Only directly infected APCs activate CD8⁺ T_EFF cells.

<p>(<b>A</b>,<b>B</b>) Mice were subjected to skin infection with HSV.CFP and 3 days later received <i>in vitro</i> activated GFP⁺ gBT-I effector cells. Intravital two-photon microscopy of infected skin 5 days post-infection. (<b>A</b>) Maximum intensity projection image corresponding to <b><a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004303#ppat.1004303.s009" target="_blank">Movie S1</a></b>. Yellow circle indicates area with slow moving gBT-I cells. 2HG, second harmonic generation signal; scale bar, 50 µm. (<b>B</b>) Mean velocities of gBT-I cells in contact with (Proximal) or distal to (Distal) virus-infected cells from one movie, representative of 8 movies from 4 individual mice. (<b>C</b>) Mice received naïve GFP⁺ gBT-I cells prior to HSV-1 skin infection. IFM analysis of skin stained with anti-HSV-1 and -IFN-γ antibodies. Yellow arrow indicates IFN-γ⁺, red arrows IFN-γ⁻ gBT-I cells. Scale bar, 50 µm. Insert (<b>C-ii</b>) from indicated area in <b>C-i</b>, without green and red channels. Photos representative of <i>n</i> = 10 sections from 2 individual mice. (<b>D</b>–<b>H</b>) Mice were subjected to skin infection with HSV.WT or HSV.GFP. (<b>D</b>) Analysis of GFP⁺ keratinocytes (KC, CD45.2⁻EpCAM⁺), MHC-II^hi cells (CD45.2⁺MHC^hiEpCAM⁻), DETCs (CD45.2⁺Vγ3⁺), Langerhans cells (LC, CD45.2⁺MHC-II^hiEpCAM⁺) and residual CD45.2⁺ cells (MHC-II⁻Vγ3⁻) from epidermal sheets 5 days post-infection. Plots representative of <i>n</i> = 5 mice. (<b>E</b>) Sorting strategy used to purify epidermal GFP⁺ (HSV) and GFP⁻ (Ctrl) DETCs, MHC-II^hi cells and keratinocytes (pooled from 20 mice) 4 days post-infection. (<b>F–H</b>) Analysis of IFN-γ⁺ <i>in vitro</i> activated T_EFF cells cultured (as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004303#ppat-1004303-g005" target="_blank"><b>Figure 5</b></a>) with GFP⁺ or GFP⁻ MHC-II^hi cells (8–13×10⁴), DETCs (7–10×10⁴) and keratinocytes (14–16×10⁴) from epidermal sheets (pooled from 20 mice) 4 days after infection. Data representative (<b>F</b>,<b>G</b>) or pooled from 2–4 experiments (<b>H</b>).