B-cell memory is short-lived in the absence of antigen.

Primary encounter with antigen stimulates specific B cells not only to differentiate into cells that produce antibody at a high rate (plasma cells), but also to give rise to populations of memory cells. These cells have many characteristics that differ from virgin B cells, including their lifespan. When re-exposed to antigen, memory cells generate secondary IgG responses that are enhanced in rate, titre and affinity. At present they are considered as small resting lymphocytes which survive for long periods in a quiescent state between each antigen encounter. However, the fact that an individual may continue to make an antibody response for many months following a single injection of antigen is often overlooked. This continued antibody production is probably due to repeated stimulation of antigen-specific B cells and raises the question of whether memory B-cell clones require antigen for their maintenance. Here we show that they do, and that following transfer, in the absence of antigen, memory B-cell populations are lost from the adoptive host after 10-12 weeks.

Differentiation of murine B cell precursors in agar culture. II. Response of precursor-enriched populations to growth stimuli and demonstration that the clonable pre-B cell assay is limiting for the B cell precursor.

We established culture conditions under which fetal liver-derived B cell progenitors divide and differentiate in semisolid agar, forming colonies containing antibody-secreting cells. An important feature of this assay system is that, under certain conditions, it is limiting for only one component. This was revealed by determining the slope of the least-squares log-log regression line generated when the initial seeding density was varied. When support for the growth of the clonable pre-B cells was provided by fetal liver-derived adherent accessory cells, the slope of the regression line was 1, consistent with the interpretation that only one component, the pre-B cell, was limiting under these conditions. This interpretation was tested in the present report by positive selection for cells expressing B220, Lyb-2, or AA4.1, surface markers known to be present on cell lines of the B lineage. In all cases, an increased incidence of clonable pre-B cells was observed. Furthermore, regression analysis of titration experiments undertaken with these enriched populations revealed that the assay was still limiting for a single component. A second set of growth conditions have been defined in which the clonable pre-B assay appears to be limiting for more than one component. These conditions are obtained when the adherent accessory cells are replaced by one of three distinct hemopoietic growth factors, including CSF-1 (macrophage growth factor), GM-CSF (neutrophil/macrophage growth factor), or Multi-HGF (multi-lineage hemopoietic growth factor, also called BPA or IL 3). The most straightforward interpretation of these data is that a second cell type, distinct from the B cell precursor and dependent on the growth factors, was limiting under these conditions. In the present report, this hypothesis was confirmed because growth factor responsiveness was completely absent in B220 and Lyb-2-enriched populations. Factor responsiveness was found in unseparated fetal liver and in AA4-enriched populations. However, the AA4-enriched populations, in contrast to the B220- or Lyb-2-enriched populations, also contained a large number of factor-responsive neutrophil/macrophage colonies, raising the possibility that the effect of factors on AA4+ clonable pre-B cells was accessory cell-mediated.

Plaque formation by B cell colonies.

A method is described for routinely detecting secretion of antibodies by intact B cell colonies grown in soft agar. Protein A-coupled sheep red blood cells and hapten-conjugated sheep red blood cells were found to be suitable targets. Using this assay we have determined that approximately 1 in 3 B cell colonies contain cells secreting IgM while approximately 1 in 6 contain cells secreting IgG. We have also determined the frequency of clones which secrete antibodies which lyse TNP3-SRBC, NIP4-SRBC, NP4-SRBC, FITC5-SRBC, SRBC, and HRBC. This assay can be used to study the mitogen-induced maturation of clonable B cells into plaque-forming cells, to follow antigen-specific clonable B cells during an immune response, and unambiguously to detect B cell colonies when other colony types are present. The method is also suitable for morphological analysis of stained colony cells.

Location of the gene for theta antigen in the mouse. II. Three-point crosses place Thy-1 in proximal region of chromosome 9.

Males of a partially inbred mouse stock homozygous for cw and d were crossed to AKR/ABom females. Progeny obtained by backcrossing heterozygous F1 females to cw d/cw d males were analyzed for the markers cw, Thy-1, d, and Mod-1. Three- and four-point recombination data are consistent with the map: cw--29--Thy-1--12--d--5--Mod-1, in which cw is nearest to the centromere. These recombination data are discussed in relation to previous multiple-point recombination studies of chromosome 9.