Allele-dependent Processing Pathways Generate the Endogenous Human Leukocyte Antigen (HLA) Class I Peptide Repertoire in Transporters Associated with Antigen Processing (TAP)-deficient Cells

Abstract

The transporters associated with antigen processing (TAP) allow the supply of peptides derived from the cytosol to translocate to the endoplasmic reticulum, where they complex with nascent human leukocyte antigen (HLA) class I molecules. However, infected and tumor cells with TAP molecules blocked or individuals with nonfunctional TAP complexes are able to present HLA class I ligands generated by TAP-independent processing pathways. These peptides are detected by the CD8 ؉ lymphocyte cellular response. Here, the generation of the overall peptide repertoire associated with four different HLA class I molecules in TAP-deficient cells was studied. Using different protease inhibitors, four different proteolytic specificities were identified. These data demonstrate the different allele-dependent complex processing pathways involved in the generation of the HLA class I peptide repertoire in TAP-deficient cells.The newly synthesized proteome is sampled continuously by CD8 ϩ lymphocytes as short peptides presented by human leukocyte antigen (HLA) class I molecules at the cell surface. The majority of the peptides presented by HLA class I molecules are produced from proteolysis by the proteasome and other cytosolic proteases, such as tripeptidyl peptidase II (1-3), puromycin-sensitive aminopeptidase (4), insulin-degrading enzyme (5), thimet oligopeptidase (6), and caspases (7,8). These peptides are transported into the endoplasmic reticulum (ER) 2 by TAP, with subsequent N-terminal trimming by the metalloaminoproteases ERAP1 and 2 frequently being required (9, 10). Peptide binding to nascent HLA class I molecules generates stable peptide-HLA complexes that are exported to the cell membrane where are they exposed to cytotoxic CD8 ϩ T lymphocyte recognition (for review, see Ref. 11). TAP Ϫ/Ϫ humans (12) and mice (13) have a reduced functional CD8 ϩ population but do not appear to have an increased susceptibility to neoplasms or viral infections. Thus, the TAPindependent pathways may be sufficient to control these diseases and allow individuals with this HLA class I deficiency to live normal life spans with only a limited susceptibility to chronic respiratory bacterial infections. In addition, evidence for TAP-independent pathways of antigen presentation by MHC class I molecules of particular but diverse pathogenic epitopes was reported previously (for review, see Ref. 14 -16). The identified proteases involved in the generation of specific ligands in TAP-deficient cells include ER signal peptidase (SPase) (17, 18), ER signal peptide peptidase (SPPase) (19,20), trans-Golgi network furin (21, 22), and lysosomal cathepsins (23). However, systematic studies of TAP-independent pathways involved in the generation of the overall peptide repertoire associated with different HLA class I molecules have not been reported. Studying the reexpression of newly synthesized complexes of different HLA class I molecules in the presence of diverse protease inhibitors allowed the determination of several allele-dependent processing pathways in ...

Diversity of Natural Self-Derived Ligands Presented by Different HLA Class I Molecules in Transporter Antigen Processing-Deficient Cells

“…In the current report, multiple sets of up to ten nested peptides were endogenously presented by different HLA class I molecules (Table S4), suggesting that amino acid trimming at both the N- and C- terminus might be a general attribute of TAP-independent peptides. The similarities between the classical and non-classical peptide repertoires of HLA class II and TAP-independent HLA class I suggest at least a partial contribution of TAP-independent peptide loading in the post invariant chain-degrading compartments as indicated the existence of uncharacterized TAP-independent peptide-HLA-B27 complexes generated by a chloroquine-sensitive pathway [43].…”

Role of Metalloproteases in Vaccinia Virus Epitope Processing for Transporter Associated with Antigen Processing (TAP)-independent Human Leukocyte Antigen (HLA)-B7 Class I Antigen Presentation

“…We observed 45% specific lysis versus 1% without virus in a standard 51 Crrelease assay. By intracellular cytokine staining (ICS) assays, we found that 32% of IFN-␥ secreting cells had a specific response versus 1% without virus.…”

“…Several proteases have been implicated in the processing of endogenously synthesized antigens independent of the classical proteasome pathway as follows: signal peptidase (43,44); furin (45,46); tripeptidyl peptidase II (47)(48)(49); lysosomal chloroquine-sensitive enzymes (50,51); and caspases (52,53). In this study, diverse proteolytic activities are required to generate the two HLA-B7-restricted epitopes studied.…”

“…In addition, 14 HLA-B7 potential ligands from a VACV proteome-based in silico prediction of high binding were also included in the study to identify new epitopes (supplemental Table 2). In a preliminary 51 Crrelease assay, only two synthetic peptides, D1R(808 -817) and J6R(303-311), were recognized by the TAP-independent CTL cell line (Fig. 1A, arrows).…”

Multiple Viral Ligands Naturally Presented by Different Class I Molecules in Transporter Antigen Processing-Deficient Vaccinia Virus-Infected Cells

“…SPase-processed peptides need further cleavage by signal peptide peptidase (SPPase) (42,68,69). Therefore, the involvement of SPPase in antigen presentation was tested by treating target cells with the SPPase-specific inhibitor (z-LL) 2 ketone (68,69).…”