SWITCH CIRCLES FROM IL-4-DIRECTED EPSILON-CLASS SWITCHING FROM HUMAN B-LYMPHOCYTES - EVIDENCE FOR DIRECT, SEQUENTIAL, AND MULTIPLE STEP SEQUENTIAL SWITCH FROM MU TO EPSILON IG HEAVY-CHAIN GENE

Ig isotype switch via deletional recombination is accompanied by excis ion of the intervening DNA between the two switch regions. The excised DNA is looped out as extrachromosomal circular DNA or switch circle. Such switch circles have been isolated and characterized in mice. We i nvestigated deletional recombination in human B cells undergoing Ig is otype switching to demonstrate whether switch circles are also excised , and to thereby gain insight into the processes involved in human iso type switching. We characterized the deleted switch circular DNA from IL-4 directed mu to epsilon switching in polycional human B lymphocyte s. By using two sets of specially designed PCR primers, we amplified s witch circle fragments representing switch circles resulting from mu t o epsilon direct switching and mu-gamma-epsilon sequential switching. The PCR-amplified products were subcloned by a TA cloning strategy and resulting clones were screened by hybridization with a 5'S epsilon pr obe. Sequence analysis of the positive clones revealed that all clones representing mu to epsilon direct switching indeed had 5' S epsilon d irectly joined to 3' S mu. Most clones representing mu-gamma-epsilon s equential switching showed 5' S epsilon joined to 3' S mu as expected. However, two clones contained S mu and S alpha 1 sequences interposed between 5' S epsilon and 3' S gamma, respectively. These data demonst rate that switch circles are excised during human B cell isotype switc hing, and that IL-4 directed epsilon class switching is accomplished b y 1) direct mu to epsilon switching, 2) sequential mu-gamma-epsilon sw itching, and 3) double sequential mu-alpha-gamma-epsilon switching.