Fig. 2

PLCγ1/PLCγ2 double deficiency impairs IL-7-mediated B cell progenitor functions. BM from Plcg1 ^+/+ Plcg2 ^+/+, Plcg1 ^−/− Plcg2 ^+/+, Plcg1 ^+/+ Plcg2 ^−/−, or Plcg1 ^−/− Plcg2 ^−/− mice were transplanted into lethally irradiated congenic wild-type CD45.1⁺ mice. Six to eight weeks after transplantation, Lin⁻ BM cells from the recipients, Rag1 ^−/− or Jak3 ^−/− mice were cultured on OP9 cells plus IL-7. a, b IL-7-dependent B cell production. The cells were stained with anti-CD45.2, anti-B220, and anti-Mac-1 after 9 days of co-culture. Numbers indicate percentages of B220⁺ and Mac-1⁺ cells in the gated CD45.2⁺ population (a). Bar graphs show the numbers of CD45.2⁺B220⁺ and CD45.2⁺Mac-1⁺ cells (b). c, d IL-7R expression. BM cells from the recipients were stained with anti-IL-7Rα. Numbers indicate percentages of IL-7Rα⁺ cells in the gated CD45.2⁺Lin⁻population (c). After 9 days of co-culture plus Flt-3L, the cells from the recipients were stained with anti-CD45.2, anti-B220, and anti-IL-7Rα. Levels of IL-7Rα expression in the gated CD45.2⁺B220⁺ population are shown (d). e Expansion in response to IL-7. Fractions B and C of B cell progenitors (YFP⁺CD45.2⁺B220⁺CD43⁺CD24⁺) were sorted from the recipients and cultured with OP9 plus IL-7. On the indicated days after co-culture, the numbers of live cells were determined by trypan blue exclusion. f, g IL-7-mediated rearrangement of the distal V_HJ558 family gene segments of IgH chain genes. YFP⁺CD45.2⁺B220⁺CD43⁺ or B220⁺IgM⁻CD43⁺ pro-B cells were sorted from the recipients (f) or from wild-type or Jak3^−/− mice (g), respectively. The frequency of V_HJ558 and V_H7183 family usage in these progenitors was determined by high-throughput sequencing of the rearranged IgH genes. Bar graphs show the ratios of percentages of rearranged V_HJ558 genes to those of rearranged V_H7183 genes in the pro-B cells. h IL-7-mediated B cell progenitor proliferation. YFP⁺CD45.2⁺B220⁺CD43⁺CD24⁺ B cell progenitors (fractions B and C) were sorted from the recipients, and co-cultured with OP9 cells plus IL-7 for 3 days. BrdU incorporation and DAPI staining were used to determine proliferation. Numbers indicate percentages of BrdU⁺ cells in the gated live cells. Error bars show ± SEM. Data shown are obtained from or representative of 6 (a, b), 4 (c, h), or 2 (d–f) independent experiments or from three wild-type and 2 Jak3 ^−/− mice (g)