Efficient Class II Major Histocompatibility Complex Presentation of Endogenously Synthesized Hepatitis C Virus Core Protein by Epstein-Barr Virus-Transformed B-Lymphoblastoid Cell Lines to CD4+ T Cells

The induction of an efficient CD4⁺ T-cell response against hepatitis C virus (HCV) is critical for control of the chronicity of HCV infection. The ability of HCV structural protein endogenously expressed in an antigen-presenting cell (APC) to be presented by class II major histocompatibility complex molecules to CD4⁺ T cells was investigated by in vitro culture analyses using HCV core-specific T-cell lines and autologous Epstein-Barr virus-transformed B-lymphoblastoid cell lines (B-LCLs) expressing structural HCV antigens. The T- and B-cell lines were generated from peripheral blood mononuclear cells derived from HCV-infected patients. Expression and intracellular localization of core protein in transfected cells were determined by immunoblotting and immunofluorescence. By stimulation with autologous B-LCLs expressing viral antigens, strong T-cell proliferative responses were induced in two of three patients, while no substantial stimulatory effects were produced by B-LCLs expressing a control protein (chloramphenicol acetyltransferase) or by B-LCLs alone. The results showed that transfected B cells presented mainly endogenously synthesized core peptides. Presentation of secreted antigens from adjacent antigen-expressing cells was not enough to stimulate a core-specific T-cell response. Only weak T-cell proliferative responses were generated by stimulation with B-LCLs that had been pulsed beforehand with at least a 10-fold-higher amount of transfected COS cells in the form of cell lysate, suggesting that presentation of antigens released from dead cells in the B-LCL cultures had a minimal role. Titrating numbers of APCs, we showed that as few as 10⁴ transfected B-LCL APCs were sufficient to stimulate T cells. This presentation pathway was found to be leupeptin sensitive, and it can be blocked by antibody to HLA class II (DR). In addition, expression of a costimulatory signal by B7/BB1 on B cells was essential for T-cell activation.Hepatitis C virus (HCV) has been known as a major etiologic agent of posttransfusion and sporadic community-acquired non-A, non-B hepatitis. Like the other members in the family Flaviviridae, HCV contains a single, positive-strand RNA genome with a single long open reading frame (ORF) coding for a polyprotein precursor of about 3,000 amino acids (aa) (12). HCV infection is frequently persistent in the majority of patients and is closely associated with the later development of liver cirrhosis and hepatocellular carcinoma (3, 12, 16, 32). The effective control of HCV infection has been limited by the high frequency of viral genetic heterogeneity (7), the low rate of response to alpha interferon (46), and inadequate production of protective immunity (44, 45). These features strongly suggest that there is a great need to establish a new, highly effective therapy.CD4⁺ T cells are considered to play a central role in the generation of protective immunity against infections, because they can provide help to B cells for antibody production (42) and to cytotoxic precursor T cells for their maturation to effectors (21). Some CD4⁺ T cells may also act as cytotoxic effectors (30). It has been recognized that CD4⁺ T-cell response to HCV antigens is important for determining the clinical course of HCV infection (17, 37). Generally, T-cell proliferation is more frequent and stronger in patients with a benign course (6, 17, 20, 33, 37) that is accompanied by the normalization of serum alanine aminotransferase and, in some cases, the clearance of viral RNA (17, 37). In contrast, patients who have a poor T-cell response tend to develop persistent infection (17, 37). These findings support the hypothesis that a sufficient CD4⁺ T-lymphocyte response is critical for limiting HCV infection.Activation of T lymphocytes depends on the recognition of processed viral peptides, but not native antigens, in the context of major histocompatibility complex (MHC) molecules that are presented by antigen-presenting cells (APCs) (56). The B cell is an important professional APC, and its role in mediating antigen-specific immune response has been described extensively (11). Epstein-Barr virus (EBV)-transformed B-lymphoblastoid cells are frequently used as APCs in in vitro analyses for antigen processing and presentation to T cells. These cells are characterized by high-level expression of class I and class II MHC molecules, along with accessory molecules such as ICAM-1, B7/BB1, and LFA-3, known to be important costimulatory molecules for T-cell activation (9, 15, 24). Importantly, transfected EBV-immortalized B cells expressing a tumor antigen have been shown to be capable of eliciting both T-helper and cytotoxic-T-lymphocyte (CTL) responses following in vivo inoculation (40). Nevertheless, dendritic cells have been shown to be critical for initiating responses by naive T cells (53), and in some situations presentation by B cells has been suggested to be toleragenic (35). To date, the role of B cells in processing and presenting HCV antigens has not been studied in detail and the mechanisms underlying T-cell–B-cell interaction are still being worked out.In the present study, EBV-transformed B-lymphoblastoid cell lines (B-LCLs) established from HCV-infected patients were transfected with an expression vector coding for the whole structural region and part of the NS2 region of the HCV genome. The capacity of transfected B-LCL APCs for presenting intracellularly synthesized peptides was assessed by in vitro induction of the HCV-specific lymphoproliferative response of autologous T-cell lines. Our results indicated that core protein was properly expressed and efficiently presented by B-LCL APCs to CD4⁺ T cells. We demonstrated that the endogenous core peptides were presented through the class II MHC pathway and that they need B7/BB1 for providing costimulatory signals.