A NEW METHOD FOR THE ENUMERATION OF ANTIGEN-REACTIVE CELLS RESPONSIVE TO A PURIFIED PROTEIN ANTIGEN

A new technique for the enumeration of antigen-reactive cells (ARC) responsive to the polymer antigen of S. adelaide flagellin (POL) is described for two strains of mice. Foci have been shown to be antibody dependent, may be mimicked by IgM as well as IgG antibodies, and contain specific antibody-forming cells (AFC). The use of POL offers a system unencumbered by relatively high numbers of background foci which, when present, appear to be basically different from those found using the SRBC antigen. The response of 1 antigen-reactive cell (ARC) focus/1 x 106 CBAT6T6 mouse spleen cells is linearly related to the injected number between 1 x 106–3 x 106 donor spleen cells and since 5% of injected cells remain in the spleen, there are an estimated 2400 ARC/spleen. The number of ARC foci does not increase significantly after the 5th postantigen day, and by the 8th day the AFC progeny of ARC have reached the maximum mean of 280 AFC/ARC focus. In response to increasing antigen concentrations, an initial rise in the number of AFC as well as ARC is observed, resulting in a relatively constant AFC/ARC ratio. This suggests that the number of ARC stimulated determines the total number of AFC produced under these conditions rather than a variable mitotic rate of the ARC offspring. The main significance of this technique is that it will allow a study of the kinetics of the ARC in the primary and secondary immune response as well as in immunological tolerance.