Journal: International Journal of Nanomedicine

Article Title: Functionalization-dependent effects of cellulose nanofibrils on tolerogenic mechanisms of human dendritic cells

doi: 10.2147/IJN.S183510

Figure Lengend Snippet: Phenotype of regulatory CD8 + CD25 + T cells induced by DC. Notes: ( A ) MACS purification and gating strategy of CD8 + T cells cocultivated with DC in the presence or absence of blocking anti-ILT-3 Ab (see also Figure S3 for the experiments with the blocking anti-ILT-4 Ab), for the analysis of IFN-γ, IL-10, CTLA-4, and TGF-β expression within CD25 + CD8 + population. ( B ) The summarized data from four different experiments are shown as mean ± SD. * P <0.05 as indicated (RM ANOVA). ( C ) The proliferation (CFSE) and cell-death (PI) analysis of the allogeneic CFSE-labeled CD3 + T cells cocultivated with DC-primed CD8 + T cells. A representative analysis and the summarized data from four independent experiments are shown as mean % of proliferated responders ± SD. ( D ) Cytotoxic activity of DC-primed CD8 + T cells toward CFSE-labeled tumor HEp-2 cells. Representative data on gated CFSE + HEp-2 cells are shown, and the summarized data from three different experiments are shown as mean ± SD. * P <0.05 compared to control (paired t -test). Abbreviations: Ab, antibody; aILT-3, blocking anti-ILT-3 antibody; APA, 3-aminopropylphosphoric acid; APAc, APA-functionalized; CFSE, carboxyfluorescein succinimidyl ester; CNF, cellulose nanofibrils; CTLA, cytotoxic T-lymphocyte-associated protein; DC, dendritic cells; IFN, interferon; +IrAb, irrelevant antibody; ILT, immunoglobulin-like transcript; LPS, lipopolysaccharide; MACS, magnetic-activated cell sorting; PBMC, peripheral blood mononuclear cells; PI, propidium iodide; RM, repeated measures; TGF, transforming growth factor.

Article Snippet: Phenotype analysis of DC and T cells after the cultures was carried out using flow cytometer (Sysmex Partec Cube 6) after staining the cells by using the following Abs (Clone) and reagents: immunoglobulin (Ig) G1a negative control-biotin (MCA928), IgG1 negative control-phycoerythrin (PE) (MCA928PE), IgG1 negative control-fluorescein isothiocyanate (FITC) (MCA928F), anti-CD1a-PE-Cy5 (NA1/34HLK) (all from Serotec, Oxford, UK), anti-human leukocyte antigen (HLA)-DR-biotin (LN3), IgG1a negative control-PECy5 (P.3.6.2.8.1), anti-CD86-PE (IT2.2), streptavidin-PECy5, anti-CD4-PECy5 (RPA-T4), anti-IL-4-PE (8D4-8), anti-ILT3-PE (ZM4.1), anti-ILT-4-PE (42D1), anti-TGF-β-biotin (eBio16TFB), anti-CD25-PE, anti-CD25-PECy5 (BC96), anti-forkhead box (Fox) P3-FITC (PCH101), anti-IL-10- PE (JES5-16E3), anti-CD39-FITC (A1), anti-CD8-PEcy5 (RPA-T8), anti-cytotoxic T-lymphocyte-associated protein (CTLA)-4-PE (14D3) (all from eBioscience), streptavidin-Alexa 488, anti-mouse IgG-Alexa 488, anti-CD1a-PE (HI149) (all from Biolegend, San Diego, CA, USA), anti CD40-allophycocyanin (APC) (5C3), anti-IL-12 (p40/p70)-PE (C11.5) (all from BD Pharmingen, San Diego, CA, USA), anti-CD83-FITC (HB15e), anti-IFN-γ-FITC (25723), anti-IL17-peridinin-chlorophyll-protein complex conjugate (PerCP) (41802), anti-IL-10-FITC (127107), anti-HLA-DR PerCP (L243), anti-CD4-FITC, anti-CD4-PerCP (11830), anti-IDO-1-APC (700838) (all from R&D Systems), anti-CD14-FITC (TUK4), IgG1 negative control-PerCP (IS5-21F5) (Miltenyi Biotec), anti-CD4-PE (MEM-241) (Partec Sysmex).

Techniques: Purification, Blocking Assay, Expressing, Labeling, Activity Assay, FACS