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

Abstract

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.

Re-evaluating the Generation of a “Proteasome-Independent” MHC Class I-Restricted CD8 T Cell Epitope

“…Because differences in presentation efficiency may be obscured by Ag overexpression (15), we conducted the same assay using a panel of vac recombinants designed to limit Ag expression in a regulated fashion. By inserting nucleotides predicted to form a thermostable duplex structure, or HP, between the promoter and start codon of the NP gene, the level of protein expression can be modulated and correlates inversely with the number of bases in the HP (15,37,(43)(44)(45). By using recombinant viruses with 14, 17, and 19 bp HP it was apparent that the presentation of NP 50 -57 could proceed in adapted cells and was only mildly compromised compared with normal cells (Fig.…”

Efficient and Qualitatively Distinct MHC Class I-Restricted Presentation of Antigen Targeted to the Endoplasmic Reticulum

“…We therefore placed the genes encoding NP and ER-NP 13-498 behind a series of sequences designed to create thermostable RNA duplexes or HP 16, 18, and 20 bp in length. These structures limit, in graded fashion, the biosynthesis of Ag and, consequently, the amount of epitope presented (28,41,42). The various HP constructs were recombined into the vac genome and the resulting viruses were tested for their ability to sensitize target cells for killing by T CD8ϩ specific for NP [147][148][149][150][151][152][153][154][155] , and two other well-defined epitopes within NP (56,57).…”

“…The Q to N substitutions inside the NP 147-155 epitopes of NP and ER-NP were conducted by one-step PCR using the downstream primer GAA CAA GGG CCC TTG TCC TGT TAT AAG TTG and upstream primer GGT AAG GAA GTA GAA TCA T, allowing the substitution of SalI/ApaI fragment. The genes were cloned into modified versions of the pSC11 plasmid, containing heteroduplexes (hairpins (HP)) of different lengths (16,18, and 20 bp) in the 5Ј untranslated region of the recombinant gene (28,41,42). Sequencing, using ␤-cyanoethyl phosphoramidities chemistry (Applied Biosystems, Foster City, CA) and conducted by the Kimmel Cancer Institute Nucleic Acid Facility, confirmed the integrity of each construct.…”

Generation of CD8+ T Cell Memory in Response to Low, High, and Excessive Levels of Epitope

“…We have developed a system to modulate Ag expression from a constant dose of rVV by inserting thermostable duplex barriers, or HP, of different sizes between the promoter and primary initiation codon of the Ag of interest (13,15,16). The larger the HP, the lower the level of Ag produced and cell-surface epitope expressed both in vitro and in vivo (13).…”