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Soluble mediators of T-B interactionsupernatants of antigen-activated T cells (ATC) were tested for their B cellactivating potential in T cell-deprived mouse lymphocyte cultures. Antigenic stimulation of ATC induced the simultaneous release of antigen-specific and nonspecific factors. The effect of the nonspecific supernatant factor was restricted to the potentiation of antibody responses against fully or partially T-independent antigens (dinitrophenylated aminoethyl dextran or heterologous erythrocytes, respectively). Expression of the nonspecific activity depended on a macrophage function, which could be replaced by 2-mercaptoethanol, whereas the specific factor required the physical presence of the macrophage.

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Molecular changes occur at the surface of hemopoietic cells during differentiation from progenitor cells to mature granulocytes and macrophages. The differential expression of surface carbohydrate residues has been probed using lectins and the results used to purify normal mouse granulocyte-macrophage progenitor cells. Ten different lectins were screened for selective interaction with mouse hemopoietic colony-forming cells (CFCs), using agglutination or a quantitative analysis of the number of fluoresceinated lectin molecules bound per cell using a fluorescence activated cell sorter (FACS). Pokeweed mitogen (PWM), Helix pomatia agglutinin (HPA), soybean agglutinin (SBA), and peanut agglutinin (PNA) preferentially bound to CFCs so that it was possible to enrich 4 to 10-fold for these progenitor cells by sorting for the highly fluorescent cells. Further analysis of the low and high angle light scattering characteristics of the CFCs indicated that these cells were polydisperse, but could be enriched ten-fold by selecting for cells with high intensity low angle (0 degrees) scatter and low intensity high angle (90 degrees) scatter. PWM gave the best enrichment (10 to 15-fold) for adult bone marrow CFCs, for CFCs from fetal sources (fetal liver, fetal blood), and for CFCs from the spleens of mice injected previously with outer membrane lipoprotein from E. coli. Three parameter sorting for CFC using the FACS (low angle scatter, high angle scatter, and PWM-fluorescence) resulted in large enrichment factors (16 to 50-fold) for CFCs from all the above sources. Over 7% of the cells sorted from bone marrow, 10% of the cells sorted from post-lipoprotein spleen, and 28% of the cells sorted from fetal peripheral blood were hemopoietic CFCs. Ninety percent of the cells in these fractions had the morphology of blast cells or myelocytes. Thus, it was possible to identify the morphological characteristics of the hemopoietic progenitor cells. Screening of other developmental systems using quantitation of fluorescence with lectins should prove of general value for the purification of selected differentiation states.