Stimulation of anti-influenza cytolytic T lymphocytes by a synthetic peptide of the influenza hemagglutinin can be modulated by at least three independent helper factors

Abstract

Secondary murine anti-influenza cytolytic T lymphocytes (CTL) could be induced in vitro by the chemically synthesized peptide HA1(181-204), corresponding to amino acids 181-204 of the hemagglutinin molecule of influenza strain A/Japan/305/57. This stimulation required the addition of exogenous helper factors, termed CHFP, which could be obtained from supernatant fluids of the WEHI-3 cell line or spleen cells stimulated by either concanavalin A or Sendai virus. Because the activities from these three sources could reflect different molecules, they were referred to as CHFPW, CHFPC, and CHFPS, respectively. When CHFPS was fractionated by using reverse-phase high performance liquid chromatography (HPLC), the CHFP activity was found to elute at two hydrophobicities. The more hydrophilic factor(s) was designated CHFPS1, while the more hydrophobic factor(s) was called CHFPS2. Similarly, CHFPW could be separated into more hydrophilic (CHFPW1) and more hydrophobic (CHFPW2) molecules. In contrast, CHFPC molecules eluted at only one hydrophobicity (CHFPC1). Based on HPLC characteristics, CHFPS1, CHFPW1, and CHFPC1 all could reflect the same molecule. That molecule could be interleukin 3 (IL 3), because IL 3, purified to homogeneity, was also found to function in the CHFP assay. In contrast, CHFPW2 and CHFPS2 had different hydrophobicities. Therefore, a possibility exists that there are a minimum number of three factors functional in the CHFP assay: IL 3, CHFPW2, and CHFPS2. Based on HPLC separations, CHFPS2 represents a molecule distinct from biochemically characterized cytokines implicated previously in the generation of CTL:CHF, CSF, interferon, and interleukins 1, 2, and 3. The killer cells stimulated are both virus-specific and H-2-restricted as is characteristic of CTL.