Journal: Immunology
Article Title: 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
doi: 10.1111/imm.13367
Figure Lengend Snippet: 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 upper right quantile. Isotype control antibodies (Rat IgG2aκ antibody–Alexa 647 and mouse IgG3 antibody–PerCP/Cy5·5) were used to decide the quantile line. In each quantile compartment, EM denotes effector memory T cells, CM central memory T cells, EF effector T cells and NA naïve T‐cell populations. These dot plots are representative of two independent experiments using two animals
Article Snippet: Rat IgG2aκ isotype control (BD Pharmingen) , R35‐95, IgG2aκ , Alexa 647(BD Pharmingen).
Techniques: Ex Vivo, Staining, Selection, Flow Cytometry
