Our study suggests a novel activity of CFZ as an immunomodulating agent

Our study suggests a novel activity of CFZ as an immunomodulating agent. shown in Supplementary Physique S1A and S1B. We also used CFZ to treat other malignancy cell types (one renal cell carcinoma and two F-TCF breast malignancy cell lines) and normal cells (CD34+ cells and monocytes), but down-regulation of HLA class I was not observed (data not shown). These results suggest the specificity of CFZ induce down-regulation of HLA class I expression on myeloma cells. Open in a separate window Physique 1 Expression of HLA class I decreased after CFZ treatment in MM cell lines and main MM cellsA. MM cells were incubated with 10 nM CFZ for 24 hours, then cells were stained Amyloid b-Peptide (1-40) (human) with FITC-HLA-ABC, APC-Annexin V and 7AAD. Circulation cytometer was used to gate the both Annexin V and 7AAD double unfavorable cells and the mean-fluorescence intensity (MFI) was recorded. Class I decrease % = 100 (MFI of control – MFI of treated cells)/MFI of control. B. The patients’ MM cells were treated with 20 to 40 nM CFZ for 24 hours. MFI was recorded to test the down-regulation of HLA. We then used different concentrations of CFZ or different durations of CFZ treatment around the H929 cell collection. We found that down-regulation of HLA class I expression was in a dose- and Amyloid b-Peptide (1-40) (human) time-dependent manner (Physique ?(Physique2A2A and ?and2B).2B). These results were Amyloid b-Peptide (1-40) (human) confirmed by using immunofluorescence analyses (Physique ?(Physique2E2E and ?and2F).2F). The kinetics analyses of apoptosis after CFZ treatment are offered in Supplementary Physique S1C and S1D. Similar results were obtained in main MM cells (Physique ?(Physique2C2C and ?and2D2D). Open in a separate window Physique 2 Down-regulation of HLA class I was in a dose- and time-dependent mannerA. H929 was treated with different doses of CFZ for 24 hours. B. H929 was treated with 10 nM CFZ for different durations. C. Main MM cells were treated with different doses of CFZ for 24 hours. D. Main MM cells were treated with 40 nM CFZ for different durations. E. and F. Immunofluorescence analysis was performed to confirm the result that down-regulation of HLA class I was in a dose- and time-dependent manner. * 0.05. HLA-C is usually a more specialized ligand for KIRs, as compared to HLA-A and -B, approximately the same level of down-regulation of HLA-C was obtained after CFZ treatment (data not shown). Then we investigated whether the exogenous HLA-C binding peptides (pointed out in Materials and Methods) could rescue the down-regulation of HLA-C caused by CFZ. The expression level of HLA-C and HLA class I remained almost unchanged in the presence of the peptides and Human 2M cocultured with the CFZ treated H929 cells (Supplementary Physique S2). The peptides experienced no effect on the HLA-C and HLA class I expression levels in the untreated H929 cells (Supplementary Physique S2). These data show that exogenous HLA-C binding peptides can stabilize HLA-C expression around the cell surface during CFZ treatment. We also decided the expression levels of other NK cell ligands on H929 cells after CFZ treatment, as shown in Physique ?Physique3A,3A, CFZ could up-regulate the expression of DR4 and DR5, but had no effect on the ligands of NKG2D (MIC A/B, ULBP 1C3) and ligands of NCRs (NKp30-L, NKp44-L and NKp46-L). Open in a separate window Physique 3 CFZ up-regulated DR4, DR5 and affected the re-expression of Amyloid b-Peptide (1-40) (human) HLA class I on cell surface, but experienced no effect on ULBP 1C3, MIC A/B, NKp30-L, NKp44-L and NKp46-LA. H929 was treated with 10 nM CFZ for 24 hours. Circulation cytometer was used to detect the expression of DR4, DR5, ULBP1C3, MIC A/B, NKp30-L, NKp44-L and NKp46-L. MFI of DR4 and DR5 were increased after CFZ treatment (DR4: 195.3 6.1 vs 44.1 2.6 and DR5: 363.2 9.2 vs 79.3 3.8) B. Acid stripping was performed to remove the HLA class I on.