Fig 2: Intracellular expression of IL-17A and IFN-? by activated bovine T cell subsets. PBMC from four cattle were stimulated with phorbol 12-myristate 13 acetate, ionomycin and brefeldin A in RPMI culture medium for 4 h. Cells were harvested and stained for viability and with mabs specific for cell-surface phenotypic markers and intracellular cytokines as described in Table 3 and “Expression of intracellular IL-17A and IFN-? by bovine and ovine T cell subsets section”. Cells were stained for CD4 with mab CC8-PE at 1:20 dilution (A, D), for CD8ß with mab CC58-PE at 1:20 dilution (B, D) and for WC-1 (?d T cells) with mab CC15-PE at 1:200 (C, E). Intracellular cytokine staining for IL-17A was conducted using mab eBioDEC17-APC at a 1:20 dilution (A–C) and for IFN-? using mab CC302-Alexafluor 647 at a 1:200 dilution (D–F). Data are shown for PBMC from one representative animal of four.

Fig 3: Relative intracellular expression of IL-17A and IFN-? by activated bovine and ovine PBMC. The data sets described in “Expression of intracellular IL-17A and IFN-? by bovine and ovine T cell subsets section” and presented in Figures 5 and 6 are summarised to compare overall intracellular expression of IL-17A (A) and IFN-? (B) by PMA/ionomycin-stimulated bovine and ovine PBMC. Each bar represents the arithmetic mean of four cattle or four sheep and the error bars represent the standard error. The data for total percentage IFN-? and IL-17A expression between species were assessed statistically using two-tailed Mann–Whitney tests allowing for ties.

Fig 4: Measurement and biological function of recombinant bovine and ovine IL-17A and detection of native ovine IL-17A by ELISA. A Detection of rbov and rovIL-17A by ELISA. The supernatants from transfected CHO cells expressing rbovIL-17A or rovIL-17A, or control parent untransfected line (UTF) were serially diluted (Log3 dilutions) and evaluated using the commercial bovIL-17A ELISA. Data presented are optical density (OD) values from the Spectrophotometer at 450 nm. The X-axis displays Dilution 1/X and the Y-axis gives the OD value. Readings from UTF supernatant were below the limit of detection. B Functional activity of rbov and rovIL-17A on bovine embryonic lung cells. Bovine embryonic lung (EBL) cells were stimulated with 100 ng/mL CHO-expressed rbovIL-17A or rovIL-17A or UTF CHO negative control supernatant. Following 24 h incubation, culture supernatants were collected from triplicate cultures then tested for CXCL8 by ELISA. The X-axis displays the bioassay treatments and the Y-axis shows CXCL8 production in pg/mL. Data are the arithmetic mean of three technical replicates with error bars representing the standard error from one of three experiments. CXCL8 expression between treatments was statistically assessed using Kruskal–Wallis test. C Functional activity of rbov and rovIL-17A on ovine ST-6 cells. Ovine ST-6 cells were stimulated with 100 ng/mL CHO-expressed rbovIL-17A or rovIL-17A or UTF CHO supernatant. Following 24 h incubation and culture supernatants collected, tested and analysed as described in Figure 2B. CXCL8 expression between treatments was statistically assessed using Kruskal–Wallis test. D Detection of native ovIL-17A by ELISA. Ovine PBMC were cultured at 2 × 106 cells/mL with or without 5 µg/mL ConA. Culture supernatants were analysed for IL-17A using the bovIL-17A ELISA. Data represent the arithmetic mean of PBMC from six ewes and error bars represent standard error. Data were analysed statistically for significance using the two-tailed Mann–Whitney test.

Fig 5: Intracellular expression of IL-17A and IFN-? by activated ovine T cell subsets. PBMC from four sheep were stimulated with phorbol 12-myristate 13 acetate, ionomycin and brefeldin A in RPMI culture medium for 4 h. Cells were harvested and stained for viability and with mabs specific for cell-surface phenotypic markers and intracellular cytokines as described in Table 3 and “Expression of intracellular IL-17A and IFN-? by bovine and ovine T cell subsets section”. Cells were then stained for CD4 with mab 44.38-PE at 1:20 dilution (A, D), CD8ß with mab CC58-PE at 1:20 dilution (B, D) and WC-1 (?d) with mab CC15-PE at 1:200 (C, E). Intracellular cytokine staining for IL-17A was conducted using mab eBio64DEC17-APC a 1:20 dilution (A–C) and for IFN-? using mab CC302-alexafluor 647 at a 1:200 dilution (D–F). Data shown is for one representative animal out of four.