A direct method for the calculation of alloreactive CD4(+) T cell precursor frequency

Background. Direct measurement of the precursor frequency of alloreactive C D4(+) T cells has been impossible due to the lack of a specific means of de termining the absolute number of daughter cells generated with each divisio n in a repertoire of stimulated T cells. Methods. Responder lymphocytes were fluorescently labeled and adoptively tr ansferred into irradiated allogeneic stimulator mice or incubated in vitro with irradiated stimulator splenocytes. After a 65- to 70-hr stimulation pe riod, responder cells were analyzed by flow cytometry. Results. The precursor frequency of dividing CD4(+) T cells was determined both in vivo and in vitro. The observed number of alloreactive daughter cel ls generated with each round of division was used to calculate the frequenc y of alloantigen-specific CD4(+) T cells. Conclusions. A novel method for the direct calculation of the frequency of alloreactive CD4(+) T cells is described. This technique allows the determi nation of changes in the frequency of alloreactive T cells that might under lie tolerance 60 alloantigens.