DC-T Cells Assays
Dendritic cells (DCs) are major antigen-presenting cells that can efficiently prime and cross-prime antigen-specific CD4+ and CD8+ T cells. DC-T cell assays are used to assess the potential immunogenicity of different product formulations that directly modulate T cell activation.
Our highly sensitive assays assess the propensity of candidate proteins or peptides to inducing immune responses by measuring activation (immune-biomarkers and cytokines) and proliferation of immune cells that may lead to anti-tumoral responses or vaccine immunogenicity using CFSE dye and flow cytometry analysis.
The DCs are differentiated in vitro from isolated CD14+ monocytes to an immature DC phenotype. These cells are then loaded with the test protein or peptides before inducing further differentiation to a mature DC phenotype. Once matured, the cells are incubated with autologous CD4+ and CD8+ T cells before measuring T cell proliferation, CD25 marker and IL-2 secretion.
Human Dendritic Cells Differentiation
Evolution of cell phenotype during 5 days of culture.
(A) Flow cytometric analysis showing that monocytes (CD14+/CD1a– cells at day 0 of culture) isolated by negative immunoselection of fresh PBMC and cultured with IL-4, GM-CSF, IFNγ and LPS acquire a mature DC phenotype (moDC). (B) The expression of CD14 (monocyte marker) CD1a, CD80, CD83 and HLA-DR (DC markers at day 5 of culture) were analysed to monitor the differenciation into DC of monocytes isolated from HLA-A2pos and HLA-A2neg healthy donors.
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DC-T Cells Assays of Immune Function
Representative data from DC-T cell assay
Monocyte-derived DCs (moDCs) were incubated with positive peptide (Melan-A) or negative peptide (Nucleoprotein of lymphocytic choriomeningitis virus) in order to obtain loaded mature DC (mDC). T cells from same donor (autologous) labeled with a cell tracer (CFSE) and moDCs were then co-cultured for 14 days. The proliferation and activation of specific CD8+ T cells were monitored by flow cytometry.
(1) CFSE-labeled T cells from HLA-A2pos healthy donor incubated with autologuos moDCs that were not loaded with antigen (only media), (2) CFSE-labeled T cells incubated with moDCs loaded with positive peptide (Melan-A26-35), (3) CFSE-labeled T cells incubated with moDCs loaded with with negative peptide control (NP-LCMV396-404) and (4) CFSE-labeled T cells from HLA-A2neg healthy donor incubated with moDCs loaded with positive peptide (Melan-A26-35).