T Cell Assays

Our T cell assays are designed to characterize T cells via T cell proliferation assays, T cell activation assays, and T cell cytotoxicity assays, or monitoring T cell differentiation via immune markers.

T cells reside in peripheral tissue or circulate in the body until they recognize their antigen on the surface of an antigen-presenting cell. The interaction with the antigen-presenting cell sets a complex response in motion. Our T cell assays are designed to understand which signals trigger a response and to characterize this response.

T cell assays at Reaction Biology include:

  • T cell proliferation assays to monitor the expansion of T cells after activation
  • T cell activation assays to monitor hallmarks for T cell activation, including cytokine release and immune surface markers
  • T cell cytotoxicity assays measuring the number of cytokine-secreting cells via ELISpot

Please reach out today and discuss directly with our scientists how we can help you best to characterize your new immunotherapeutic agent.

T Cell Assays

T cell assays help understand what is happening after T cells engage with antigen-presenting cells and how an immune-modulating drug skews this response, such as

  • Immune cell proliferation
  • Cytokine release
  • Activation marker expression
  • Exhaustion marker expression.

Example Studies of T Cell Characterization Available at Reaction Biology

T Cell Proliferation Assay

Tracking of T cell division via CFSE after activation via plate-bound anti-CD3 antibody 

Assay principle

T cell proliferation is one of the hallmarks of successfully activated T cells. Carboxyfluorescein succinimidyl ester (CFSE) is a cell-permeable dye that binds covalently to intracellular molecules and is retained within cells for very long periods. The intensity of CFSE in cells can be measured by flow cytometry. With each cell division, the intensity halves, enabling the identification of proliferating cells.  

Setup

PBMCs are plated in multi-well plates coved with anti-CD3 antibodies, which engage the T cell receptor, triggering T cell activation and expansion. CFSE dye is used to track cell division by measuring CFSE intensity in the cells. 

Results

T cell proliferation assay with CFSE

Healthy donor PMBCs were labeled with CFSE and either cultured in the presence of two different concentrations of plate-bound anti CD3 (OKT3) or left untreated. After 5 days, cells were harvested, stained with a fixable viability dye and surface markers (CD3, CD4, and CD8), and fixed with 4% PFA. Cells were acquired using a Cytoflex S (Beckman Coulter).

Characterization of donor PBMCs

Peripheral blood mononuclear cells (PBMCs) are a mixture of immune cell populations developed in the bone marrow from hematopoietic stem cells. The PBMCs include particularly those with a single round nucleus, composed mainly of lymphocytes, dendritic cells, and monocytes. We isolate PBMCs out of buffy coats, which entail the entire cellular fraction of one blood donation unit.

The material is ideal for the isolation of a large number of immune cells from one healthy donor and allows us to have enough material to isolate rare cell types (like NK cells or monocytes) and to have several frozen vials from the same donor in order to perform different tests with the same cell batch if needed.

Since the frequency of different cell types within PBMCs varies from donor to donor, we performed a broad characterization of the main immune cell populations. With this information, we can choose the best donor for a project depending on the particular need for a certain immune cell population.

Setup

Healthy donor PBMC were isolated from buffy coats after density gradient centrifugation and stored in liquid nitrogen until further use. PBMC were thawed, labeled with a fixable viability dye, and stained for surface markers for T cells (CD3, CD4, CD8), monocytes (CD14), B cells (CD19), and NK cells (CD56).

We also included a Human Leucocyte Antigen Serotype (HLA-A2) marker and stained for HLA-DR, which is expressed on the surface of antigen-presenting cells (macrophages/monocytes, B cells, and dendritic cells) and is upregulated in response to stimuli.

Results

pbmc characterization

Immune cell population Frequency of viable cells (% of viable single cells)
HLA-A2+ 97 %
HLA-DR+  54 %
CD3+  30 %
CD3+ CD4+ 16 %
CD3+ CD8+ 10 %
CD3- 70 %
CD3- CD19+ 6 %
CD3- CD56+ 11 %
CD3- CD14+ 39 %

 

The frequencies of several immune cell populations in the PBMCs of one donor are shown. Please reach out to inquire about the profiles of other PMBC donors or sources to ensure we align the experiment setup with your research questions.