In vitro characterization of chB6 positive and negative cells from early avian embryos

Density gradient-separated embryonic spleen and bone marrow cells were enriched for chB6(+) cells with positive and negative selection procedures and magnetic cell sorting. The majority of chB6(+) cells, consisting of small, dense, strongly chB6(+) cells, were prone to apoptosis, which was further accentuated after exposure to monoclonal antibodies directed against chB6 alloantigen, but was largely inhibited by PDBU, leading to maintenance and frequently numerical increases of chB6(+) cells after an initial decline. sIgM(+) cells within that population followed a very similar pattern, suggesting a PDBU-induced upregulation of sIgM expression on a proportion of chB6(+) cells. The protective effect of PDBU on anti-chB6-exposed cells was confirmed with bursal lymphocytes and shown to be entirely PDBU concentration dependent. It was calculated that each 14-day embryonic spleen contained a minimum of 250,000 chB6(+) and 3000-4000 sIgM(+) cells, respectively. Endogenous apoptosis appeared to be increased with embryonic age, reaching a peak with bursal cells in the posthatching period. A second population of larger, less dense, and weak chB6(+) cells, often with vacuoles in a more abundant cytoplasm, differed functionally, expanding numerically in unstimulated cultures and being inhibited by PDBU. No sIgM(+) cells developed within this population. It is proposed that this chB6(+) fraction may represent progenitors of a previously suggested chB6(+) subset of macrophages, in contrast to the dense chB6(+), small cells, viewed as B cell progenitors. chB6(-) cells, consisting predominantly of granulocytes, proliferated vigorously in unstimulated cultures, but were consistently inhibited by PDBU. Coculture of age-matched embryonic bursal stroma with positively and negatively enriched chB6(+) cells revealed enhanced protection from apoptosis for chB6(+) cells and a PDBU-induced upregulation of sIgM expressing cells. Bursal stroma also had a pronounced positive effect on the proliferation of chB6(-) cells.