Number of interleukin-4- and interferon-γ-secreting human T cells reactive with tetanus toxoid and the mycobacterial antigen PPD or phytohemagglutinin: distinct response profiles depending on the type of antigen used for activation

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Abstract
The enzyme-linked immunospot (ELISPOT) assay has been proven to be an efficient and sensitive method for the enumeration of single cells secreting antibodies or cytokines. Here we have used this method to determine the number of interleukin-4 (IL-4)- and interferon-γ (IFN-γ)-producing cells in in vitro secondary responses to tetanus toxoid (TT) and the mycobacterial antigen (purified protein derivative; PPD) or the mitogen phytohemagglutinin (PHA). PHA-induced IL-4 and IFN-γ secretion was well correlated suggesting polyclonal activation of cells. This was not the case with the specific antigens, where PPD preferentially induced IFN-γ- and very few IL-4-producing cells, while TT-induced both IL-4 and IFN-γ. These differences are probably a reflection of the types of immunity the two antigens induce, mycobacteria preferentially inducing a cell-mediated T helper type 1 (Th 1) type of immunity, while immunity to tetanus is an antibody-dependent, Th 2 type of response. In individuals recently boosted with TT, a significant increase in both IL-4- and IFN-γ-producing cells in response to TT was seen at day 7 after boost, followed by decline. This was in contrast to what was seen in response to PPD where an increase of IFN-γ-producing cells after the TT boost at day 7 persisted for at least 14 days. These results suggest that after an in vivo boost both antigen-specific and nonspecific T cells are activated and that antigen-specific cells home to other organs and therefore may be difficult to demonstrate in the circulation. Our data show that the ELISPOT assay is a powerful tool for determining the frequency of cells secreting cytokines. The assay has several advantages over other assays since it is sensitive, measures the number of actually secreting cells, and avoids the problems of binding of cytokines to their cell-bound or soluble receptors.