The interaction of specific T-cell help and non-specific B-cell growth factors in the production of anti-tetanus antibody by human B cells grown in serum-free microcultures

Apart from a brief period after in-vivo immunization, only a minority of human donors provide peripheral lymphocytes that synthesize specific antibody on stimulation with tetanus toxoid in vitro. A 20 μl hanging drop microculture technique using serum-free medium has been adapted to analyse the conditions under which B cell mature into antibody-secreting cells. Multiple permutations of antigen dose, cell concentration and T:B cell ratios have been examined. The results indicate that in-vitro failure of antigen response by the majority of donors is not due simply to an inappropriate choice of culture conditions. The addition to antigen-stimulated cultures of lectin-free conditioned medium derived from pokeweed mitogen-stimulated peripheral lymphocytes, enables B cells from the majority of donors to produce high titres of specific antibody, in a T-dependent manner, for up to 24 months after immunization. The observed failure of prolonged antigen responsiveness in vitro thus appears to represent a failure to expand a population of antigen-specific B cells, rather than indicating an absence of such clones.