Fig. 2.

CCP2 deficiency potentiates ILC3 differentiation from CHILPs. a Total RNAs were extracted from hematopoietic populations. Indicated gene expression levels of Ccp1, Ccp2, Ccp3, Ccp4, Ccp5, and Ccp6 were examined by real-time qPCR. b Gating strategies and flow cytometry analysis of CLP (Lin⁻IL-7Rα⁺Sca-1^lowc-Kit^low), αLP (Lin⁻IL-7Rα⁺Sca-1⁺c-Kit⁺α₄β₇ ⁺), CHILP (Lin⁻IL-7Rα⁺Flt3⁻CD25⁻α₄β₇ ⁺), and ILCP (Lin⁻IL-7Rα⁺α₄β₇ ⁺PLZF⁺) in BM from Ccp2 ^+/+ and Ccp2 ^−/− mice. n = 6 per group. Numbers of indicated cells in Ccp2 ^+/+ and Ccp2 ^−/− mice were calculated as shown means±s.d. c, d CHILPs were isolated from Ccp2 ^+/+ or Ccp2 ^−/− mice and cultured on OP9 cells in the presence of SCF and IL-7 for 12 days. Percentages c and total numbers d of ILC3s were examined by flow cytometry, gated on CD45⁺Lin⁻RORγt⁺. The CCP agonist CoCl₂ (10 μm) and antagonist Phen (2 μm) were added for in vitro differentiation assays. e Apoptosis of CHILPs from Ccp2 ^+/+ and Ccp2 ^−/− mice was analyzed with Annexin V/PI. Apoptotic cells were calculated and shown as means±s.d. f Active caspase 3 in CHILPs from Ccp2 ^+/+ and Ccp2 ^−/− mice was detected by flow cytometry. g Schematic representation for BM transplantation assays. h–j 5 × 10⁴ CD45.2⁺ LSK from Ccp2 ^+/+ or Ccp2 ^−/− mice with 5 × 10⁶ CD45.1⁺ helper cells were transplanted into lethally irradiated CD45.1⁺ recipients. After 8 weeks, percentages of CHILPs h, ILCP i, and ILC3s j in chimeras were checked by FACS. n = 6 for each group. k A 50/50 mixture of CD45.1⁺ wild-type and CD45.2⁺ Ccp2 ^+/+ or Ccp2 ^−/− bone marrow was transplanted into lethally irradiated CD45.1⁺ recipients. The ratio of CD45.1⁺ to CD45.2⁺ ILC3s in chimeras (n = 6) were analyzed by gating on CD45.2⁺Lin⁻RORγt⁺ (Ccp2 ^+/+ or Ccp2 ^−/−) and CD45.1⁺Lin⁻RORγt⁺ (WT). *P < 0.05, **P < 0.01 (Student’s t-test). Data are representative of three independent experiments. Error bars in a–f and h–k indicate s.d.