SIAT^® DC-T Cell Proliferation Service

SIAT® DC-T cell Proliferation Assay Workflow Published Data FAQ Resources

Antigen-presenting cells (APCs), typically dendritic cells (DCs), can process antigens and present peptide epitopes in conjugation with human leukocyte antigen (HLA) class II molecules to specific naïve helper-T (Th) cells, which results in the activation of Th cells. The activated Th cells can stimulate B cells to produce antibodies against antigens. For instance, anti-drug antibodies (ADAs) are generated in this way. Therefore, detection of T cell responses is frequently employed to monitor the ability of biotherapeutic candidates to elicit immune system activation, i.e., the immunogenicity. The most widely used method is the ex vivo DC-T cell proliferation assay.

SIAT® DC-T cell Proliferation Assay

Creative Biolabs provides SIAT^® ex vivo DC-T cell proliferation assay service to assess the immunogenicity of biotherapeutic drug candidates using cells from HLA-typed donors. The SIAT^® DC-T cell proliferation assay allows the use of whole proteins, and more importantly, it can assess the factors other than protein sequence that contribute to immunogenicity. High-resolution HLA II-typed donor blood samples are used to mimic the allele frequencies found in the global population or to provide representative coverage of a particular population of interest without the need of animal models. Information provided by this assay is very useful during the early development of biotherapeutic drugs in terms of lead selection, humanization, and/or de-immunization.

Proliferation and differentiation of naïve T cells upon stimulation by DCs. (Idris-Khodja et al. 2014)

Workflow of SIAT® DC-T cell Proliferation Assay

SIAT^® DC-T cell proliferation assay uses Flow Cytometry to detect T cell proliferation after co-culture with loaded DCs, which is extremely sensitive and accurate. Specifically, T cells are labeled with 5,6-carboxyfluorescein diacetate succinimidyl ester (CFSE) dye. Those that proliferate in response to peptide antigen presented by DCs show a CFSE dye dilution, which can be measured directly by Flow Cytometry. In combination with detection of cell surface markers, the percentage of proliferating CD4⁺ cells, as well as the detailed phenotype of T cell responses are all monitored. Below is a brief description of our workflow for SIAT^® DC-T cell proliferation assay service.

Customers provide protein samples or the information of protein of interest so that we can obtain samples in house. Diverse protein expression strategies and high-quality recombinant protein synthesis platform are available.
Obtain human blood samples from our blood bank of healthy and high-resolution HLA class II-typed donors, which covers representative populations in the globe.
Isolate PBMC from blood samples and differentiate into to DCs by culturing in media supplemented with specific cytokines.
DCs are pulsed with protein samples and then harvested.
CD4⁺ T cells are enriched from the same donors who provide monocytes by a negative selection system. Unwanted cells are removed with antibody complexes recognizing non-T cells fixed on dextran-coated magnetic particles.
Co-culture the loaded DCs and CD4⁺ T cells derived from the same donors.
T cell proliferation is determined using a CFSE staining assay with Flow Cytometry.
Statistical analysis of the data and interpretation of data in a report.

With years of experience in biotherapeutic drug development, scientists of Creative Biolabs are confident in offering the best service to meet our clients’ specific requirements.

More SIAT^® Immunogenicity Related Services at Creative Biolabs

Published Data

Characterization of Functional Changes in MSC Immunomodulation by T-cell Proliferation Assays

Fig. 2 Inflammatory priming and/or exposure to inflammatory macrophages does not impair MSC immunomodulatory function. (Jennifer M. Cassano, 2018)

The study demonstrated the potential of inflammatory licensed mesenchymal stem cells (MSCs) to enhance immunomodulation and exhibit chondroprotective effects in an inflammatory environment. This was achieved by priming MSCs with IFN-γ or poly I, which notably improved their ability to suppress T-cell proliferation, particularly after exposure to pro-inflammatory macrophages. The research underscored that IFN-γ primed MSCs had a superior ability to suppress T-cell proliferation compared to naïve MSCs and maintained this ability even after interaction with proinflammatory macrophages. This enhancement in immunomodulatory function could contribute to more effective MSC therapies in inflammatory conditions. The ex vivo DC-T cell proliferation assay helped demonstrate that MSCs primed with IFN-γ and exposed to inflammatory macrophages retained their capacity to modulate T-cell proliferation effectively, highlighting a crucial aspect of MSCs' functionality that could be harnessed in therapeutic applications to regulate immune responses in diseased or injured tissue environments.

References

Idris-Khodja, Noureddine, et al. "Dual opposing roles of adaptive immunity in hypertension." European heart journal 35.19 (2014): 1238-1244.
Cassano, Jennifer M., et al. "Inflammatory licensed equine MSCs are chondroprotective and exhibit enhanced immunomodulation in an inflammatory environment." Stem cell research & therapy 9 (2018): 1-13.

FAQ

What is an ex vivo DC-T cell proliferation assay?
An ex vivo DC-T cell proliferation assay is a laboratory technique used to evaluate the immunogenic potential of biotherapeutic drug candidates. This assay involves co-culturing dendritic cells with T cells in the presence of the drug candidate to observe T cell activation and proliferation, indicative of an immune response.
Why use dendritic cells in immunogenicity assessment?
Dendritic cells are pivotal in the immune response as they act as antigen-presenting cells that can initiate and modulate T cell responses. Using these cells in assays helps in mimicking the in vivo interaction and response to a biotherapeutic, providing insights into its potential immunogenicity.
How are dendritic cells obtained for the assay?
Dendritic cells used in these assays are typically derived from peripheral blood mononuclear cells (PBMCs) of healthy donors. These PBMCs are cultured with specific growth factors like GM-CSF and IL-4 to differentiate them into dendritic cells.
What markers are used to measure T cell proliferation in this assay?
T cell proliferation can be assessed using several markers, such as the expression of activation markers like CD25 and CD69, and through incorporation of DNA synthesis markers like BrdU or EdU during cell division.
How does this assay help in predicting the immunogenicity of biotherapeutics?
By evaluating T cell proliferation in response to a biotherapeutic, this assay provides direct evidence of the potential for the therapeutic to elicit an immune response in patients. High levels of T cell proliferation suggest higher immunogenicity, which could impact the safety and efficacy of the therapy.
Can this assay be used for all types of biotherapeutic drugs?
The ex vivo DC-T cell proliferation assay is versatile and can be used to assess a wide range of biotherapeutic drugs, including monoclonal antibodies, fusion proteins, and other recombinant proteins. However, the specific conditions and readouts may be optimized based on the type of drug being tested.
How long does it take to perform an ex vivo DC-T cell proliferation assay?
The duration of the assay can vary, but typically it takes about 1 to 2 weeks. This includes the time for dendritic cell differentiation, drug exposure, co-culturing with T cells, and subsequent analysis of T cell proliferation.
How does this assay compare with other immunogenicity testing methods?
Compared to in vivo studies, ex vivo assays are less expensive, faster, and require no ethical approvals, which makes them suitable for early-stage drug development. However, they do not fully replicate the complexity of an organism's immune system. Other methods like in silico prediction and in vivo animal studies are often used alongside ex vivo assays to provide a comprehensive assessment of a biotherapeutic's immunogenicity.