In vitro–differentiated TH17 cells traffic to GVHD target organs and secondary lymphoid organs. Naive CD4⁺ T cells from eGFP⁺ mice were differentiated into TH17 cells as in Figure 1. TH17 cells (1.35 × 10⁶) were transferred with T cell–depleted bone marrow cells into lethally irradiated B6D2 recipients. Seven days after transfer, animals were anesthetized with avertin, and organs were imaged with a Zeiss SteREO Lumar.V12 microscope with eGFP bandpass filter. Brightfield images (left) and GFP images (middle) were taken for each organ. GFP intensities (right) were determined by software analysis: liver (A), lung (B), colon (C), spleen (D), inguinal lymph node (E), and mesenteric lymph node (F). eGFP⁺ TH17 cells were transferred into irradiated B6D2 recipients. Eight days after transplantation, lymphocytes were extracted from the lung and stimulated as in Figure 1 followed by intracellular cytokine staining for IL-17A (G) and IL-17F (H). Plots derived from eGFP⁺ gate. We verified that the eGFP signal documented was not due to background autofluorescence by transfer of non–GFP-expressing T cells and imaging of GVHD target organs and lymphoid tissues as described previously (Figure S2). Original magnification liver = ×40, lung = ×25, colon = ×20, spleen = ×40, ILN = ×45, MLN = ×45.