Co-incubation with IL-18 potentiates antigen-specific IFN-γ response in a whole-blood stimulation assay for measurement of cell-mediated immune responses in pigs experimentally infected with Lawsonia intracellularis

The whole-blood interferon-gamma (IFN-γ) assay is a quantitative in vitro assay for a direct read-out of Ag-specific cell-mediated immune (CMI) responses to infectious diseases. The IFN-γ assay is robust in severe intracellular infections like Brucella or mycobacteria, but more difficult to evaluate for less severe or immunocompromising infections. Here we investigated the performance of the assay when recombinant co-stimulatory cytokines IL-12 and/or IL-18 were added along with Ag or PBS to cultures of whole-blood from pigs infected with Lawsonia intracellularis. In pigs recovering from a natural infection, addition of rIL-12 or rIL-18 alone increased the Ag-specific IFN-γ release while addition of both cytokines resulted in increased IFN-γ release also in PBS cultures. In analyses after experimental infections with L. intracellularis, significant increased levels of Ag-specific IFN-γ production were measured in Ag + rIL-18 cultures from infected pigs compared to the background response in PBS + rIL-18 control samples ( p < 0.01) or to Ag + rIL-18 cultures from non-inoculated control pigs ( p < 0.05). Flow cytometry identified two lymphocyte subsets as the Ag-specific IFN-γ producers. The highest IFN-γ production was by CD4 +CD8 + cells while a more numerous population of CD4 −CD8 + cells produced lower amounts of IFN-γ in response to rIL-18 and L. intracellularis Ag.