Isolation of antigen-binding cells from unprimed mice: demonstration of antibody-forming cell precursor activity and correlation between precursor and secreted antibody avidities

Abstract

Cells binding DNP groups conjugated to fluoresceinated mouse gamma globulin ((F)DNP-MGG) were isolated from spleens of unprimed mice using a fluorescence-activated cell sorter (FACS). The isolated cells were specifically enriched at least 100-fold for anti-DNP precursor activity in an adoptive transfer assay as compared to unfractionated spleen. The fraction depleted of binding cells, although depleted of anti-DNP precursor activity, responded as well as unfractionated spleen when assayed for anticarrier (keyhole limpet hemocyanin [KLH]) precursor activity. High avidity binding cells were stained using low concentrations of (F)DNP-MGG. Medium and low avidity binding cells were stained using high concentrations of (F)DNP-MGG in the presence of free hapten which selectively blocked staining of the high avidity binding cells. Cells were supplemented with an excess of carrier-primed (KLH), nylon-purified splenic T cells and transferred to irradiated recipients. DNP-KLH was given at transfer and 5 days later. The anti-DNP plaque-forming cell (DNP-PFC) response and the avidities of the DNP-PFC in the irradiated recipients were measured by hapten inhibition of direct PFC plaque formation 12 days after transfer. At this time, very few indirect PFC were found. There was a positive correlation between the avidity of the DNP-binding cells and the avidity of the anti-DNP antibody secreted by their progeny. High avidity DNP-binding cells gave rise to predominantly high avidity anti-DNP-PFC. Medium and low avidity binding cells gave rise to medium and low avidity DNP-PFC.