Different response requirements for IFNgamma production in ELISPOT assays by CD4+ T cells from mice early and late...

Antigen specific immune responses that occur early after antigen exposure differ from those that are present late in the response. The present study focused on detecting changes in production of IFNgamma by CD4+ T cells over time during chronic antigen exposure. (C3HxCB17)F1 mice were primed with recombinant hepatitis B core antigen (rHBcAg) in incomplete Freund's adjuvant to allow persistent antigen exposure. To assay the CD4+ T cell response to HBcAg, splenocytes from immunized mice were restimulated with rHBcAg for 24 or 48 h in vitro and tested for IFNgamma and IL-5 secreting cells by ELISPOT. Results showed that early after antigen exposure (7 days for primary and 3 days for secondary exposures), the maximal number of IFNgamma secreting cells was detected in the ELISPOT after 24 h of restimulation. However, late after antigen exposure (28 days for primary and 14 days for secondary exposures), the maximum number of IFNgamma secreting cells was not detected until 48 h of restimulation in this assay. This delay in IFNgamma production was related to the availability of IL-2, since addition of IL-2 allowed the delayed cells from late responses to develop peak IFNgamma production in vitro by 24 h, equivalent to that of cells from early responses. This IL-2 dependent delay occurred in Th1-type IFNgamma responses but not in Th2-type IL-5 responses. These observations indicate that, when detecting IFNgamma secreting cells it is important to screen responses at different times of restimulation or in the presence and absence of IL-2 to ensure optimal detection. This approach should prove critical, particularly when evaluating patients with chronic infections and in determining the effectiveness of vaccines since these deal with both early and late responses.