Clinical application of responder cell frequency estimates with four years of follow up

Limiting dilution cultures have been used to estimate the frequency of T cells which respond to antigen stimulation in vitro, but nothing is known of the reproducibility of this assay when applied to human blood. We developed a simplified form of limiting dilution culture in which blood mononuclear cells were diluted in round bottom 96-well plates and cultured for 10 days. The assay was used to estimate the frequency of blood mononuclear cells proliferating in response to varicella-zoster virus antigen. 250 subjects aged 25-87 years were studied: 95 of these subjects had annual measurements repeated over a 4 year period. The coefficients of variation for intra-assay replicates was 5%, and for inter-assay comparisons was 17.6% and 26% for tests at a 1 or 3 month interval respectively. For subjects studied at 1 year intervals the coefficient of variation was 42% with the total variability equally distributed between the variation between subjects and the variation within subjects. These data provide the first quantitative data to validate limiting dilution cultures for the long term in vitro measurement of human T cell responses.