At RoukenBio, we have developed a standardised protocol for the generation of DCs. CD14+ monocytes are magnetically isolated from cryopreserved PBMCs and incubated with GM-CSF and IL-4 over a 7-day period. The phenotype and purity of these moDCs is confirmed by flow cytometry after which the cells can be used in assays. Where appropriate magnetic isolation techniques can be applied to isolate DCs from PBMCs directly however this is limited due to the low recovery rate.

DCs become activated when exposed to certain soluble mediators (such as TLR agonists) in the inflammatory environment. This leads to an upregulation of MHCII, costimulatory molecules (CD80/86) and the release of inflammatory cytokines. In DC activation assays, DCs are incubated with a stimulus in the presence of a candidate therapeutic with flow cytometry and cytokine analysis used to assess activation. These assays are well suited for screening candidate therapeutics for their ability to drive or repress DC activation.

The ability of DCs to activate T cells can be assessed using a mixed lymphocyte reaction (MLR). In this assay, naïve or mature DCs are incubated with T cells from an allogenic donor. The HLA mismatch between donors drives T cell activation, which can be assessed by flow cytometry and cytokine analysis. Candidate therapeutics that alter the activation state of DCs can be readily tested within this system. 

Human donor selection

Donor pairing is crucial for effective MLR. With an onsite bank of ~70 recallable cryopreserved PBMCs and three onsite phlebotomists, RoukenBio can regularly screen donor pairs to ensure optimal assay conditions. Pairings in which Nivolumab can enhance the levels of INFγ production in a dose-dependent manner provide favourable pairings for assessment of test articles.

Pre-screening of donor combinations - four PBMC donors (A-D), 12 allogenic combinations, INFγ ELISA. Donor pairs driving a dose-dependent increase in INFγ production are selected for our Customer studies.  

Mixed lymphocyte reaction readouts:

Additional readouts can be included in an MLR based upon the needs of your study including T cell proliferation, assessment of activation markers and the measurement of additional cytokines aside from INFγ.

Antigen presentation can be indirectly assessed using common pathogen derived (CMV, tetanus toxoid, Candida albicans) or cancer (MART-1) antigens. DCs are generated from appropriate donors and are pulsed with the relevant antigen in the presence of a candidate therapeutic prior to co-culture with T cells from the same donor. T cell activation, as measured by flow cytometry and cytokine analysis, acts an indirect indicator of antigen presentation. Alternatively, reporter cell lines, such as NFAT reporters can be used with luminescence acting as an indication of T cell activation and therefore antigen presentation. Candidate therapeutics that influence antigen presentation can be readily assessed within this system.