CD4+ T-cell responses to foot-and-mouth disease virus in vaccinated cattle

Abstract

We have performed a series of studies to investigate the role of CD4(+) T-cells in the immune response to foot-and-mouth disease virus (FMDV) post-vaccination. Virus neutralizing antibody titres (VNT) in cattle vaccinated with killed FMD commercial vaccine were significantly reduced and class switching delayed as a consequence of rigorous in vivo CD4(+) T-cell depletion. Further studies were performed to examine whether the magnitude of T-cell proliferative responses correlated with the antibody responses. FMD vaccination was found to induce T-cell proliferative responses, with CD4(+) T-cells responding specifically to the FMDV antigen. In addition, gamma interferon (IFN-γ) was detected in the supernatant of FMDV antigen-stimulated PBMC and purified CD4(+) T-cells from vaccinated cattle. Similarly, intracellular IFN-γ could be detected specifically in purified CD4(+) T-cells after restimulation. It was not possible to correlate in vitro proliferative responses or IFN-γ production of PBMC with VNT, probably as a consequence of the induction of T-independent and T-dependent antibody responses and antigen non-specific T-cell responses. However, our studies demonstrate the importance of stimulating CD4(+) T-cell responses for the induction of optimum antibody responses to FMD-killed vaccines.

Fig. 1.

Intravenous administration of CD4 mAbs results in a transient depletion of circulating CD4⁺ T-cells in peripheral blood and loss of proliferative response to BHV-1 antigen. Two groups of three calves were vaccinated with commercial BHV-1 vaccine, and subsequently vaccinated with A22 Iraq FMD commercial vaccine (2–3 weeks later). Each animal received intravenous injections of either an isotype-matched control TRT 3 (a and c) or anti-CD4 (b and d) mAb for a period of 4 days, starting the day before FMD vaccination. At various time points prior to and following FMD vaccination, the percentage of CD4⁺ T-cells in PBMC was determined by flow cytometry after staining the cells with either an anti-CD4 (black bars) or TRT1 isotype control (grey bars) mAb (a and b) and the proliferation of PBMC to heat-inactivated BHV-1(1/100, black bars) or EBTr cell lysate (control antigen, 1/100, grey bars) was assessed by [methyl-³H] thymidine incorporation (c and d). Results are expressed as the mean of determinations from individual calves in each group±sem, n = 3.

Fig. 2.

Effect of the CD4⁺ T-cell depletion on VNT and proliferation to FMDV vaccine antigen. Two groups of three calves were treated as described in Fig. 1. (a) VNTs are shown for anti-CD4 mAb (triangles) and TRT3 mAb-(squares) treated calves at time points pre- and post-vaccination. Results are expressed as the mean of determinations (log₂ titres) from individual calves in each group±sem. (b) Total PBMC proliferative responses are shown for TRT3 mAb-treated calves to A22 FMDV vaccine antigen (0.1 µg ml⁻¹, black bars) or BHK-21 cell lysate (0.1 µg ml⁻¹, grey bars) at time points pre- and post-vaccination, as assessed by [methyl-³H] thymidine incorporation. Results are expressed as the mean of determinations from individual calves in each group±sem. (c) Similar to (b), but showing the antigen-specific proliferation of PBMC from CD4 mAb-treated calves. Results are expressed as the mean of determinations from individual calves in each group±sem.

Fig. 3.

Proliferation of T-cell subsets monitored by CFDA SE labelling in response to in vitro restimulation with FMDV vaccine antigen and p252. PBMC from cattle previously vaccinated with O1 Manisa FMD commercial vaccine were labelled with CFDA SE prior to their culture in vitro for 6 days in the presence of medium alone (panel 1), inactivated O1 Manisa FMDV vaccine antigen (panel 2) or FMDV p252 (panel 3). At the end of the culture period, the cells were stained for expression of cell surface differentiation antigens without (a) or with APC-conjugated cc8 (CD4⁺ T-cells, b), cc15 (WC1⁺ γδ T-cells, c) and cc63 (CD8⁺ T-cells, d) mAbs, and analysed by flow cytometry. The percentages of cells in each quadrant are illustrated. One representative dataset (animal FMD 4) from three independent experiments using two animals is shown.

Fig. 4.

Proliferation post-vaccination monitored by thymidine incorporation in response to in vitro restimulation with FMDV vaccine antigen and p252. Calves were immunized with Ova in incomplete Freund’s adjuvant, and subsequently vaccinated with O1 Manisa FMD commercial vaccine (6 weeks later). Total PBMC (a), PBMC depleted of CD4⁺ T-cells (b) and purified CD4⁺ T-cells+APCs (c) were cultured in vitro in the presence of medium alone or optimal concentrations of PWM, ovalbumin (Ova), inactivated O1 Manisa FMDV vaccine antigen (FMDV, 0.001 µg ml⁻¹), FMDV VP1 peptide (p252, 0.01 µg ml⁻¹) or BHK-21 cell lysate (BHK-21, 0.001 µg ml⁻¹). After 6 days, proliferation was assessed by [methyl-³H] thymidine incorporation. Results are expressed as the mean c.p.m. of triplicate determinations±sd. One representative dataset from five different animals is shown.

Fig. 5.

Quantification of IFN-γ production assessed by ELISA in response to in vitro restimulation with FMDV vaccine antigen. Five calves were vaccinated with commercial BHV-1 vaccine, and subsequently vaccinated with O1 Manisa FMD commercial vaccine (6 weeks later). Post-FMD vaccination, PBMC (a), PBMC depleted of CD4⁺ T-cells (b) and purified CD4⁺ T-cells+APCs (c) were cultured in vitro in the presence of medium alone or optimal concentrations of PWM, heat-inactivated BHV-1, EBTr cell lysate (EBTr), inactivated O1 Manisa FMDV vaccine antigen (FMDV, 0.1 µg ml⁻¹) or BHK-21 cell lysate (BHK-21, 0.1 µg ml⁻¹). After 5 days, supernatants were harvested and assayed for IFN-γ by sandwich ELISA. Results are expressed as the mean IFN-γ concentration (ng ml⁻¹) of duplicate determinations±sd for each of the five vaccinated calves. For each culture condition, the five columns represent animals FMD 6–FMD 10.

Fig. 6.

IFN-γ production assessed by intracellular flow cytometry in response to in vitro restimulation with FMDV vaccine antigen. Post-vaccination with O1 Manisa FMD commercial vaccine, PBMC (panel 1, 14 weeks post-vaccination) and purified CD4⁺ T-cells+APCs (panel 2, 24 weeks post-vaccination) were cultured in vitro for 24 (a and b), 48 (c and d), 72 (e and f) and 96 (g and h) h in the presence of medium+IL-12 (a, c, e and g) or optimal concentrations of inactivated O1 Manisa FMDV vaccine antigen (FMDV)+IL-12 (b, d, f and h). At the end of the culture period, the cells were permeabilized, stained intracellularly with either a mouse anti-bovine IFN-γ (cc302) or isotype-matched control mAb (TRT 1, data not shown) followed by incubation with FITC-conjugated goat anti-mouse IgG1 antibody (IgG1 FITC), and analysed by flow cytometry. The percentages of cells in each quadrant are illustrated. [Cells were only stained with FITC-labelled antibodies, the negative FL4-H fluorescence channel (y-axis) was only used for the purpose of creating the dot plots]. One representative dataset (animal FMD 7) from five (panel 1) and two (panel 2) different animals is shown.