FIG. 3.

Presentation of HPLC Fraction 28 by H-2K^d and lysis of P815 cells transfected with LLO. a, P815 cells were coated with HPLC fraction 28 peptides and exposed to LmT CTL in the presence of monoclonal antibodies 34-4-20s and 34-4-21s, which are specific for H-2D^a, and 31-3-4s²⁵ and SF1-1.1.1, which are specific for H-2K^d. b, P815 cells transfected with LLO (PHem3) were used as targets for LmT CTL in the presence and absence of anti-H-2^d antibodies as in a.

METHODS. P815 and PHem3 cells were labelled with ⁵¹Cr and suspended in culture supernatants from hybridomas 34-4-20s, 34-4-21s, 31-3-4s, SF1-1.1.1 and control medium. Fraction 28 peptides were added to the P815 targets. LmT effectors were added to targets at a ratio of 20:1 and the specific lysis was determined after 3 h. PHem3 was obtained by transfecting P815 cells with linearized pHβAPr-1-neo²⁶ containing the entire LLO gene¹⁷. Bases 1-1,587 of the hlyA gene encoding LLO were cloned into the BamHI site of the pHβAPr-1-neo expression vector after polymerase chain reaction (PCR) amplification from L. monocytogenes DNA. The oligonucleotides used for priming were 5′-CCCGGGATCCACCATGAAAAAAATAATGCTAG-3′ and 5′-GGATCCGGATCCTTATTCGATTGGATTATC-3′ which introduced flanking BamHI sites. Electroporation of PvuI linearized plasmid containing the insert into P815 cells and selection of G418 resistant clones was performed as described²³. P815 cells transfected with the vector without the insert were not lysed by the LmT CTL line (not shown).