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Tracking membrane and secretory immunoglobulin alpha heavy chain mRNA variation during B-cell differentiation by
S D Xiang1, E M Benson, I S Dunn
1The Institute for Immunology and Allergy Research, Westmead Hospital, New South Wales, Australia.
Primary transcripts for all Ig heavy chain isotypes are alternatively processed to encode either secreted or membrane forms of the same antibody and, in plasma cells, a shift towards the secreted form occurs. In principle, measuring the relative quantities of secreted and membrane forms for a particular isotype could monitor B-cell plasmacytoid differentiation. Ratios of alpha heavy chain mRNA secreted (alphas) to membrane (alpham) form were assessed by quantitative reverse transcriptase-polymerase chain reaction (RT-PCR; TaqMan) using an IgA plasma cell line (NCI-H929), a surface IgA+ line (Dakiki) and human tonsillar B cells. While NCI-H929 cells showed the highest alphas: alpham ratio as expected, alphas mRNA predominated for all unstimulated B cells and Dakiki cells. Treatment of B cells and Dakiki cells with IL-2 and IL-10 resulted in a further progression towards the alphas form, correlating with increased human plasma cell antigen-1 (HPC1) mRNA levels. However, alpha mRNA processing and HPC1 expression were independently regulated, as IFN-gamma treatment suppressed HPC1 levels while increasing alphas: alpham ratios. Cytokine-mediated increases in the alphas: alpham ratio resulted from strongly enhanced levels of alphas with relatively constant alpham values. Differentiation-related changes in mRNA processing can thus be tracked by automated quantitative PCR.
Primary transcripts for all Ig heavy chain isotypes are alternatively processed to encode either secreted or membrane forms of the same antibody and, in plasma cells, a shift towards the secreted form occurs. In principle, measuring the relative quantities of secreted and membrane forms for a particular isotype could monitor B-cell plasmacytoid differentiation. Ratios of alpha heavy chain mRNA secreted (alphas) to membrane (alpham) form were assessed by quantitative reverse transcriptase-polymerase chain reaction (RT-PCR; TaqMan) using an IgA plasma cell line (NCI-H929), a surface IgA+ line (Dakiki) and human tonsillar B cells. While NCI-H929 cells showed the highest alphas: alpham ratio as expected, alphas mRNA predominated for all unstimulated B cells and Dakiki cells. Treatment of B cells and Dakiki cells with IL-2 and IL-10 resulted in a further progression towards the alphas form, correlating with increased human plasma cell antigen-1 (HPC1) mRNA levels. However, alpha mRNA processing and HPC1 expression were independently regulated, as IFN-gamma treatment suppressed HPC1 levels while increasing alphas: alpham ratios. Cytokine-mediated increases in the alphas: alpham ratio resulted from strongly enhanced levels of alphas with relatively constant alpham values. Differentiation-related changes in mRNA processing can thus be tracked by automated quantitative PCR.