ANTI-CD40 PLUS INTERLEUKIN-4-ACTIVATED HUMAN NAIVE B-CELL LINES EXPRESS UNMUTATED IMMUNOGLOBULIN GENES WITH INTRACLONAL HEAVY-CHAIN ISOTYPE VARIABILITY

Combination of anti-CD40 antibody and interleukin-4 (IL-4) induces B cell clonal expansion reminiscent of the T-dependent proliferation following antigenic challenge in vivo. We have analyzed the usage of C-H genes and the presence or absence of somatic mutations within the progeny of a single human naive B cell activated with anti-CD40-IL-4. To address this issue, single-cell cultures of naive (sIgD(+)) tonsillar B lymphocytes expressing the VH1-restricted G8 idiotype were set up. After culture and RNA extraction, VH1(+) Ig mRNA were reverse-transcribed, amplified by polymerase chain reaction and sequenced. A single sIgD(+) B cell could generate clones expressing mu, gamma 1, gamma 3 or epsilon, illustrating that the progeny of a single cell can express different isotypes in response to the same stimulus in vitro. The rate of somatic mutations affecting the immunoglobulin variable heavy chain gene was indistinguishable from the background of errors introduced by Tag polymerase.