Development of CD8- a Positive Dendritic Cells from a Common Myeloid Progenitor David Traver, Koichi Akashi, Markus Manz, Miriam Merad, Toshihiro Miyamoto, Edgar G. Engleman, and Irving L. Weissman

Supplementary Material

Supplemental Figure 1. CMP-derived CD8a⁺ DCs are functional APCs. (A) DC function as measured by IL-2 release from OVA-specific T cells. CMP-derived splenic CD8A⁺DCs were placed in overnight cultures containing 10 ng/ml granulocyte-macrophage colony-stimulating factor (GM-CSF; Peprotech) and 300 mg/ml ovalbumin (OVA; Sigma). Cells were extensively washed then cultured in the presence of 5 × 10⁵ OVA-specific, class I-restricted B3Z T cells for 24 hours. IL-2 production from B3Z cells was measured by enzyme-linked immunosorbent assay (ELISA; Pharmingen). (B) DC activation as measured by IL-12 secretion. IL-12 production by sorted DCs was similarly measured by ELISA after 24-hour cultures containing lipopolysaccharide (100 ng/ml; Sigma). (C and D) Allostimulatory potentials of sorted DCs in the MLR. (C) MLR with splenic CMP-derived CD8a⁺ DCs. CD8A⁺DCs were sorted from recipients transplanted with C57Bl/Ka (H-2^b) CMPs, irradiated (30 Gray), and mixed with whole splenocytes from Balb/c (H-2^d) mice in U-bottom 96-well plates (Falcon). The noted numbers of DCs sorted from transplanted spleens were cultured in the presence of 2 × 10⁵ allogeneic splenocytes for 4 days before quantification of splenocyte proliferation as measured by adding 1mCi of [³H]-TdR per well after 3 days. Cells were harvested 16 hours later and [³H]-TdR incorporation was counted in a b plate counter (Wallac). For positive controls, BMDCs were generated as described [K. Inaba et al., J. Exp. Med. 176, 1693 (1992)] with the addition of 10 ng/ml IL-4 (Genzyme), and allogeneic whole splenocytes were incubated with phorbol 12-myristate 13-acetate (PMA) and ionomycin as a maximum proliferation control. For negative controls, autologous whole splenocytes were mixed with allogeneic whole splenocytes (mixed splenocytes) or autologous splenocytes alone were used. (D) MLR with thymic CMP-derived CD8a⁺ DCs. Methods were as described in (C). Representative experiments of two are shown in (A) and (B) and of three are shown in (C) and (D).

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