Complex mechanisms for inhibition of immunoglobulin gene expression in a germinal center B cell line.

CD40 ligation and IL-4 stimulation are critical Th2 cell-derived signals that act on germinal center B cells to stimulate immunoglobulin isotype switching. In addition to this well-known effect, these same Th2 signals have also been reported to inhibit ongoing immunoglobulin synthesis in germinal center B cells. To study the mechanism of this inhibition, we have investigated which immunoglobulin gene regulatory regions might be affected by IL-4 and CD40 Ligand (CD40L). CL-01 cells, a human B cell line of germinal center phenotype, were transiently transfected with luciferase reporter constructs containing various light and heavy chain enhancers and promoters; the cells were then incubated with or without CD40L and IL-4 and then assayed for luciferase expression. We find that the intronic enhancer of the kappa light chain (but not the heavy chain) is upregulated by CD40 ligation, but that VH and Vkappa promoters and the 3' enhancers of both the kappa and heavy chain loci are inhibited by CD40 ligation and the Th2 cytokines IL-4 and IL-10. The inhibitory response of the 3'alpha enhancer can be observed with a 130 bp core fragment of the enhancer, and remains unaffected by mutations in several motifs known or suspected to contribute to enhancer function. The ultimate effects of cytokines and CD40 ligation on immunoglobulin gene transcription therefore represent a complex integration of positive and negative stimuli acting on enhancers and promoters.

Detection of isotype switch rearrangement in bulk culture by PCR.

When a B lymphocyte changes from synthesizing IgM to synthesizing IgG, IgA, or IgE, this isotype switch is generally accompanied by a unique DNA rearrangement. The protocols in this unit describe two polymerase chain reaction (PCR)-based strategies for detecting switch rearrangements in bulk culture. The first involves direct PCR across the switch junctions, providing the opportunity for characterizing the recombination products by nucleotide sequence analysis; however, because of characteristics inherent to the PCR methodology this strategy cannot easily be used as a quantitative assay for recombination. A support protocol details the preparation of the 5' Su PCR probe for this protocol. The second basic protocol describes a method known as digestion-circularization PCR (DCPCR) that is more amenable to quantitation but yields no information on structure of the recombination products. Both techniques should be capable of detecting reciprocal deletion circles as well as functional recombination products remaining on the expressed chromosome.