Impacts of STAT3 and STAT5 on BM conditioning. (A) Representative intracellular staining for pSTAT3 (pY705) versus pSTAT5 (pY694) following various step 1 conditioning regimens, either at end of 6-day step 1 culture (left) or after an additional 48-hour step 2 culture in GMCSF + IL-4 (right). Numbers in parentheses in each histogram display mean fluorescence specificity indices. Results are representative of 4 (left) and 3 (right) separate experiments, respectively. (B-D) Capacities of STAT5 or STAT3 knockout mice to respond to various conditioning regimens. (B) BM was prepared from STAT5^KO mice (St5 KO) or from littermates expressing wildtype STAT5 (St5 LM) and subjected to step 1 conditioning with Ft3L+IL-6 or Flt3L+IL-6+GMCSF. Results shown for Flt3L+IL-6+GMCSF are also highly representative of GMCSF or Flt3L+IL-6+GMCSF conditioning outcomes (not shown). Proliferation kinetics during step 1 culture were comparable for KOs versus LMs (not shown). Histograms display mean fluorescence specificity indices for DC maturation markers following 48 hours of step 2 culture in GMCSF+IL-4, with CpG+LPS added during the final 18 hours. Results shown are representative of 3 experiments. (C) BM was prepared from STAT3^KO mice (“St3 KO”) or from LMs expressing wild-type STAT3 (“St3 LM”) and subjected to step 1 conditioning and step 2 culture as in panel A. Results shown for Flt3L+IL-6+GMCSF are also highly representative of GMCSF or Flt3L+GMCSF conditioning. Proliferation kinetics during step 1 culture were comparable for KOs versus LMs, except that abject culture failure occurred when “St3 KO” BM was conditioned with Flt3L+ IL-6 (not shown). Results shown are representative of 2 experiments. (D) ELISA comparisons of supernatants for IL12p70 at end of step 2 cultures. BM was obtained from KO mice or from matched wildtype LMs, placed in standard step 1 cultures with Flt3L+IL-6, Flt3L+IL-6+GMCSF, or GMCSF alone as conditioning agents, and finally placed in 48-hour step 2 cultures with GMCSF+IL-4, and CpG+LPS added during the final 18 hours. Culture failure was observed only when STAT3^KO BM was conditioned with Flt3L+IL-6. Data are presented as means plus or minus SD of 3 replicates and are representative of 3 (STAT5^KO vs LM) or 2 (STAT3^KO vs LM) independently performed experiments.