Multifunctional CD4+ T cells persist for at least 10 weeks post immunization with Vreps.

<p>Mice were immunized twice at weeks 0 and 3 with Vrep-Luc or Vrep-Acr/Ag85B vaccines as described in Materials and Methods and shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0136635#pone.0136635.t001" target="_blank">Table 1</a>. A) Representative dot plots showing lymphocytes gated as CD3+ CD4+ T cells secreting IFN-γ, TNF-α or IL-2 in response to recall antigen. After gating, Boolean analyses allow determination of concurrent expression of these cytokines. Splenocytes were harvested at either 3 or 10 weeks post-immunization and Acr-specific (B) and Ag85B-specific (C) CD4+ T cells producing IFN-γ, TNF-α and/or IL-2 were assessed by ICS after subtraction of values for media controls in each case. Data shown are numbers of cytokine secreting CD4+ T cells per million CD4+ T cells in splenocytes from individual mice (n = 5) as mean ± SEM in a single experiment that is representative of three such experiments. * p < 0.05, + p < 0.01, ++ p < 0.001 by Student’s t-test. Pie charts under the bar graphs depict relative percentages of CD4+ T cells in each vaccine group that produced all three of these cytokines (3+), two of these cytokines (2+) or a single cytokine (1+), after exposure to recall antigen and were created using the SPICEv4.1.5 and PESTLEv1.5.2 software programs (a gift from M. Roederer, Vaccine Research Center, NIAID/National Institutes of Health).