is not known whether posttranslationally modified peptides are also presented by class I MHC molecules in vivo. Many different posttranslational modifications occur on cytoplasmic proteins, including a cytosolic O-β-linked glycosylation of serine and threonine residues with N-acetylglucosamine (GlcNAc). Using synthetic glycopeptides carrying the monosaccharide O-β-GlcNAc substitution on serine residues, we have shown that glycopeptides bind efficiently to class I MHC molecules and elicit a glycopeptide-specific cytotoxic T lymphocyte response in mice. In this study, we provide evidence that peptides presented by human class I MHC molecules in vivo encompass a small, significant amount of glycopeptides, constituting up to 0.1% of total peptide. Furthermore, we find that carbohydrate structures present on glycopeptides isolated from class I MHC molecules are dominated by the cytosolic O-β-GlcNAc substitution, and synthetic peptides carrying this substitution are efficiently transported by TAP (transporter associated with antigen presentation) into the endoplasmic reticulum. Thus, in addition to unmodified peptides, posttranslationally modified cytosolic peptides carrying O-β-linked GlcNAc can be presented by class I MHC molecules to the immune system

Murine Transporter Associated with Antigen Presentation (TAP) Preferences Influence Class I–restricted T Cell Responses

The transporter associated with antigen presentation (TAP) complex shuttles cytosolic peptides into the exocytic compartment for association with nascent major histocompatibility complex class I molecules. Biochemical studies of murine and human TAP have established that substrate length and COOH-terminal residue identity are strong determinants of transport efficiency. However, the existence of these specificities in the intact cell and their influences on T cell responses have not been demonstrated. We have devised a method for studying TAP- mediated transport in intact cells, using T cell activation as a readout. The approach makes use of a panel of recombinant vaccinia viruses expressing peptides containing the Kd-restricted nonamer influenza nucleoprotein residues 147–155. The COOH terminus of each construct was appended with a dipeptide composed of an internal threonine residue followed by a varying amino acid. Synthetic peptide versions of these 11-mers exhibit vastly different transport capabilities in streptolysin O–permeabilized cells, in accordance with the predicted influence of the COOH-terminal residues. Presentation of the endogenously expressed version of each construct requires TAP-mediated transport and cooexpression with a vac-encoded exocytic COOH-terminal dipeptidase, angiotensin converting enzyme, to allow liberation of the minimal epitope. Recognition by epitope-specific CTLs therefore signifies TAP-mediated transport of a complete 11-mer within the target cell. Under normal assay conditions no influences of the COOH-terminal residue were revealed. However, when T cell recognition was limited, either by blocking CD8 coreceptor interactions or by decreasing the amount of transport substrate synthesized, significant COOH-terminal effects were revealed. Under such conditions, those peptides that transported poorly in biochemical assays were less efficiently presented. Therefore, TAP specificity operates in the intact cell, appears to reflect previously defined rules with regard to the influence of the COOH-terminal residue, and can strongly influence T cell responses

HIV's evasion of the cellular immune response

Despite a strong cytotoxic T-lymphocyte (CTL) response directed against viral antigens, untreated individuals infected with the human immunodeficiency virus (HIV-1) develop AIDS, We have found that primary T cells infected with HIV-1 downregulate surface MHC class I antigens and are resistant to lysis by HLA-A2-restricted CTL clones. In contrast, cells infected with an HIV-1 in which the nef gene is disrupted are sensitive to CTLs in an MHC and peptide-specific manner. In primary T cells HLA-A2 antigens are downmodulated more dramatically than total MHC class I antigens, suggesting that nef selectively downmodulates certain MHC class I antigens. In support of this, studies on ceils expressing individual MHC class I alietes have revealed that nef does not downmodulate HLA-C and HLA-E antigens, This selective downmodulation allows Infected cells to maintain resistance to certain natural killer cells that lyse infected cells expressing low levels of MHC class I antigens. Downmodulation of MHC class I HLA-A2 antigens occurs not only in primary T cells, but also in B and astrocytoma cell lines. No effect of other HIV-1 accessory proteins such as vpu and vpr was observed. Thus Nef is a protein that may promote escape of HIV-1 from immune surveillance.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/75570/1/j.1600-065X.1999.tb01283.x.pd

Rhesus macaque MHC class I molecules show differential subcellular localizations

The MHC class I gene family of rhesus macaques is characterised by considerable gene duplications. While a HLA-C-orthologous gene is absent, the Mamu-A and in particular the Mamu-B genes have expanded, giving rise to plastic haplotypes with differential gene content. Although some of the rhesus macaque MHC class I genes are known to be associated with susceptibility/resistance to infectious diseases, the functional significance of duplicated Mamu-A and Mamu-B genes and the expression pattern of their encoded proteins are largely unknown. Here, we present data of the subcellular localization of AcGFP-tagged Mamu-A and Mamu-B molecules. We found strong cell surface and low intracellular expression for Mamu-A1, Mamu-A2 and Mamu-A3-encoded molecules as well as for Mamu-B*01704, Mamu-B*02101, Mamu-B*04801, Mamu-B*06002 and Mamu-B*13401. In contrast, weak cell surface and strong intracellular expression was seen for Mamu-A4*1403, Mamu-B*01202, Mamu-B*02804, Mamu-B*03002, Mamu-B*05704, Mamu-I*010201 and Mamu-I*0121. The different expression patterns were assigned to the antigen-binding α1 and α2 domains, suggesting failure of peptide binding is responsible for retaining ‘intracellular’ Mamu class I molecules in the endoplasmic reticulum. These findings indicate a diverse functional role of the duplicated rhesus macaque MHC class I genes

TAPBPR: a new player in the MHC class I presentation pathway.

In order to provide specificity for T cell responses against pathogens and tumours, major histocompatibility complex (MHC) class I molecules present high-affinity peptides at the cell surface to T cells. A key player for peptide loading is the MHC class I-dedicated chaperone tapasin. Recently we discovered a second MHC class I-dedicated chaperone, the tapasin-related protein TAPBPR. Here, we review the major steps in the MHC class I pathway and the TAPBPR data. We discuss the potential function of TAPBPR in the MHC class I pathway and the involvement of this previously uncharacterised protein in human health and disease.C.H was supported by a Wellcome Trust PhD Studentship (Grant 089563) and L.H.B was funded by a Wellcome Trust Career Development Fellowship (Grant 085038).This is the author accepted manuscript. The final published version is available via Wiley at http://onlinelibrary.wiley.com/doi/10.1111/tan.12538/abstract;jsessionid=3D6AF64F5BD8C64E84634A4303842BE2.f04t01