Sequential use of hapten‐gelatin fractionation and fluorescence‐activated cell sorting in the enrichment of hapten‐specific B lymphocytes

Abstract

Spleen cells from normal unimmunized adult mice were subjected to a 2-stage fractionation procedure designed to prepare a subpopulation highly enriched for the capacity to form antibody to the hapten fluorescein (FLU).First, the spleen cell suspension was rocked in a petri dish coated with a thin layer of FLU-gelatin (1 5 min at 4 "C), the nonadherent cells washed away and the adherent cells recovered by melting the gelatin and treated with colleenase. The cells were relabeled with a fluorescent protein, usually FLU-gelatin at 100 pg/ml, (25 "C, 15 min). After washing, the cells were sorted into cohorts of relatively homogeneous fluorescence intensity in the fluorescence-activated cell sorter (FACS). Finally, the sorted cells were again collagenase-treated and stimulated with FLU-coupled polymerized flagellin in a limit dilution microculture system capable of generating single clones of anti-hapten plaque-forming cells (PFC).It was found that the cohort of cells representing the 10 % most intensely fluorescent cells, yielded PFC clones with a frequency of 1 in 8, about 5 times higher than the hapten-gelatinenriched cells. Less fluorescent cohorts gave progressively lower frequencies of clonable PFC precursors. Some evidence was obtained, suggesting that the most highly fluorescent cells produced antibody of higher median avidity than the FLU-gelatin-fractionated cells, which in turn showed higher avidity than unfractionated spleen cells, findings consistent with the clonal selection hypothesis.Somewhat surprisingly, cells with high fluorescence intensity prepared by FACS sorting of FLU-gelatin-labeled unfractionated spleen cells, yielded much lower PFC precursor frequencies than cells of equivalent fluorescence intensity derived through the 2-stage fractionation procedure.Spleen cells from newborn mice were subjected"t0 2-stage fractionation. The method proved equally applicable to them, and preparations with a 1 in 11 PFC precursor frequency could be readily generated. However, the neonatal cells differed from the adult cells in responding significantly better to the polyclonal B cell activator, lipopolysaccharide, than to antigen.The theoretical implications of these findings and the potential uses of pure populations of hapten-binding cells are briefly discussed.

Antibody production by single, hapten-specific B lymphocytes: an antigen-driven cloning system free of filler or accessory cells.

“…FLU Conjugates. FLU-protein conjugates were prepared by allowing the protein to react with fluorescein isothiocyanate in 0.2 M carbonate/hydrogen carbonate buffer as described (23,24). The FLU-POL used had a substitution ratio of 0.7 mol of FLU per mol of monomeric flagellin; and the FLU-gelatin had 2 molecules of FLU per 100,000 daltons of gelatin.…”

“…Spleen cells were fractionated on thin layers of hapten-gelatin as described, using the method of Haas and Layton (20) as modified by ourselves (3,23,25). Adherent antigen was removed from the recovered binding population prior to culture by collagenase treatment.…”

“…This. method yields populations of 97% B cells (21), which are over 100-fold enriched for in vitro reactivity to hapten-POL conjugates (3,23,25 …”

“…The adherent population, 97% surfaceimmunoglobulin-positive (21), was stimulated with FLU-polymerized flagellin (FLU-POL) and media rich in various interleukins. Single cells generated antibody-forming clones almost as efficiently as in our usual thymus filler cell-containing system.…”

Single cell studies on the role of B-cell stimulatory factor 1 in B-cell activation.

“…Specific pathogen-free CBA/CaH/WEHI mice were used at 8-10 weeks of age. Hapten-speciflic B cells were prepared from spleen cell suspensions by fractionation on plastic dishes coated with fluorescein-gelatin as described (18)(19)(20). Adherent fluorescein-gelatin was removed from the recovered binding cells by collagenase.…”