388 THIRD INTERNATIONAL LYMPHOKINE WORKSHOP

E&cts of Monoclonal Antibodies Raised against Antigen-Specific Factors. ROGER JAMES, ERIC CUL- BERT,SIRKKA KONTIAINEN,AND MARC FELDMANN, ICRFTumourImmunologyUnit,Department of Zoology, University College, London WCIE 6BT, England.

Antigen-eluted antigen-specific helper and suppressor factors, obtained from supernatants of in vitro activated helper or suppressor cells by antigen colonan absorption, were repeatedly injected into rats and mice, and fusions to NS-1 performed. A rat anti-mouse helper monoclonal factor was found to bind to helper hybridomas and TCGF lines, but not suppressor hybridomas. This antibody increased the primary or secondary antibody response in vitro, but only if antigen was present in the cultures. No effect on suppressor cell induction or suppression was detected. T-Cell proliferation and MLR and killer cell induction were augmented, and immunoadsorbents bound antigen-specific helper factor. A mono- clonal BALB/C anti-BlO.DZ suppressor factor (to KLH) was generated, and this antibody was found to bind to suppressor hybridomas or tumours of C57Bl but not CBA origin, and not to C57Bl helper cell clones. Functionally it stimulated suppressor ceils and reduced responses to sheep red cells and TNP KLH in vitro. These reagents offer a new approach to selectively manipulate the immune response and to isolate components of the immune system.

Biochemical Characterization of Human B-Cell Growth Factor (BCGF). T. KASAHARA, A. MURA- GUCHI, J. J. OPPENHEIM, AND A. S. FAUCI, Laboratory of Microbiology and Immunology, NIDR, and Laboratory of Immunoregulation, NIAID, NIH, Bethesda, Maryland.

Compared with considerable available data on T-cell growth factor, i.e., interleukin 2 (IL-2), factors interacting with B cells are as yet ill defined. In this study, we employed a B-cell costimulator assay system for the detection of BCGF activity, using highly purified human peripheral blood which was prestimulated with killed Staphylococcus aureus Cowan I bacteria for 3 days. The purified B cells were prepared from E-rosetted, adherent cell-depleted mononuclear cells, which were treated with anti-leu 1 antiserum and rabbit complement. BCGF was obtained from human peripheral blood lymphocyte culture supernatant incubated with PHA (2 *g/ml) or Con A (IO pg/ml) plus PMA (10 rig/ml) for 2 to 4 days. BCGF copurified with IL-2 on Sephacryl S-200 (MW 20,000-25,000) and also chroma- tofocused with IL-2 at PI’S of 7.8 and 6.9. Hydrophobic phenyl-sepharose column chromatography also failed to separate IL-2 and BCGF. However, in a preliminary study of elimination of IL-2 by absorption at 37°C with IL-2-dependent CT-6 cells, most of the BCGF activity still could be detected in the crude supernatant and in fractions eluted from S-200 and phenyl-Sepharose columns. Thus human BCGF and IL-2 are rather difficult to separate by conventional column chromatography, but these studies suggest that these two factors although biochemically similar seem to be different molecular entities.

SESSION IV: LYMPHOKINE-INDUCED DIFFERENTIATION

OF TARGET CELLS

Chairmen: H. REMOLD, S. WAHL

Difirentiation between MAF and MIF Activities in LK Preparations. W. E. FOGLER, J. S. BRANDHORST, AND I. J. FIDLER, NCI-Frederick Cancer Research Facility, P.O. Box B, Frederick, Maryland 21701.

We determined whether the activities of macrophage-activating factor (MAF) and migration-inhi- bition factor (MIF) in culture supernatants of Con A-stimulated lymphocytes could be distinguished by physicobiological criteria. Four days after the ip injection of Marco1 52, murine peritoneal macro- phages (PEM) were harvested and incubated in suspension for 24 hr with lymphokines (LK) or control supernatants. The MIF activity was assayed by the agarose microdroplet assay, and MAF activity was determined by macrophage-mediated cytolysis of radiolabeled tumor cells. A 2 log difference between titers of MIF (1:lOOO dilution) and MAF (1:lO dilution) activities occurred. Treatment of LK at 56, 70, or 100°C for 30, 10, or 2 min, respectively, resulted in a linear decay to extinction of MIF activity. MAF activity was retained even after boiling. PEM cultures incubated for 2 hr with liposomes containing LK in the presence of 0.1 M a-L-fucose followed by a 22-hr incubation in medium became highly tumoricidal. Presentation of LK in liposomes, however, did not generate MIF activity. We conclude that the activities of MIF and MAF can be distinguished on the basis of their dose-response characteristics, thermal stability, and mode of presentation to the macrophage. (Research supported by the National Cancer Institute, DHHS, under Contract NOl-CO-75380 with Litton Bionetics, Inc.)