Dendritic Cell Assays

The ability of therapeutics to alter the activation profile of moDC can be evaluated using flow cytometry.

The addition of LPS to moDC results in their maturation, as evidenced by upregulation of both MHC Class I and II molecules and costimulatory or activation markers such as CD86, CD83, CD80, and CD40 (Figure 2). These phenotypic changes are known to enhance the ability of moDC to stimulate a T cell response and therefore can be used as a surrogate for predicting dendritic cell function.

Figure 2: Ddendritic cell assay: phenotypic profiles of LPS-matured and immature moDC by flow cytometry. Histograms show the raw flow cytometry data for surface marker expression from 6 day differentiated moDC, cultured overnight as immature dendritic cell (red) or matured with LPS + IFNγ (blue).

Therapeutics that induce a similar change in the dendritic cell maturation profile are likely to show efficacy in an infection setting while those that block the LPS-induced changes are likely to be suitable for autoimmune disease intervention.

Confocal imaging or HCS can provide both a greater understanding of mechanism of action and evaluate the sub-cellular localization of targets of interest.

For the example data shown in Figure 3, moDC have been co-cultured with fluorescently labelled IgG as a surrogate for immune complex uptake. The moDC endocytose the IgG which then partially co-localizes with LAMP1, a lysosomal marker, suggesting that the IgG has entered the lysosomal degradation pathway. This uptake mechanism could therefore drive responses to self or pathogenic antigens depending on the disease context.

Figure 3: Uptake of immunoglobulin by moDC and sub-cellular localization. Images taken of moDC exposed to fluorescently labelled IgG. On the left-hand side the cells were stained with Actin (green), IgG (purple), and nuclear stain (blue). On the right-hand side, the cells were stained with IgG (green), LAMP1 (red), and nuclear stain (blue). These images show that IgG is internalized by moDC and subsequently partially co-localizes with lysosomes (LAMP1+).

LPS treatment of moDC induces both inflammatory cytokines such as IL-12, IL-6, and TNFα, and the anti-inflammatory cytokine, IL-10 (Figure 4). The absolute quantities of the cytokines can be used as a functional readout as they will influence the nature of the T cell response.

Figure 4: Cytokine production by moDC. moDC were cultured overnight with (blue bars) and without (red bars) LPS. The production of cytokines (IFNα, IL-1β, IL-10, IL-6, IL-12p70, TNFα) were then quantified from supernatants by Luminex.

T cell activation can be used as a readout for modulation of dendritic cell antigen presenting function.

MLR assays using allogenic CD4 T cells co-cultured with therapeutically targeted dendritic cell provide a straightforward screening assay to assess dendritic cell antigen presenting function. Readouts of dendritic cell function include T cell proliferation and cytokine production as well as phenotyping of the dendritic cell and T cells by flow cytometry.

The use of mature moDC enhances the ability of CD4 T cells to proliferate and is accompanied by upregulation of MHC Class II and ICAM on the dendritic cells (Figure 5A). In addition, inflammatory cytokines including TNFα, IL-6, and IL-12 go from undetectable levels to a robust response upon maturation of dendritic cell (Figure 5B).

Figure 5: DC and CD4 T cell MLR comparing immature and mature dendritic cell function. The moDC were cultured with and without dendritic cell maturation conditions (LPS + IFNγ). Allogenic CD4 T cells were added for several days before measuring cytokines by Luminex and CD4 proliferation by flow cytometry.

This offers a platform for testing therapeutics that will modify the interaction between the dendritic cell and T cells to either increase, decrease, or repolarize the T cell response.

Agents that induce a tolerogenic dendritic cell profile can be assessed as demonstrated below

Levels of IL-10 in the culture supernatant were increased in cultures where the therapeutically modulated dendritic cell were present, which could be desirable for the treatment of autoimmune disease.

Figure 6: Tolerogenic dendritic cell generation and induction of IL-10-secreting T cells. Monocytes were differentiated into moDC in the presence or absence of a bacterial product. Following this the different moDC populations were co-cultured with autologous naïve CD4+ T cells in the presence of anti-CD3/28 antibodies. Levels of IL-10 secretion in co-cultures were determined by Luminex.

Levels of IL-10 can then be quantified in the culture supernatant, and were increased in cultures where the therapeutically modulated dendritic cell were present, suggesting the treatment could have efficacy in autoimmune disease.

Using nucleofection with a plasmid or mRNA, lentiviral transduction, or siRNA transfection, genetic manipulation can be used to overexpress antigens or to target specific genes associated with dendritic cell function.

