IMMUNE CELL POLARISATION ASSAYS

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.

 (day 0)

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).

cd25_cd8_edited.png

Monocyte Polarization Assays

Monocytes are highly abundant circulatory effector immune cells and play a vital role in driving or resolving immune responses depending on their activation phenotype. In response to stimuli present in their local microenvironment or in circulation, monocytes modulate their gene expression towards either a pro-inflammatory (M1), an anti-inflammatory (M2) macrophages or dendritic cells (DCs).

Our assays provide a panel of monocyte polarization protocols of blood-derived monocytes or cell lines to achieve a stable, optimal and effective regimen for in vitro induction to drive antigen specific immune responses or immunosuppressive status. Our protocols allow the assessment of cell surface receptor expression, cytokine profiles, scavenging functions and ability to activate or suppress T-cell proliferation.

Human Macrophage Polarization 

CD16-PE

CD80-PE-Cy7

CD206-BV-421

CD163-AF-647

Macrophage_polarization_assay_v4_edited.