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

Abstract

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.

Comprehensive Flow Cytometric Characterization of Bronchoalveolar Lavage Cells Indicates Comparable Phenotypes Between Asthmatic and Healthy Horses But Functional Lymphocyte Differences

“…Co-expression of CD154 and the Th subset differentiating cytokines IFN-γ, IL-4, and IL17A was usually observed in BAL cells and PBMC, but was incomplete, i. e. not all cytokine expressing Th cells simultaneously expressed CD154 ( Supplementary Figures 7A–C ), as previously described for equine PBMC ( 25 ). The frequencies of CD154 + lymphocytes co-expressing different cytokines in BAL and PBMC samples ( Supplementary Figures 7A–C ) showed distributions between the groups comparable to all cytokine-positive lymphocytes ( Figure 6 ).…”

“…Beyond demonstrating similar frequencies of T helper cells, cytotoxic T cells and B cells in samples from all groups ex vivo , we aimed to functionally characterize these lymphocytes by analysis of the T helper cell activation marker CD154 (CD40L) ( 25 , 31 ) and expression of five different cytokines after in vitro stimulation with PMA and ionomycin ( Figures 6A, B ). In vitro stimulation was performed for samples from 26 horses (n=4–7 per group) with sufficient yield of BAL cells, and only groups with five or more values (healthy, mEA, sEA) were compared statistically.…”

“…Apart from the composition of BAL cells from healthy and asthmatic horses, functional characterization of lymphocytes was carried out to elucidate mechanisms contributing to the sustained inflammation present in EA. We analyzed lymphocytes’ expression of cytokines, and the T helper cell activation molecule CD154 ( 25 , 41 ) after stimulation with PMA and ionomycin. Lymphocytes expressing the pro-inflammatory cytokines TNF-α and IFN-γ after stimulation were overall present at similar frequencies in BAL cells from horses of all groups, except for a trend of increased Th1 activation (CD4 + IFN-γ + ) in BAL lymphocytes from horses with sEA.…”

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“…Co-expression of CD154 and the Th subset differentiating cytokines IFN-g, IL-4, and IL17A was usually observed in BAL cells and PBMC, but was incomplete, i. e. not all cytokine expressing Th cells simultaneously expressed CD154 (Supplementary Figures 7A-C), as previously described for equine PBMC (25). The frequencies of CD154 + lymphocytes coexpressing different cytokines in BAL and PBMC samples (Supplementary Figures 7A-C) showed distributions between the groups comparable to all cytokine-positive lymphocytes (Figure 6).…”

“…Apart from the composition of BAL cells from healthy and asthmatic horses, functional characterization of lymphocytes was carried out to elucidate mechanisms contributing to the sustained inflammation present in EA. We analyzed lymphocytes' expression of cytokines, and the T helper cell activation molecule CD154 (25,41) after stimulation with PMA and ionomycin. Lymphocytes expressing the proinflammatory cytokines TNF-a and IFN-g after stimulation were overall present at similar frequencies in BAL cells from horses of all groups, except for a trend of increased Th1 activation (CD4 + IFN-g + ) in BAL lymphocytes from horses with sEA.…”