Fig. S1. Livers and spleens were harvested from human carboxyfluorescein succinimidyl ester (CFSE)-injected NOD.Cg-Prkdcscid Il2rgtm1Sug/JicTac (NOG) mice at the time of 20% weight loss. (a) Liver leucocytes from naive or humanized NOG were isolated using 33·75% isotonic Percoll gradient (Amersham Biosciences) and counted. (b) Liver leucocytes and splenocytes were analysed by flow cytometry for the indicated surface markers. hCD45+ cells are shown as the percentage of live and single cells based on forward/side-scatter (FSC/SSC) properties and exclusion of dead cells by LIVE/DEAD® Fixable Near-IR Dead Cell Staining (Invitrogen). CD3+ and CD19+ cells are shown as the percentage of hCD45+ and CD4+ and CD8+ cells are shown as te percentage of CD3+ cells.


Fig. S2. Plasma was collected from groups of human peripheral blood mononuclear cell (PBMC)-injected NOD.Cg-Prkdcscid Il2rgtm1Sug/JicTac (NOG) mice on the indicated days post-injection of PBMCs. Plasma samples were analysed for hIgG using Easy-Titer® human immunoglobulin (Ig)G (H + L) assay kits (Thermo Scientific), according to the manufacturer's instructions. Plasma samples from naive NOG mice were all below detection in the assay (data not shown).


Fig. S3. Splenocytes isolated from human blood mononuclear cell (PBMC)-injected NOD.Cg-Prkdcscid Il2rgtm1Sug/JicTac (NOG) mice at the time of 20% weight loss or untouched human PBMCs (frozen) were labelled with carboxyfluorescein succinimidyl ester (CFSE). Cells were stimulated in vitro for 72 h with plate-bound anti-CD3 and soluble anti-CD28, or the indicated cytokines and proliferation were measured as percentage CFSElow per number of cell divisions. (a) Histograms show the gating of each cell division in unstimulated (left) and stimulated (right) CD4+ and CD8+ T cells from PBMCs. (b) Bar plots show the mean ± standard deviation (s.d.) of the percentage of CFSElow cells per number of cell divisions of cells from PBMCs or humanized spleens in duplicate wells and is representative of two individual experiments. The horizontal bar in (b) indicates the level of CFSE dilution in cells from unstimulated wells. (c) Magnetically purified CD8+ and CD4+ T cells isolated from spleens of humanized mice at the time of graft-versus-host disease (GVHD) were stimulated with the indicated cytokines. Cells were incubated for 72 h and proliferation was assayed by [3H]-thymidine incorporation for the last 18 h. Data were measured on a top-counter (Perkin-Elmer) and the graph shows mean ± standard error of the mean (s.e.m.) of triplicate wells. Representative of four independent experiments.

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