DC Assays

The type of dendritic cell and the context in which it was activated will shape the dendritic cell response and this in turn helps shape the T cell response in terms of T cell polarisation and function. As the tumour micro-environment is often sterile and the microenvironment is often suppressive this can result in a lack of DC maturation, migration to the draining lymph node and cross presentation of antigen. By developing strategies to enhance or manipulate dendritic cell uptake of tumour antigen, their subsequent maturation and their trafficking to the lymph node would allow activation of the naïve CD8 T cell repertoire in the draining lymph node generating anti-tumour responses, as well as generating DC which remain in the tumour microenvironment where they can interact with any infiltrating TILs.

Dendritic cells can be largely grouped into three major types, plasmacytoid (pDC), conventional (cDC1 and cDC2) DC which can be identified using a panel of surface markers and flow cytometric analysis. These populations can be isolated in small numbers from blood and the effect of therapeutics on their ability to produce cytokines, express surface molecules required for T cell activation and ability to take up antigen and activate T cells. Alternatively, if larger numbers of DC are required these can be differentiated from peripheral blood monocytes. Together these provide the tools to examine how your therapeutic acts to modulate DC function.

Dendritic cells and macrophage can be transduced with lentivirus expressing your gene of interest and GFP.  Transduced cells can be detected and sorted by FACS based on GFP expression.
Figure 1: Primary Immune Cell Transduction. Dendritic cells and macrophage can be transduced with lentivirus expressing your gene of interest and GFP. Transduced cells can be detected and sorted by FACS based on GFP expression. This provides a mechanism of manipulating DC and macrophage function and for introducing tumour antigens. DC can then be used in a range of assays such as assessment of APC function using T cell proliferation and cytokine production as a readout.

 

The effect of test therapeutics and their ability to enhance the ability of DC to activate T cells and to produce pro-inflammatory cytokine can be measured.
Figure 2: Dendritic cells can be matured using a variety of maturation signals: Cells can be phenotyped by flow cytometry, cytokine production measured and their ability to drive T cell responses assessed. Expression levels of ICAM-1 and MHCII are dependent on how DC are matured and allogenic CD4 T cell proliferation is higher in cultures where DC have received maturation signals. The cytokine profile is also altered by the type of maturation signal. The effect of test therapeutics and their ability to enhance the ability of DC to activate T cells and to produce pro-inflammatory cytokine can be measured using this system.

 

 

Dendritic Cell Based Assays Frequently Asked Questions (FAQs)

 

  • Why use monocyte derived DC rather than cDC isolated directly from blood?

    pDC can be isolated in sufficient number from blood to allow some direct testing of therapeutics on this cell type. However cDC populations are rare in the blood and insufficient numbers are often isolated to allow testing of multiple conditions. The use of monocyte DC provides a larger number of cells which can be used to assess many aspects of DC function.

  • How can lentiviruses be used in DC research and therapeutic development?

    Lentiviruses offer the ability to transduce non-dividing primary immune cells with high efficiency. Using this technology we can introduce stable genomic insertions to modulate the function of DC or express specific protein to enhance or diminish the adaptive immune response.