FIGURE 6.

c-Kit⁺CD41⁺cells are the Th17 inducers in the Lin⁻c-Kit⁺CX3CR1⁻ cell subset. (A) Isolated Lin⁻c-Kit⁺CX3CR1⁻ cells were further sorted into CD41⁺cells, HSC (Sca-1⁺CD150⁺CD48⁻) and MPP1 (Sca-1⁺CD150⁺CD48⁺), MPP2 (Sca-1⁺CD150⁻CD48⁺), and CFU-E (Sca-1⁻CD150⁻ FcγRIII/II⁻ CD105^+low/−). (B) c-Kit⁺CD41⁺cells differentiated into megakaryocytes in 3 day culture with IL-3, TPO, EPO and SCF (right bottom panel). Phase contrast of each subset on 3 day culture (top and middle panels) and Giemsa staining of MPP2 cells (left bottom. Bar = 10μm) and cultured CD41⁺cells (right bottom. Bar = 20μm). Culture conditions for determining hematopoietic lineage potential are in Methods. (C) Sorted c-Kit⁺CD41⁺cells (from A) exclusively induced Th17 response to nucleosomes when co-cultured with SNF1 T cells. The sorted subsets of Lin⁻c-Kit⁺CX3CR1⁻ cells are designated by color key, and Th17 responses are shown by bars. Mean ± s.e.m. of 5 independent experiments. (D–E) Lin⁻c-Kit⁺_pure cells differentiate into CD42⁺cells and produce platelet related molecule, Pf4. FACS shows Lin⁻c-Kit⁺pure cells differentiated into CD42⁺ cells in the presence of TPO but not in SCF alone (D). Nucleosomes feeding increased the production of Pf4 in Lin⁻c-Kit⁺_pure cells (intracellular staining). PBS (black line) = 14.0% Pf4⁺ cells vs. Nucleosome (blue) = 55.4% Pf4⁺ cells in c-Kit⁺ (CD117⁺) cell gated population (E). Dotted line = isotype control.