Clonal endurance and clonal burst size: novel indices of helper T cell heterogeneity

This report presents a method for examining the behavior of individual antigen-responsive helper T cell precursors (pHTL), following their repeated encounter with antigen. In our model system, lymph node cells from mice immunized with keyhole limpet hemocyanin (KLH) are cultured in vitro under limiting dilution conditions, using responder cell doses chosen to ensure that each culture well contains, at the outset, at most a single KLH-reactive helper T cell precursor. Those wells which in fact did receive a KLH-reactive pHTL are identified by the accumulation of the helper T cell product, interleukin 3 (IL3), during a 6-day culture interval. The lymphocytes generated during this culture interval are washed, divided into replicate aliquots, and then further cultured (with and without additional KLH) to see if the original pHTL has generated cells which are capable of further lymphokine production. Using this approach, we found a surprising amount of variation from clone to clone in two measurements. Clones differed greatly in "endurance", that is in the ability of progeny cells to continue to secrete IL3 in the absence of additional antigenic stimulation. Among 148 clones tested, 90 were found not to continue lymphokine secretion in antigen-free secondary cultures, while IL3 production among the other 58 clones varied from 1 to 128 arbitrary units. Individual clones also varied greatly with respect to their ability to secrete additional IL3 in response to additional antigen, and in "burst size", that is in the number of new, antigen-responsive helper cell precursors which they can generate during the initial culture interval; 43% of the clones produced no new pHTL, while others produced as many as 800 new pHTL cells in an 8-day period. Neither measure correlates strongly with the amount of lymphokine originally produced by the initial "parental" pHTL. This method will allow us to examine the factors which influence the allocation of clonal progeny cells into lymphokine producing effectors and antigen-sensitive "memory" T cells.