ABSTRACT
It is becoming increasingly apparent that the majority of tumours display defects in the MHC class I antigen processing pathway, particularly low levels of the transporters-associated with antigen processing (TAP) and tapasin. Thus, immunotherapy approaches targeting such tumours with CD8+ cytotoxic T lymphocytes (CTL) requires strategies to overcome these defects. Previously we had identified an antigen processing pathway by which cytosolically derived hydrophobic peptides could be presented in the absence of TAP. Here we show in the tapasin-negative cell line 721.220 that a number of these hydrophobic TAP-independent peptides can also be presented in a tapasin-independent manner. Yet when these experiments were extended to tumour cell lines derived from small cell lung cancer (SCLC), which we show to be tapasin deficient in addition to TAP-negative, the TAP-, tapasin-independent peptides were not presented. This lack of presentation could be rectified by pre-treatment of SCLC cells with IFNgamma. Alternatively, by directing the TAP-, tapasin-independent peptides into the endoplasmic reticulum (ER) via an ER signal sequence, these peptides were presented efficiently by SCLC cells. We infer from this data that the TAP-independent pathway for presentation of hydrophobic peptides generates a low concentration of peptide in the ER and, for tumour cells which also lack tapasin, this concentration of antigenic peptide is insufficient to load onto MHC class I molecules. Thus, for immunotherapeutic approaches to target SCLC and other tumours with defects in the MHC class I antigen processing pathway it will be important to consider strategies that address tapasin-defects.
Subject(s)
ATP-Binding Cassette Transporters/physiology , Antigen Presentation , Antigens, Viral/immunology , CD8-Positive T-Lymphocytes/immunology , Endoplasmic Reticulum/metabolism , Histocompatibility Antigens Class I/immunology , Membrane Transport Proteins/physiology , Peptide Fragments/immunology , ATP Binding Cassette Transporter, Subfamily B, Member 2 , ATP Binding Cassette Transporter, Subfamily B, Member 3 , ATP-Binding Cassette Transporters/genetics , Antigens, Viral/metabolism , Carcinoma, Small Cell/pathology , Cell Line, Transformed , Cell Line, Tumor , Cytosol/metabolism , HLA-A Antigens/immunology , HLA-A2 Antigen/immunology , HLA-A24 Antigen , Herpesvirus 4, Human/immunology , Humans , Hydrophobic and Hydrophilic Interactions , Immediate-Early Proteins/immunology , Immediate-Early Proteins/metabolism , Intracellular Membranes/metabolism , Lung Neoplasms/pathology , Membrane Transport Proteins/deficiency , Peptide Fragments/chemistry , Peptide Fragments/metabolism , Phosphoproteins/immunology , Phosphoproteins/metabolism , Protein Sorting Signals/physiology , Protein Transport , T-Cell Antigen Receptor Specificity , Trans-Activators/immunology , Trans-Activators/metabolism , Transfection , Viral Matrix Proteins/immunology , Viral Matrix Proteins/metabolism , Viral Proteins/immunology , Viral Proteins/metabolismABSTRACT
For recognition by CD8(+) lymphocytes, peptides derived from cytosolically processed antigen need to access MHC class I molecules en route to the target cell surface. This normally requires peptide transport into the endoplasmic reticulum via the transporter associated with antigen presentation (TAP) complex. However, as recent work with Epstein-Barr virus illustrates, TAP-independent presentation pathways also exist and are growing in number.
Subject(s)
Antigen Presentation , Antigens, Viral/immunology , Herpesvirus 4, Human/immunology , Histocompatibility Antigens Class I/metabolism , ATP Binding Cassette Transporter, Subfamily B, Member 2 , ATP-Binding Cassette Transporters/physiology , Cysteine Endopeptidases/metabolism , Epitopes/immunology , Epstein-Barr Virus Nuclear Antigens/metabolism , Humans , Hydrophobic and Hydrophilic Interactions , Interleukin-10/metabolism , Models, Biological , Multienzyme Complexes/metabolism , Proteasome Endopeptidase Complex , Viral Matrix Proteins/metabolism , Viral Proteins/metabolismABSTRACT
We have identified an HLA-A2-restricted CD8(+) T-cell epitope, FLYALALLL, in the Epstein-Barr virus (EBV) latent membrane protein 2 (LMP2), an important target antigen in the context of EBV-associated malignancies. This epitope is TAP independent, like other hydrophobic LMP2-derived epitopes, but uniquely is dependent upon the immunoproteasome for its generation.
