The detection of long‐lasting memory foot‐and‐mouth disease (FMD) virus serotype O‐specific CD4⁺ T cells from FMD‐vaccinated cattle by bovine major histocompatibility complex class II tetramer

Figures

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  FIGURE 1. Identification of DRA/DRB3*020:02 molecule‐restricted CD4+ T‐cell epitopes within the FMDV capsid. PBMCs from animal FMD9, heterozygous for the MHC II DRB3*020:02 allele, were stimulated with inactivated, intact FMDV (strain O1/Manisa) antigen for 6 days and then incubated for a further 6 to 10 days in the presence of rboIL‐2. PBMCs were then stained with pooled (a) or individual (b) peptide‐loaded tetramers and analysed by flow cytometry. Cells were gated on lymphocytes, singlets and then live cells. Black circles indicate CD4+ tetramer+ cell populations, confirming the recognition of a FMDV capsid epitope–tetramer complex by CD4+ T cells. Data sets for VP1 to VP3, and for VP4, FMDV structural proteins were acquired on two separate days. These data are representative of two independent experiments using two animals

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  FIGURE 2. Position of identified epitopes on the protomer of FMDV capsid. Structures (a‐d) are based on the protein data bank: 5nej.1, FMDV‐O1/Manisa model [30] and depict the externally viewed capsid. The structural proteins VP1, VP2, VP3 and VP4 are shown in blue, green, red and grey, respectively. Yellow surface regions indicate the locations of identified epitopes. (a) p220 (VP3: 116‐130), (b) p245 (VP1: 32‐45), (c) p248 (VP1: 46‐60) and (d) p432 (VP4:71‐85)

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  FIGURE 3. Tetramer staining was specific and correlated with CFSE proliferation results. CD4+ T‐cell‐enriched cell populations were stained with CFSE, then stimulated with individual peptide (p220, p248 or p432) or intact, inactivated FMDV and cultured for 6 days. After staining with individual peptide‐loaded tetramer–PE and anti‐CD4‐Alexa 647, data were acquired by flow cytometry. (a) Dot plots showing the gating strategy employed for CD4+ T‐cell identification. (b) Histograms and dot plots showing CFSE proliferation assay results gated on CD4+ T cells. Histogram percentages represent the ratio of CFSElow cells in CD4+‐gated cell populations. Each row of dot plots represents the analyses of cells stimulated with individual peptide or intact capsid antigen using the indicated peptide‐loaded tetramer. The x‐axis represents CFSE staining, with proliferating cells exhibiting a lower intensity of CFSE. Black circles indicate proliferating CD4+ T‐cell populations for each peptide. These data are representative of three independent experiments using two animals

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  FIGURE 4. Intracellular cytokine staining of peptide‐expanded CD4+ T‐cell‐enriched populations. CD4+ T‐cell‐enriched populations were expanded over a 4‐week period and then cultured with autologous PBMCs overnight in the presence or absence of respective peptide. Cells were then treated with brefeldin A for 4 hours to prevent cytokine release prior to staining for the detection of cytokines (IFN‐gamma, IL‐4 and IL‐17A) by flow cytometry. Isotype controls were used to decide the quantile line. Cytokine production by (a) p220‐, (b) p248‐ and (c) p432‐specific CD4+ T‐cell populations expanded from FMD9 PBMCs. Cells were gated on lymphocytes, singlets and then CD4+ cells. The left plots in each panel show the staining results obtained in the absence of stimulation and the right plots after stimulation with the indicated peptide

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  FIGURE 5. Frequency and phenotype of non‐expanded FMDV epitope‐specific CD4+ T‐cell populations ex vivo. Non‐expanded PBMCs were stained with loaded tetramer in combination with a cocktail of mAbs for negative selection (CD8, TCR1‐N24 delta chain, CD14 and CD21), mAbs for the detection of CD4, CD45RO and CCR7, and the fixable viability dye eFluor 780. Data were then acquired by flow cytometry. (a) Dot plots, using data obtained with p220‐loaded tetramer–PE, showing the gating strategy for receptor phenotyping of non‐expanded FMDV‐specific CD4+ T cells ex vivo. (b‐h) The memory phenotype of individual peptide‐specific CD4+ T cells is shown for each animal (FMD7 or FMD9) and peptide‐loaded tetramer. Red dots indicate FMDV peptide‐specific CD4+ T cells in PE‐enriched PBMC aliquots, and grey dots indicate CD4+‐gated cells in pre‐enriched PBMC aliquots. The frequency of peptide‐specific CD4+ T cells, per million CD4+ T cells in the respective PBMC population, is shown below each animal‐peptide name. The x‐axis represents CCR7‐Alexa 647 and y‐axis CD45RO‐PerCP/Cy5·5 staining. The ratio of CCR7 to CD45RO double‐positive population in peptide‐specific CD4+ T cells is shown as a red letter in the…