Monoclonal antibodies to T-cell defined HLA-DR4 subtypes: peptide-dependent, antibody-binding epitopes on HLA-DR [beta] chains associated with rheumatoid arthritis

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Study of a monoclonal antibody to human B cells

The purpose of this work was to characterize and determine the specificity of a mouse monoclonal antibody (NFLD.M1) which was derived from a fusion between SP2/0-Ag14 and spleen cells from a Balb/c mouse that had been hyperimmunized with B-cells from a chronic lymphatic leukemic patient. Cloning was done by limiting dilution and positive clones were selected by screening on a panel of viable cells using the cellular enzyme-linked immunosorbent assay (CELISA). This assay was shown to be more specific and sensitive than an ELISA that used glutaraldehyde-fixed cells. -- Two sources of the antibody (purified IgG1 from ascites fluid and supernatant from overgrown cultures) appeared to be identical in their serological pattern on several B-cell lines. Specificity testing using the CELISA and several different cell types revealed that NFLD.M1 recognized some B-cells, but failed to react with any of the T-cells tested. A Frequency Distribution plot of the data showed that NFLD.M1 reacted with the cells in a bimodal fashion compared to the normal distribution observed with the monomorphic monoclonal antibody, NEI anti-Ia. Furthermore when NFLD.M1 antibody was expressed as a percent of the NEI anti-Ia it was found that all the DR4 positive cells produced values greater than 50% whereas DR4 negative cells gave values less than 30%. Using 30% as a cutoff point a correlation analysis was done on the CELISA results for 42 cell lines. The r value obtained for DR4 and NFLD.M1 was 1 with a p value of 2 x 10⁻¹⁰. In addition significant r values were obtained for DRw53 and DQw3 which are in linkage disequilibrium with DR4. -- One-dimensional electrophoresis of the immunoprecipitated molecules from a DR4 cell produced a banding pattern that was compatible with that of the alpha and beta subunits of DR

E₂-ERα signaling pathway interferes with CIITA pIV activity in MC2.

<p>VC5 and MC2 cells were cultured in E₂-depleted media followed by transfection with CIITA pIV luciferase constructs. On the following day, cells were treated with vehicle (ethanol), E₂ (10⁻⁹ M) and/or ICI (10⁻⁶ M), and stimulated or not with IFN-γ (100 U/ml) for 12 hours. Data are expressed as fold induction over the PGL2 Basic empty plasmid after controlling for transfection efficiency using cells dual transfected with GFP (Green Florescent Protein). The effect of ERα on the transcription activation of CIITA PIV was determined from relative luciferase activities in transfected MC2. Error bars represent the mean ± SEM of three independent experiments (**p<0.01).</p

Coordinate downregulation of IFN-γ inducible HLA-II expression by E₂ is reversed by ICI-mediated degradation of ERα in MC2 cells.

<p>VC5 and MC2 cells were cultured in E₂-depleted media, treated with vehicle (ethanol), E₂ (10⁻⁹ M) or/and ICI (10⁻⁶ M) followed by stimulation with IFN-γ (100 U/ml) for 96 hours. HLA-II expression was analyzed by surface flow cytometry using (A) anti-DR, (L243), and intracellular flow cytometry using (B) anti-DM (Map.DM1) and (C) anti-Ii (LN2). Bar graphs represent the MFI ± SEM of three independent experiments. (*p<0.05, **p<0.01). (D) Western blot analysis was performed on whole cell extracts using for HLA-DRα (TAL 1B5), HLA-DM (TAL18.1) and Ii (LN2); GAPDH (Ab8245) is the protein loading control. Bar graphs show the ratio of band intensities, normalized to GAPDH band intensities and represent the mean ± SEM ratio of three independent experiments: (E) HLA-DRα/GAPDH (F) HLA-DM/GAPDH, and (G) Ii/GAPDH (* p<0.05, ** p<0.01).</p

Mutation of putative ERE sites in CIITA pIV does not enhance CIITA pIV activation in MC2.

<p>(A) CIITA pIV nucleotide sequence from −346 to +50 with the GAS and IRF1 binding sites (shaded hexagon) and the predicted ERE (clear rectangles) were identified using online transcription factor prediction software, (<a href="http://tfbind.hgc.jp/" target="_blank">http://tfbind.hgc.jp/</a>, <a href="http://alggen.lsi.upc.es/" target="_blank">http://alggen.lsi.upc.es/</a> and <a href="http://www.cbrc.jp/index.eng.html" target="_blank">http://www.cbrc.jp/index.eng.html</a>). Site directed mutagenesis was used to perform deletion of the predicted ERE. (B) VC5 and MC2 were transfected with CIITA pIV constructs, then treated with vehicle (ethanol) or E₂ (10⁻⁹ M) and stimulated with IFN-γ (100 U/ml) for 12 hours, followed by determination of luciferase activity. Bar graphs represent the mean ± SEM of three independent experiments (**p<0.01, ***p<0.001).</p

E₂ differentially down regulates IFN-γ signaling and IFN-γ induced proteins in endogenous ER⁺ breast cancer cell lines.

<p>(A) MCF-7, (B) BT-474, (C) T47D, (G) MDA-MD-231, and (H) SK-BR-3 were cultured in E₂-depleted media, transfected with 8 X GAS binding sequence construct, then treated with vehicle (ethanol), E₂ (10⁻⁹ M) and stimulated or not with IFN-γ (100 U/ml) for 6 hours. Firefly luciferase activities in samples were normalized to Renilla luciferase activities in the same samples and expressed as fold induction over the un-stimulated mock. (D) MCF-7, (E) BT-474, (F) T47D, (I) MDA-MB-231 and (J) SK-BR-3 were cultured in E₂-depleted media, treated with vehicle (ethanol), or E₂ (10⁻⁹ M) and stimulated or not with IFN-γ (100 U/ml) for 96 hours. Western blot analysis of cytoplasmic extracts was performed for expression of IFN-γ inducible proteins: STAT1 (06-501), IRF1 (BD-20), IRF9 (C-20), GILT (T-18). Each figure represents one of three independent experiments.</p

E₂ differentially modulates inducible HLA-DR expression in ERα⁺ and ERα⁻ breast cancer cell lines.

<p>MDA-MB-231, SK-BR-3, MCF-7, BT-474, and T47D were cultured in E₂-depleted media, treated with vehicle (ethanol) or E₂ (10⁻⁹ M) and stimulated or not with IFN-γ (100 U/ml) for 96 hours. (A & E) HLA-DR cell surface expression (L243) was analyzed by flow cytometry: grey line, isotype control; black line, constitutive expression; shaded histogram, IFN-γ induced expression. (B & F) Bar graphs represent the MFI (mean florescence intensity) ± SEM for HLA-DR expression of three independent experiments. (C & G) Western blot analysis was performed on cytoplasmic and nuclear extracts for ERα expression (HC-20) and on cytoplasmic extracts for HLA-DRα (TAL 1B5). Protein loading controls included α-tubulin (B-7) and P84 (5E10) for cytoplasmic and nuclear proteins, respectively. (D & H) Bar graphs show the ratio of band intensity for HLA-DRα, normalized to the α-tubulin band intensity and represent the mean ± SEM of three independent experiments (*p<0.05, **p<0.01, ***p<0.001).</p