Growth of B cell colonies independent of T cells: a new perspective.

Growth of human peripheral blood B cells in a B cell colony assay system is a useful technique to study the function of B cell biology. In the initial reports, T and B cells were admixed in the culture system, prior to which the T cells were treated with mitomycin or irradiation to prevent their proliferation. There were reports that optimal growth of B cell colonies required T cells to be in contact with the B cells. However, we were able to grow B cell colonies physically separated from T cells which were placed on a filter. We speculated then that T cells contacted B cells via pseudopods through the pores of the filter. We now report the growth of B cell colonies independent of T cells and conclude that B cell colony growth depends upon a critical number of B cells plated rather than on T cell help.

Feedback suppression of B cell colony formation by supernatants of B colony cells: role of immunoglobulin.

We have reported previously that CD5+ B cells from mature B cell colonies provide a negative feedback signal to the growth of autologous B cell colonies. Now we have observed that supernatants from mature B cell colonies also provide a negative feedback signal to the growth of autologous B cell colonies. We investigated the mechanism of this effect by growing B cell colonies physically separated by a 0.45 micron filter from T cells in millicell-CM chambers. Addition of colony supernatants to the T cell compartment reduced the number of B cell colonies by 28 +/- 6%. Colony numbers were reduced by 11 +/- 2 and 17 +/- 5% when the supernatants were added to the B cell or to both compartments, respectively. Pulsing T cells with the B cell colony supernatants before adding them to the colonies also decreased colony numbers by 33 +/- 13%. The addition of exogenous Ig classes and IgG subclasses to B cells decreased B cell colony numbers, although the effect was variable. In the presence of T cells, IgG had the greatest suppressive activity and the subclass IgG4 was most suppressive. In the absence of T cells, high concentrations of IgG almost abolished B cell colony formation. We conclude that these supernatants provide a negative feedback signal either directly to B cells, or via T cells which may be mediated at least in part by Ig.

Feedback suppression of B cell colony formation in healthy individuals.

Proliferation and differentiation of B cells has been extensively studied and the study of feedback suppression of B cell proliferation has been limited to humoral factors. However, very little is known about feedback suppression of B cell proliferation by cellular influences. We have previously reported on the role of T cells and their subsets on B cell proliferation in that we did not observe suppression of B cell colony growth by T cells. We now report on the role of B cells in limiting B cell proliferation. B cell colonies were grown in methyl cellulose for either 3 days or 5-6 days utilizing 2 x 10(5) T cells irradiated with 9,000 rads, and 2 x 10(5) B cells. The B cells were then obtained from these colonies and increasing numbers of cells were added to fresh autologous B cells that were further cultured for 5 days to form new B cell colonies. At the end of this period, B cell colony numbers were determined. Our data show that addition of CD19- and CD20- positive B cells recovered from mature colonies after 5 days to fresh B cells suppressed further B cell colony growth in all cases tested, whereas addition of CD19-positive B cells recovered from immature colonies after 3 days of culture did not suppress further B cell colony growth. Elimination of CD 19- or CD20-positive cells with monoclonal antibody to CD19 and complement or by the technique of panning enhanced colony growth. Supernatants obtained from B cell colonies did not suppress B cell colony formation. Our data suggest that there is feedback suppression of normal progenitor B cell proliferation by constituent B cells and that this effect develops during maturation of colonies during the growth phase.

Growth of B cell colonies independent of T cell contact.

One of the methods of obtaining B cell colonies involves physically admixing B cells in growth medium containing mitogens with accessory T cells treated with mitomycin C or low dose irradiation to prevent T cell colony formation. However, under these conditions T cells still have the capacity to form colonies in methylcellulose, which is most frequently used for colony assays, and B cells recovered from the colonies are contaminated by the large number of added T cells. Therefore, we have developed a method of growing B cell colonies by physically separating the enriched B cells from the T cells by using millicell-HA inserts (Millipore). The requirement for mitogens, culture conditions, development, and number of B cell colonies formed were similar to those observed with admixture of T and B cells. The advantage of this system is that it permits growth of B cell colonies without the confounding influence of T cell proliferation or treatment of T cells with mitomycin or irradiation, and relatively pure B cell colonies can be recovered for further characterization or functional studies.