How Can Dendritic Cell Function Be Modified to Help Treat Autoimmune and Inflammatory Diseases?

When exposed to inflammation or pathogen-associated molecular patterns (PAMPs), dendritic cells (DC) are activated and migrate towards lymphoid organs through which naïve T cells circulate. Activated (mature) dendritic cell express high levels of costimulatory molecules and release cytokines which co-ordinate with antigen presentation via MHC to activate CD4 and CD8 T cells, initiating or reactivating an existing immune response.

These pathways play a critical role in protective immunity to pathogens or tumors yet can be dysfunctional in autoimmune disease. As such, there is considerable scope to target various aspects of dendritic cell function therapeutically.

Key Assays to Target Dendritic Cell Function

Charles River has developed a range of in vitro dendritic cell assays which enable characterization of dendritic cell phenotype and function to evaluate the effect of therapeutics in a variety of disease settings:

  • MLR assays: co-culturing dendritic cells with allogeneic T cells allows assessment of the effect of a therapy on dendritic cell phenotype and function. This is assessed using flow cytometric analysis, proliferation of T cells, and production of cytokines as readouts.
  • By feeding dendritic cell with a tumor-associated antigen or an autoantigen associated with autoimmune responses, we have also developed sophisticated systems to screen for both antigen and therapeutic specificity, and can evaluate the ability of dendritic cell to drive either CD4 or CD8 T cell responses.
  • By transducing primary dendritic cell with a bespoke lentivirus containing the protein or antigen relevant to the disease or autoimmune condition, antigen-specific T cells can be expanded even when endogenous expression levels are low or absent in dendritic cell.
  • Tolerogenic dendritic cells are important in driving regulatory T cell (Treg) development and differentiate into this phenotype in the presence of high levels of IL-10 or certain bacterial components. Our models can drive the differentiation of this dendritic cell subset in vitro and the ability of a therapeutic to drive a similar polarization can be assessed; readouts include the ability of these tolerogenic cells to alter CD4 T cell polarisation. This readout is specifically targeted for evaluating therapeutics for autoimmune conditions, such as colitis, Crohn’s disease, or rheumatoid arthritis.

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Working With Dendritic Cell Assays In Vitro

Dendritic cells comprise three main subsets, conventional (cDC); types 1 and 2, and plasmacytoid (pDC). These cells are identified by their characteristic surface markers and can be isolated in small numbers from the blood to test the effect of therapeutics on d differentiation and function (Figure 1).

Alternatively, when larger numbers of dendritic cell are required, these can be differentiated from monocytes. Immature monocyte-derived dendritic cell (moDC) can be matured by exposing them to a variety of stimuli which in turn dictate the phenotype of the matured cell (Figure 2). This assay can be used to screen infection targeted therapeutics for their ability to promote immature moDC activation, whereas therapeutics that suppress this response in the presence of activating stimuli may be autoimmune targeted.

Dendritic cell assay; frequency in PBMC by flow cytometry

Figure 1: Assessment of dendritic cell subset frequency in peripheral blood mononuclear cells (PBMC) by flow cytometry. A representative gating strategy is shown for the identification of dendritic cell subsets from healthy human PBMC. From single viable cells, gates for CD45+, CD4-, CD8-, Lineage-, and HLA-DR+ were used to identify dendritic cell. Within the dendritic cell population, cDC1 were identified as CD11c+ CD141+ CD1c-, and cDC2 were identified as CD11c+ CD141- CD1c+. pDC were identified as CD11c- HLA-DR+CD123+. The frequency of each subset within total PBMC is shown on the right-hand side of the figure.

 

Dendritic Cell Assays: Phenotyping

  • Flow cytometry analysis for 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.

    Phenotypic profiles by flow cytometry in a dendritic cell assay.

    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 analysis for dendritic cell assays

    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.

    Confocal imaging analysis for dendritic cell assays.

    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+).

Functional Dendritic Cell Assays

There are several ways to probe the functionality of moDC using in vitro assays.

  • Dendritric Cell maturation cytokine profiles

    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.

    Analyze of cytokines production in immature and mature moDC culture, overnight.

    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.

  • Functional Dendritic Cell and T cell co-cultures

    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).

    Analyze of cytokines production in immature and mature moDC +/- CD4 T cells culture by flow cytometry or Luminex readout.

    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.

  • Tolerogenic dendritic cells

    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.

    DC and T cell coculture generating IL-10 schema and IL10 results

    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.

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Specialist Dendritic Cell Assays

  • Genetic manipulation of primary moDC

    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.

    Results of the efficiency of transfection/transduction of moDC by flow cytometry.

    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.

  • Human dendritic cell activation of autologous antigen specific T 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.

    Results of detection of antigen specific T cells and IFNγ expression by flow cytometry in a T cell & moDC coculture.

    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.

Murine Functional Dendritic Cell Assays

Like humans, the frequencies of primary circulating dendritic cell in rodents are very low. Bone marrow-derived dendritic cell (BMDC) therefore provide a suitable source of dendritic cell for screening assays.

BMDC can be used in similar assays to those described for human cells. The use of TCR transgenic T cells provides the advantage of being able to study naïve T cell responses and to investigate how intervening in dendritic cell differentiation and polarization can alter T cell responses to cognate antigen.

The example data here shows a model utilizing OT-I CD8 TCR transgenic T cells, which are specific for the SIINFEKL peptide derived from ovalbumin. The ability of therapeutically treated BMDC that have been pulsed with SIINFEKL peptide or fed with whole OVA protein to drive antigen-specific proliferation, cytokine production, and effector function of OT-I CD8 T cells can then be assessed.

Experimental workflow details and differentiation phenotype by flow cytometry.

Figure 9: Murine bone marrow-derived dendritic cell and T cell coculture. Bone marrow (BM) progenitor cells were isolated from C57BL/6 mice and cultured in vitro with IL-4 and GM-CSF to generate BMDC. Representative data for measuring the differentiation of these cells by flow cytometry is shown. Splenocytes from OT-I TCR transgenic mice were isolated and CD8 T cells were enriched by magnetic selection. The T cells were then labelled with CTV dye and co-cultured with BMDC that had been pulsed with SIINFEKL. Dilution of CTV was used as a readout for CD8 T cell proliferation.

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Dendritic cell are a critical player in the activation and polarization of T cell and other lymphocyte responses, as well as in the modulation of the tissue microenvironment. With these dendritic cell assays, which can be used to accurately measure and manipulate inflammatory or tolerogenic responses, our expertise provides dendritic cell assay platform for determining MOA or efficacy of dendritic cell targeting therapeutics.