For instance, introducing an antigen associated with autoimmune responses that is not typically expressed by dendritic cell allows endogenous processing and presentation to T cells to test modulation of immunogenicity in a range of sophisticated assays.

Genetically modified dendritic cell assay can be combined with any other assay platform to offer a bespoke system, targeting a gene of interest and/or therapeutic.

Figure 7: Dendritic cell assay: genetic modification of DC. moDC were subjected to nucleofection with GFP mRNA or lentiviral transduction with a GFP vector. The efficiency of transfection/transduction was determined two days later by flow cytometry compared to mock transduced cells.

Naïve CD8 T cells can be co-cultured with matured autologous moDC alongside an antigen of interest. This antigen could be a known target, such as a tumor-associated antigen (TAA), an autoimmune antigen, or even a novel target to test its immunogenicity.

The example data here shows a TAA inducing the strong expansion of antigen specific T cells following a two-week co-culture with moDC (Figure 8).

This platform can therefore be utilized to test the efficacy of a compound in the context of rare IO or auto-antigen specific T cell responses.

Figure 8: Antigen specific T cell expansion in an autologous moDC co-culture. Naïve T cells and CD14+ monocytes were isolated from healthy human blood samples. Following the differentiation of dendritic cells, naïve T cells were added for a two-week co-culture with tumor-associated antigen peptide and cytokine stimulation. Antigen specific T cell expansion was then assessed. A) T cells were stained with tetramer to detect antigen specific T cells by flow cytometry. B) CD8 T cells were restimulated with peptide alongside autologous CD14+ monocytes in the presence of Golgi plug for four hours, and IFNγ expression was then measured by flow cytometry.

Dendritic cells are a key intersection between the innate and adaptive immune system. Their phenotype and function are shaped by the microenvironment or context in which they were activated. Dendritic cell play a critical role in shaping the ensuing adaptive immune response by either enforcing tolerance or enhancing T cell responses, which are largely determined by the activation status of the Dendritic cell. Dendritic cell patrol peripheral sites, such as the skin or the intestines, and are continuously sampling their environment to process and present antigens via MHC molecules to lymphocytes. Dendritic cells also play a critical role in activating other lymphocyte subsets, including innate subsets such as NK cells, through the secretion of IL-12 and IL-15.

Coutant, F., Miossec, P. Altered dendritic cell functions in autoimmune diseases: distinct and overlapping profiles. Nat Rev Rheumatol 12, 703–715 (2016)

Audiger C. & al., The Importance of Dendritic Cells in Maintaining Immune Tolerance. J Immunol 198(6):2223-2231 (2017)

Yes, we have experience translating many of our assays for in vitro dendritic cell assay work with both murine and human cells.

We can isolate particular dendritic cell subsets based on surface marker expression. However, the limited availability of differentiated dendritic cell subsets from blood often means it is more practical to use established methods for differentiating monocytes into dendritic cell and applying dendritic cell maturation or inhibitory stimulus to these cells to differentiate them into a given phenotype for the most appropriate functional dendritic cell assay.

A model using dendritic cell offers a more physiologically relevant system, allowing an assessment of both T cell function and the ability of the key immune priming cell to stimulate a T cell response. Numerous well-known receptor: ligand interactions, commonly targeted in drug discovery programs, will not occur without the presence of dendritic cell. The inclusion of dendritic cell assay also allows for antigen-specific responses to be assessed. This opens up the possibility to work with an antigen specific to the disease model, making it more translationally relevant, and to determine whether a novel peptide is immunogenic.

Through lentiviral transduction or nucleofection we can introduce an antigen of interest, allowing for an assessment of physiologically relevant primary cell systems to screen for therapeutics that modulate antigen processing and presentation by the dendritic cell to T cells. Alternatively, we can introduce gene knockdown to validate pathway mechanisms within a dendritic cell assay.

We are happy to discuss and customize any of our dendritic cell assays to meet the requirements of your project. Our expert immunologists can help by suggesting what parameters can be changed to tailor the setup and readouts of the dendritic cell assays to suit your needs.

This is obviously a complex question to answer, however as a starting point, a therapeutic that enhanced dendritic cell activation, and subsequently T cell function, might be targeted towards vaccinology or immune oncology. One that suppressed dendritic cell function and did not initiate T cell responses, or even inhibited existing T cell responses, may be more appropriately targeted towards autoimmune disease or inflammatory conditions.

Wculek, S.K. et al. Dendritic cells in cancer immunology and immunotherapy. Nat Rev Immunol 20, 7–24 (2020)

Bieber, K. et al. Dendritic cell development in infection. Mol Immunol, 121, Pages 111-117 (2020)