ANALYSIS OF SOMATIC MUTATION IN 5 B-CELL SUBSETS OF HUMAN TONSIL

Using a series of phenotypic markers that include immunoglobulin (Ig)D , IgM, IgM, IgG, CD23, CD44, Bcl-2, CD38, CD10, CD77, and Ki67, human tonsillar B cells were separated into five fractions representing diff erent stages of B cell differentiation that included sIgD(+) (Bm1 and Bm2), germinal center (Bm3 and Bm4), and memory (Bm5) B cells. To esta blish whether the initiation of somatic mutation correlated with this phenotypic characterization, we performed polymerase chain reaction an d subsequent analysis of the Ig heavy chain variable region genes from each of the B cell subsets. We studied the genes from the smallest VH families (V(H)4, V(H)5, and V(H)6) in order to facilitate the mutatio nal analysis. In agreement with previous reports, we found that the so matic mutation machinery is activated only after B cells reach the ger minal center and become centroblasts (Bm3). Whereas 47 independently r earranged IgM transcripts from the Bm1 and Bm2 subsets were nearly ger mline encoded, 57 point mutations within the VH gene segment. gamma tr anscripts corresponding to the same VH gene families were isolated fro m subsets Bm3, Bm4, and Bm5, and had accumulated an average of 9.5 som atic mutations. We conclude that the molecular events underlying the p rocess of somatic mutation takes place during the transition from IgD( +), CD23(+) B cells (Bm2) to the IgD(-), CD23(-), germinal center cent roblast (Bm3). Furthermore, the analysis of Ig variable region transcr ipts from the different subpopulations confirms that the pathway of B cell differentiation from virgin B cell throughout the germinal center up to the memory compartment can be traced with phenotypic markers. T he availability of these subpopulations should permit the identificati on of the functional molecules relevant to each stage of B cell differ entiation.