A Role for Intercellular Antigen Transfer in the Recognition of EBV-Transformed B Cell Lines by EBV Nuclear Antigen-Specific CD4+ T Cells

Abstract

The CD4+ T cell response to EBV may have an important role in controlling virus-driven B lymphoproliferation because CD4+ T cell clones to a subset of EBV nuclear Ag (EBNA) epitopes can directly recognize virus-transformed lymphoblastoid cell lines (LCLs) in vitro and inhibit their growth. In this study, we used a panel of EBNA1, 2, 3A, and 3C-specific CD4+ T cell clones to study the route whereby endogenously expressed EBNAs access the HLA class II-presentation pathway. Two sets of results spoke against a direct route of intracellular access. First, none of the clones recognized cognate Ag overexpressed in cells from vaccinia vectors but did recognize Ag fused to an endo/lysosomal targeting sequence. Second, focusing on clones with the strongest LCL recognition that were specific for EBNA2- and EBNA3C-derived epitopes LCL recognition was unaffected by inhibiting autophagy, a postulated route for intracellular Ag delivery into the HLA class II pathway in LCL cells. Subsequently, using these same epitope-specific clones, we found that Ag-negative cells with the appropriate HLA-restricting allele could be efficiently sensitized to CD4+ T cell recognition by cocultivation with Ag-positive donor lines or by exposure to donor line-conditioned culture medium. Sensitization was mediated by a high m.w. antigenic species and required active Ag processing by recipient cells. We infer that intercellular Ag transfer plays a major role in the presentation of EBNA-derived CD4 epitopes by latently infected target cells.

Nuclear location of an endogenously expressed antigen, EBNA1, restricts access to macroautophagy and the range of CD4 epitope display

“…2C shows the results when the above dox-induced targets (and standard LCLs matched and mismatched for the same restricting alleles) were assayed using T cell clones against the four epitopes. Cells induced to express the Ii-tagged antigen were recognized very strongly by the HPV-specific clone (in line with earlier work showing efficient MHC I processing of Ii-tagged constructs) (14,17) and also by clones against all three CD4 epitopes including PQC; this clearly demonstrates that PQC can be processed from endogenously expressed EBNA1 providing the antigen is directly targeted to the endolysosomal system. However, high overexpression of the EBNA1 or E1Δ nuclear proteins led to only a 2-to 3-fold increase in SNP and VYG epitope display above the low baseline values seen on unmanipulated LCLs; furthermore there was still no detectable presentation of the PQC epitope, suggesting that antigen delivery into the MHC II pathway was still limited.…”

“…Whereas EBNA2, -3A, and -3C, or antigenic fragments thereof, are shed into LCL culture supernatant and then acquired and processed as exogenous antigen by coresident cells (14), such intercellular antigen transfer is never detectable for EBNA1, whether expressed at physiologic levels or hyperexpressed as native or even as cytoplasmically targeted protein. We infer that, in LCL cells, endogenously expressed EBNA1 must access the MHC II presentation pathway by an intracellular route.…”

“…Turning to how endogenously expressed EBNA1 was accessing the MHC II presentation pathway in LCL cells, we first conducted a series of cross-feeding experiments with concentrated culture supernatants to show that access must be occurring by an entirely intracellular route and not, as has been observed with the other EBV-coded nuclear antigens EBNA2 and EBNA3C (14), through the release of antigenic species into culture medium and their uptake as exogenous antigen by neighboring cells (Fig. S3A).…”

The latent membrane protein 1 oncogene modifies B-cell physiology by regulating autophagy

“…LMP1's induction of autophagy may facilitate the clearance of its own aggregates and allow cells to escape from direct immune recognition by CD4 þ T cells. While the processing of one EBV protein, EBNA1, apparently requires autophagy for delivery to MHC class II molecules, that for the more commonly recognized antigens, EBNA2 and EBNA3C does not (Paludan et al, 2005;Taylor et al, 2006).…”

Definition of the peptide binding motif within DRB1*1401 restricted epitopes by peptide competition and structural modeling

“…Residues Epitope VP16 (18) 477-487 LMFINGSLTVR P53 (8) 193-204 LIRVEGNLRVE Actin (19) 206-216 REIVRDIKEKL Histone H4 (Mouse) (19) 31-45 TKPAIRRLARRGGVK Mycobacterium leprae (20) 1-16 VAKVKIKPLEDKILVQ Mycobacterium leprae (20) 41-56 GTVVAVGPGRWDEDGA Mycobacterium tuberculosis (20) 65-80 KRIPLDVAEGDTVIYS Mycobacterium tuberculosis (20) 73-88 KYGGTEIKYNGEEYLI Mycobacterium tuberculosis (20) 81-100 YNGEEYLILSARDLAVVSK Synaptonemal complex protein 1 (21) 635-649 QLNVYEIKVNKLELE Herpesvirus 4 B95-8 (22) 529-543 PQCRLTPLSRLPFGM Plasmodium falciparum (23) 239-249 LKIRANELDVL Plasmodium falciparum (23) 249-263 LKKLVFGYRKPLDNI Plasmodium falciparum (23) 338-350 IDTLKKNENIKEL…”