How to fully exploit the mixed leukocyte reaction assay?

Immunological synapse that occurs between T lymphocytes and dendritic cells (DCs) is a key component for an effective immune response, where DCs specialize in the initiation of immune responses by directing the T cell fate. For their function, either immunogenic or tolerogenic, DC subpopulations are generated through the engagement of many signaling cues, and the nature of these signals only determines their functional properties. Indeed, DCs can stimulate the polarized outgrowth of T cell subsets but, depending on their maturation context, generated DCs may have an adverse effect, thereby underlining the ability of the microenvironment to control their function.

Hence, interventions through a fine-tuning of these signal cues are key so as to elicit or boost an anti-tumor immune response, for instance.

The data shown here illustrate our DC differentiation and mixed leukocyte reaction (MLR) assay as a valuable and robust tool to address DC function and evaluate the modulation of their mediated immune response. Based on a co-culture of allogenic CD4 T cell (responder) and monocyte-derived DCs (stimulator), our assay is specifically configured to get either basal MLR (Fig 1) or impaired response (Fig 2) due to an immunosuppressive condition, e.g. Adenosine, leading to tolerogenic and functionally dysregulated DCs. This thus makes it well suited for evaluating biologics for their modulating ability to either i) amplify an immune response or ii) alleviate an immunosuppressive condition and trigger DC-mediated T cell-stimulating activity.

Figure 1: Mixed leukocyte reaction (MLR) in allogenic human mDC/CD4 T cell co-cultures is enhanced upon PD1 inhibition. Isolated peripheral blood monocyte-derived DCs are co-cultured with allogenic CD4+ T cells. IL2 (A) and IFNg (B) levels released in the supernatants are measured by HTRF. While mature DCs (mDC) display T-cell stimulating ability underlined through the MLR response, this response is further optimized following PD1 blockade with Nivolumab.

Figure 2: Adenosine impairs DC phenotype and function in allogenic human mDC/CD4 T cell co-cultures. Isolated peripheral blood monocyte-derived DCs are challenged with Adenosine (Ado) in the presence and absence of an Adenosine Receptor (R) antagonist. Adenosine-mediated effect is then profiled by flow cytometry on DC differentiation and maturation (A), and supernatants from DC cultures or allogenic CD4+ T / DC co-cultures (B) are processed, respectively, for IL10 (a) and IFNg (b) HTRF quantifications. (A) Adenosine is shown to skew DC differentiation and maturation toward a CD1a low CD14+ cell population, thereby inducing phenotypically dysregulated DCs. (B) While control mDC displays a MLR response – as expected, Adenosine strongly alters the allostimulatory ability of DCs to elicit T cell response, as shown through both the increase of IL10 levels released by DCs (a) and decrease of IFNg released by T cells (b). All these immunosuppressive effects are mediated through Adenosine receptors since relieved in the presence of an Adenosine R antagonist.