Identification of H-2K(b)-restricted T-cell epitopes within the nucleocapsid protein of hantaan virus and establishment of cytotoxic T-cell clones

Although neutralizing antibodies against Hantaan virus (HTV) can protect ho sts from viral infection, T-cell responses to HTV are also important in hos t defense against HTV. However, much less is known about cytotoxic T lympho cyte (CTL) responses to HTV. To identify CTL epitopes in the HTV nucleocaps id protein (NP), we selected 7 H-2K(b)-motif-fitting peptides. Of these pep tides, 3 peptides (NP3, NP4, and NP7) were recognized by CTL responses deri ved from HTV-immunized mouse splenocytes. NP3 and NP4 peptides were also re cognized by HTV-immunized splenocytes after secondary in vitro stimulation with the relevant peptide, but NP7 could not be recognized after in vitro s timulation. These results agree well with peptide immunization studies show ing that peptide-specific CTL responses could be induced with NP3 and NP4 b ut not with NP7 peptide. Furthermore, CTL activity assay using targets, pre pared to express the antigen (NP) endogenously, demonstrated that NP3 and N P4 peptides could be presented endogenously. CTL elicited with NP4 peptide retained some cross-reactivity and was difficult to long-term culture. Howe ver, NP3-elicited CTL was very specific for NP3 peptide and was stable enou gh to be cloned. Among many CTL lines elicited with HTV or HTV NP peptides, 6 NP3-specific CTL clones were established and have been maintained more t han 2 years. All 6 CTL clones were characterized to be CD3+, CD4-, CD8+, CD 25+, CD62L-, and NK1.1-, and to use TCR V beta 6. This preferential usage o f TCR V beta 6 indicates that TCR V beta 6 regions are important for recogn ition of the HTV NP3 epitope (NP221-228, SVIGFLAL) on H-2K(b) molecule. Our data demonstrate the definition of mouse CTL epitopes in HTV and the gener ation of HTV-specific mouse CTL clones. J. Med. Virol. 60: 189-199, 2000. ( C) 2000 Wiley-Liss, Inc.