Only directly infected APCs activate CD8+ TEFF cells.

(A,B) Mice were subjected to skin infection with HSV.CFP and 3 days later received in vitro activated GFP+ gBT-I effector cells. Intravital two-photon microscopy of infected skin 5 days post-infection. (A) Maximum intensity projection image corresponding to Movie S1. Yellow circle indicates area with slow moving gBT-I cells. 2HG, second harmonic generation signal; scale bar, 50 µm. (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. (C) 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 (C-ii) from indicated area in C-i, without green and red channels. Photos representative of n = 10 sections from 2 individual mice. (D–H) Mice were subjected to skin infection with HSV.WT or HSV.GFP. (D) Analysis of GFP+ keratinocytes (KC, CD45.2−EpCAM+), MHC-IIhi cells (CD45.2+MHChiEpCAM−), DETCs (CD45.2+Vγ3+), Langerhans cells (LC, CD45.2+MHC-IIhiEpCAM+) and residual CD45.2+ cells (MHC-II−Vγ3−) from epidermal sheets 5 days post-infection. Plots representative of n = 5 mice. (E) Sorting strategy used to purify epidermal GFP+ (HSV) and GFP− (Ctrl) DETCs, MHC-IIhi cells and keratinocytes (pooled from 20 mice) 4 days post-infection. (F–H) Analysis of IFN-γ+ in vitro activated TEFF cells cultured (as in Figure 5) with GFP+ or GFP− MHC-IIhi 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 (F,G) or pooled from 2–4 experiments (H).