Rapidly recruited antigen-specific cells confer protection and are not derived from central memory cells stimulated by challenge.

<p>(<b>A–F</b>) CD4⁺CD44⁺CD62L⁺ (T_CM) and CD4⁺CD44⁺CD62L⁻ T cells were FACS sorted from chronic congenic mice, labeled with VIOLET proliferation dye and co-transferred into naïve UB-gfp mice (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004538#ppat.1004538.s002" target="_blank">Fig. S2</a>). One day post-transfer recipient mice were challenged with <i>L.m</i>. Ears or purified CD4⁺ T cells from the spleen or dLNs of recipient mice were analyzed at the indicated time-points post-challenge by flow cytometry (histograms and dot-plots gated on TCRβ⁺CD4⁺ donor cells). (<b>A</b>) Representative FACS plots of CD44 and CD62L expression on CD4+ T cells from chronic mice pre- and post-sorting. (<b>B and C</b>) Recipient mice were co-transferred with equivalent numbers (3×10⁶) of T_CM and CD4⁺CD44⁺CD62L⁻ T cells. Recipient mice were analyzed on day 3 post-challenge. *, p = 0.028. (<b>D–F</b>) Sorted cells were co-transferred at their physiological pre-transfer ratio (1–1.5×10⁶ T_CM; 3–4×10⁶ CD4⁺CD44⁺CD62L⁻). At the indicated times post-infection cells from recipient mice were subjected to short-term (8 hours) Ag re-stimulation. (<b>D</b>) Representative dot-plots of IFN-γ production and dilution of VIOLET proliferation dye from a single-mouse. Numbers in dot plots represent frequencies. (<b>E</b>) Pooled analysis of the proliferation history of transferred, IFN-γ⁺, cells on day 5 p.i. Data is pooled from three independent experiments. ***, p = 0.005; **, p<0.008; *, p = 0.012. (<b>F</b>) Total number of transferred cells per ear over time. Data is pooled from three independent experiments. (<b>G</b>) Sorted cells were independently transferred into naïve recipient mice at their physiological ratio. Three weeks post challenge with 500 <i>L.m.</i> metacyclic promastigotes the ears and dLN were analyzed for parasite burden by limiting dilution analysis. *, p = 0.022 (ears) or p = 0.028 (dLN) versus the naïve group. Data is representative of two or more independent experiments.