To investigate the role of CD40 ligand in the delivery of help to B cells, we examined the Ag-specific interaction of B cells from CD40-deficient mice with a Th2 cell line in vitro. Small resting B cells from normal mice are stimulated to synthesize DNA when they present monovalent Ag (rabbit Fab anti-Ig) to a rabbit Ig-specific Th cell line. This response, which is independent of a signal through the B cell Ag receptor (sIg), is nearly absent in B cells from CD40-deficient mice. The CD40-deficient B cells are not defective in Ag presentation because they induce T cell IL-4 synthesis as well as normal B cells. Also, CD40-deficient B cells respond to T cell help with DNA synthesis almost as well as normal B cells if an additional signal is provided through sIg. In conjunction with a sIg signal, cell contact with helper T cells induces DNA synthesis more effectively than soluble cytokines. CD40-independent T cell help can also be measured as an early increase in c-myc mRNA levels in CD40-deficient B cells presenting Ag to helper T cells, although the levels of c-myc RNA expression are lower than those in normal B cells. However, c-myc RNA induced by noncognate interaction with anti-CD3-activated T cells is completely CD40 dependent. We conclude that early growth signals from activated Th cells are received by CD40-/- B cells, but that CD40 and/or sIg signals are required for efficient induction of DNA synthesis.

The regulation of the synthesis of membrane-bound and secreted IgA was investigated in the murine B lymphoma I.29 during the differentiation from IgA-bearing lymphocytes to IgA-secreting cells, as caused by treatment with lipopolysaccharide (LPS). LPS induced a threefold to fivefold increase in the amount of IgA synthesized, and induced a shift from the synthesis of the membrane form of alpha-chain (alpha m) to the synthesis of the secreted form of alpha-chain (alpha s), resulting in a 60-fold increase in the amount of IgA secreted. In vitro translation of sucrose gradient-fractionated RNA indicated that two mRNA molecules, 3.1 and 2.1 kilobase pairs (kb), encode alpha m-chains, whereas a smaller RNA molecule, 1.7 kb, encodes alpha s. Analyses by RNA blotting showed that the relative amounts of the three alpha mRNA changed rapidly during LPS-induced differentiation. The amount of the 3.1 and 2.1 kb alpha mRNA decreased, and the amount of the 1.7 kb alpha s mRNA increased in LPS-stimulated cells as compared with controls. These observations suggest that the regulation of alpha m/alpha s synthesis is controlled mostly at the pretranslational level.

Three different forms of alpha-chains are synthesized by BF0.3 and 615.2, two cloned cell lines derived from the murine B lymphoma 1.29. The three forms of alpha-chains differ in size, pI, cellular location, and rate of turnover. They were identified by means of lactoperoxidase-catalyzed radioiodination, internal 14C or 35S labeling, and immunofluorescence techniques as membrane-bound(alpha m), secreted (alpha s), and intracellular (alpha ic) proteins. Comparison of immunoglobulin products of the two lymphoma lines with those of a hybridoma cell line, Id 150, which secretes IgA of the 1.29 idiotype but lacks membrane IgA, confirmed the assignments of alpha m, alpha s, and alpha ic. Results of biosynthetic labeling of BF0.3, 615.2, and Id 150 in the presence and absence of tunicamycin suggest that the difference in m.w. and charge observed between alpha m and alpha s can be attributed to differences in primary amino acid structure rather than different degrees of glycosylation.

We have compared the synthesis and processing of immunoglobulin alpha chains in two murine cell lines, a B cell lymphoma that expresses membrane-bound IgA and a hybridoma that secretes IgA. Results of biosynthetic labeling experiments demonstrated that membrane-bound and secreted alpha chains have two distinct intracellular precursors, of different molecular weights and isoelectric points. RNAs from both of these cell lines direct the synthesis in vitro of two alpha polypeptides of Mr 59,000 and 62,000, the larger one being the precursor for membrane-bound alpha chain and the smaller one being the precursor for secreted alpha chain. These cell lines each contain three RNAs, 1.7, 2.1, and 3.1 kilobases in length, which hybridize with cDNA for the alpha constant region and which are present in different concentrations. Our results suggest that the smallest RNA encodes the secreted alpha chain and one or both of the larger RNAs encode(s) the membrane-bound alpha chain.