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.
SummaryImmature and mature B cells differ in the signals generated and transduced through their antigen receptor, surface immunoglobulin M (slgM). Whereas signals generated through slgM on mature B cells initiate a program leading to the positive activation of these cells, signaling through this receptor at the immature stage of development leads to a state of induced unresponsiveness or tolerance. Our previous studies have described developmental differences in slgM transmembrane signaling that are independent of ligand-receptor affinity. In an attempt to understand the molecular basis for signaling differences between immature and mature B cells, we have analyzed the slgM receptor complex in neonatal and adult mouse splenic B cells. While previously described components of this complex do not exhibit marked developmentally regulated differences in their association with slgM, we have identified a 56-kD protein that associates with slgM in mature (antigenresponsive), but not immature (tolerance-sensitive) B cells. This protein (p56) associates with slgM as a homodimer, is constitutively phosphorylated on tyrosine, and is coimmunoprecipitated with IgM but not IgD. The observed inability to iodinate p56 suggests it is an intracellular component of the receptor complex. Based upon its migration in one-and two-dimensional gel electrophoresis we show, however, that p56 is distinct from the blk, lyn, or fyn src family kinases that have been shown to be associated with slgM in mature B cells. The developmentally regulated participation of p56 in the B cell antigen receptor complex suggests a role in the differential signaling mediated via slgM on immature and mature B cells. (7), while all populations of immature B cells manifest the transmembrane signaling defect. Indeed, this membrane-associated signaling difference appears to be the primary mechanism accounting for the unresponsiveness of neonatal splenic B cells to anti-/~ antibody stimulation (5).
CD8+ T cells (T(CD8+)) recognize viral Ags as short peptides (epitopes) displayed at the cell surface by MHC class I molecules. Using a panel of recombinant vaccinia viruses, we show that single-point mutations flanking either side of an H-2Kd-restricted epitope, residues 147-155, within full-length influenza nucleoprotein (NP) can impact, even ablate, presentation of that epitope, while having no effect on presentation of distal epitopes. The most severe blocking mutation (Ala to Pro at position 146) did not inhibit NP(147-155) presentation in the context of a truncated minigene, implying that this peptide is not a functional processing intermediate. An amino-terminal proline replacement also significantly reduced presentation of NP(50-57) (H-2Kk restricted), while the same mutation did not affect a third NP epitope. Thus, while trends in processing specificity may exist, the epitope itself contributes to flanking sequence effects. These findings were paralleled by in vivo priming experiments in which, depending on viral dose, subtle in vitro blocking effects were absolute. Proteasome/synthetic peptide coincubation studies support a role for enhanced epitope destruction in preventing presentation, as did the effect of the peptide aldehyde, LLnL, which restored presentation of NP(147-155) from the mutated constructs. This reagent did not inhibit epitope presentation, even from wild-type NP, suggesting that its production may be proteasome independent. These results support the notion that point mutation of epitope flanking sequence can serve as a mechanism for viral immune evasion, shed light on the mechanisms involved, and suggest that in vitro assays may not be sensitive indicators of flanking sequence effects.
Influenza A/Puerto Rico/8/34 nucleoprotein (NP) contains an H-2Kd-restricted CD8+ T cell (T CD8+) epitope spanning amino acid residues 147-155. It was previously demonstrated that expression of NP147-155 and NP147-158 in isolation via "minigene"/recombinant vaccinia virus (vac) technology leads to sensitization of target cells for NP-specific killing while expression of 147-158 lacking the arginine at position 156 (termed here as 147-155TG) does not. The presentation block was overcome by placing this fragment into the context of full length NP. We show that addition of a single amino acid, Met159, to the C terminus of the blocked peptide (creating 147-155TGM) restores presentation. Presentation of 147-155TGM was not due to trimming in the exocytic compartment, consistent with severe limitations on C-terminal trimming activity in this location. Rescued presentation was also achieved when the blocked construct was extended in the N-terminal direction only, but in this case more than 55 amino acids of flanking sequence were required. The transition to presentation was abrupt, with 91-155TG and shorter constructs showing little or no detectable presentation and 90-155TG showing full level presentation. Presentation could not be attributed to acquisition of conventional targets for ubiquitination since mutation of all Lys residues, to which the ubiquitin moiety is conjugated, does not abrogate presentation. Rescued presentation was not inhibited by the peptide aldehyde N-acetyl-L-leucinyl-L-leucinal-L-norleucinal, suggesting that the added elements may be recruiting nonproteasomal activity. We have therefore identified and begun to characterize protease targeting of regulatory elements, both local and distal to an epitope, which strongly influence the ability of the epitope to be excised.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.