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Blood, Vol. 112, Issue 5, 1832-1843, September 1, 2008
STAT3- and STAT5-dependent pathways competitively regulate the pan-differentiation of CD34pos cells into tumor-competent dendritic cells
Blood Cohen et al.
112: 1832
Supplemental materials for: Cohen et al
Files in this Data Supplement:
- Figure S1. Contrasting surface expression profiles of BM cells following 6 day exposure to various Step 1 conditioning treatments (JPG, 175 KB)
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(A) Same experiment as described in Fig 1D, but all treatment groups shown. Individual treatments are listed in far left column. 10 different conditioning treatments were synchronously compared. Number within each histogram plot indicates the “mean fluorescence specificity index” for the molecule tested, defined as the geomean fluorescent intensity of all cells after staining with the specified mAb (filled histogram), divided by geomean staining intensity for isotype control mAb (unfilled histogram). Results shown are representative of three comprehensive comparisons. (B) Percentage of total cells displaying CD11cpos/B220neg versus CD11cpos/B220pos phenotype following each Step 1 conditioning regimen, tabulated from the same experiment, determined by multicolor FACS analysis. CD11cpos/B220neg cells were also strongly CD11bpos (not shown). Results shown are representative of three comprehensive comparisons.
- Figure S2. Maturation profiles of BM cells following Step 2 culture in DC1-polarizing stimuli (JPG, 134 KB)
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(A) Same experiment as described in Fig 1E, but all treatment groups shown. (B) Percentage of total cells displaying Gr-1pos/MHC Class IIneg or Gr-1pos/MHC Class IIlow phenotype following various conditioning regimens, tabulated from the same experiment at the end of Step 2 culture, determined by multicolor FACS analysis. Representative of three comprehensive comparisons.(C) Relative production of IL12p70 heterodimer, IFNα and IFNβ following Step 2 culture treatment with various TLR agonist combinations. Culture method as described in Fig 1E. ELISA results for culture supernatants shown, representing pg of cytokine/106 cells/24 hours following TLR agonist addition. “b.d.” indicates below the level of detection, <20 32 pg/ml depending on the assay.
- Figure S3. Spontaneous maturation of BM cells following Step 2 culture in the absence of exogenous maturational stimuli (JPG, 146 KB)
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(A) Same experiment as described in Fig 2B, but all treatment groups shown. Following the Step 1 conditioning treatments listed in far left column, each group was replated in fresh medium with only GMCSF added for 72 hours, after which FACS analyses were performed. Number within each histogram is the calculated “mean fluorescence specificity index” at culture’s end. Results shown are representative of three comprehensive comparisons. (B) A similar but even more pronounced pattern of spontaneous maturation was observed during 48 hour Step 2 culture in GMCSF+IL4. Results shown are representative of three comprehensive comparisons. (C) Expression of DEC 205 following 48h hour Step 2 culture only in GMCSF+IL4. Each figure shown is representative of three comparisons for the shown Step 1 conditioning regimens.
- Figure S4 (JPG, 107 KB)
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(A) Uniform expression of Toll like receptors in Flt3L+IL6 conditioned immature DCs. BM cultures were conditioned in Flt3L+IL6 (Step 1), then cultured for 24 hour in GMCSF+IL4 (Step 2). At the usual timepoint for stimulation with TLR agonists, cells were instead fixed in Cytoperm, washed in Permwash, then stained with FITC conjugated mAb to TLR3, TLR4, TLR8 and TLR9 (filled histograms), or isotype ctrl Ig (empty histogram) (Imgenex). Essentially uniform staining for TLR protein on the individual cell level was observed. Number within each histogram is the calculated “mean fluorescence specificity index.” Similarly uniform staining was also observed for TLR7 performed with unconjugated mAb (Imgenex) with indirect immuno fluorescent staining (not shown). (B) Persistent expression of interferon regulatory factors (IRFs) at early and late stages of maturation following Flt3L+IL6 conditioning. BM cultures were conditioned in Flt3L+IL6 (Step 1), then DC1-polarized as in Fig 2 with GMCSF and IL4, then CpG+LPS, during Step 2 culture. Cells were harvested at the ends of Step 1 or Step 2 culture, then fixed in Cytoperm, washed in Permwash, stained with polyclonal goat Ab to IRF4, IRF8 or ctrl Goat Ig, then counterstained with PE conjugated, mouse adsorbed F(ab)′2 fragments of donkey anti goat Ab. Number within each histogram is the calculated “mean fluorescence specificity index.”
- Figure S5. Comparison of impacts when PGE2 or tumor lysate was added to Flt3L+IL6 conditioned BM cells at either 0 hr or 18 hr of Step 2 culture (JPG, 111 KB)
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Methods as in Fig 2C except that PGE2 or 4 million killed tumor lysate cells (MCA 205 sarcoma or B16 melanoma) were also added at 0 hr or 18hr of Step 2 culture. CpG+LPS were then added at 24 hr, and FACS analyses performed at 44 hr. It will be observed that DCs were variably resistant to PGE2 or tumor inhibition when exposed at 0 hr of Step 2 culture, but that resistance became a uniform DC property within 18 hr of initiation of Step 2 culture.
- Figure S6. T cells from tumor bearing mice are effective therapy against advanced tumors following in vitro stimulation with Flt3L+IL6 conditioned DCs (JPG, 66.9 KB)
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DCs and T cells from MCA 203 bearing mice were co cultured as in Fig 3b/Fig 4. After 12 day culture expansion, T cells were harvested for therapy. Syngeneic mice bearing 10 day established subcutaneous MCA 203 tumors were treated with adjunct nonmyeloablative total body irradiation (500 cGy), and optionally received 10 million culture expanded T cells, 5 mice per group. Cure rates were 0/5 (A, 500 cGy only) or 5/5 (B, 500 cGy, then 10 million “Flt3L+IL6” DC driven T cells). Treatment outcome A vs B p1<0.008. This is representative of 3 experiments.
- Figure S7. Graphic rendering of pSTAT3 vs pSTAT5 staining at end of Step 1 cultures (JPG, 53.7 KB)
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Each bar shows average fluorescence specificity index ±sd of four side by side experimental runs for each conditioning regimen. Staining was significantly different for Group A vs. all other groups both for pSTAT3 (p1 range <0.004 to <0.007) and for pSTAT5 (p1 range <0.001 to <0.011). No significant differences in pSTAT3 or pSTAT5 staining were observed among GMCSF containing groups (B), (C) and (D). (p1 <0.190 to <1.0).
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