Journal: OncoImmunology

Article Title: Immune independent crosstalk between lymphoma and myeloid suppressor CD14+HLA-DRlow/negmonocytes mediates chemotherapy resistance

doi: 10.1080/2162402x.2014.996470

Figure Lengend Snippet: Figure 4. Heat-shock protein 27 (Hsp27) is associated with LYM resistance to DOX and decreased HLA-DR expression on monocytes. (A) LYM co-cultured with monocytes with and without DOX have increased intracellular Hsp27 expression. Quantitative analysis of LYM (Granta-519) intracellular Hsp27 expression by immunoblot is shown (mean § SD, n D 3). Representative immunoblot images are shown above each condition. (B) Quantitative analysis of LYM intracellular Hsp27 expression by flow cytometry is shown for OCI-Ly3 (n D 4) and OCI-Ly10 (n D 6; mean § SD). Representative histogram of OCI-Ly3 intracellular Hsp27 expression are shown above the corresponding condition (shaded D isotype control; line D Hsp27). (C) Hsp27 can directly induce decreased HLA-DR expression on monocytes in a dose-dependent manner (left panel, n D 4). LYM cells can secrete Hsp27. Supernatants from LYM cells and monocyte co-culture with and without DOX have significantly higher soluble Hsp27 compared to media (right panel, white bars, left axis). Culture supernatant from LYM co-culture high in Hsp27, but not media or supernatant from normal B cell co-culture, induced decreased HLA-DR expression on normal CD14CHLA-DRC monocytes (right panel, black bars, right axis: LYM D Granta-519; n D 6; * p < 0.05 compared to control media; D p < 0.05 compared to normal B cell and monocyte co-culture supernatant.) (D) More importantly, Hsp27 is detectable in plasma of patients with lymphoma. Increased plasma level of Hsp27 correlated with increased percentage of CD14CHLA-DRlow/neg

Article Snippet: When specified, stock solution of DOX (SelleckChem, Houston, TX) in DMSO was diluted into desired con- D ow nl oa de d by [ U ni ve rs ity o f O ta go ] at 0 1: 55 2 8 Ju ly 2 01 5 For culture with recombinant human Hsp27 (R&D Systems) at specified concentration, monocytes were cultured in RPMI1640 media with 10% fetal bovine serum and M-CSF (10 ng/ mL) for 3 d. Dendritic cell differentiation To differentiate monocytes into dendritic cells, TNFa (1100 U/mL) and PGE2 (1 mg/mL) were added after 3 d of culture for additional 2 d as previously described.5,8 Cell viability and phenotype To assess cell viability, cultured cells were collected with- out further isolation, immediately stained with Annexin-V, 7- AAD, monoclonal antibodies (mAb) and analyzed by flow cytometry.

Techniques: Expressing, Cell Culture, Western Blot, Cytometry, Control, Co-Culture Assay, Clinical Proteomics