CD154 Expression Indicates T Cell Activation Following Tetanus Toxoid Vaccination of Horses

Despite the relevance of adaptive immunity against equine pathogens antigen-specific T cell responses of horses are not well characterized and the lack of insight into T cell responses hampers the understanding of the pathogeneses of important diseases. In this study we used tetanus toxoid (TT) as a well-defined antigen to characterize antigen-reactive T cells. Six healthy adult horses received a routine booster against tetanus with an immune stimulating complex (ISCOM)-based vaccine and were followed for 28 days. TT-specific serum antibodies were quantified by ELISA and increased in all horses by day 7 after vaccination. CD154 is an established indicator of antigen-reactive T helper cells in other species, but has not been characterized in horses. CD154 detection in equine PBMC by an anti-human CD154 antibody (clone 5C8) was confirmed by Western blots and then applied for flow cytometry. As a common indicator of equine T cell activation, cytokine induction was studied in parallel. T cells were analyzed by multicolor flow cytometry of PBMC after re-stimulation with TT in vitro. Reactive T helper (Th) cells were characterized by increased frequencies of CD4(+)CD154(+) lymphocytes in in vitro TT-re-stimulated PBMC on day 14 after vaccination of the horses compared to pre-vaccination. The majority of all CD154(+) cells after TT re-stimulation were CD4(+) Th cells, but CD154 was also induced on CD4(-) cells albeit in lower frequencies. CD154(+)CD4(+) Th cells were enriched in cytokine-expressing cells compared to CD154(-)CD4(+) Th cells. Similar to the CD4(+)CD154(+) frequencies, CD4(+)IL-4(+), CD4(+)IFN-γ(+) and CD4(+)TNF-α(+) were increased after vaccination, but IL-4(+) increased later than IFN-γ(+) and CD4(+)TNF-α(+), which already exceeded pre-vaccination frequencies on day 7. CD4(+)CD154(+) frequencies correlated positively with those of CD4(+)IL-4(+) (Th2) on day 14, and negatively with CD4(+)IFN-γ(+) induction on day 7, but did not correlate with CD4(+)TNF-α(+) frequencies or TT-specific antibody concentrations. CD154 appears to be a useful marker of antigen-reactive equine Th cells in combination with cytokine expression. The T cell analyses established here with TT can be applied to other antigens relevant for infections or allergies of horses and in horse models for translational research.