Description

About iQ Biosciences’ Human Treg Suppression Assay Kit

Regulatory T cells (Tregs) are currently being targeted as a modality to reshape the disease environment to modulate T cell function. In cases of autoimmunity, therapeutics are being developed to enhance Treg function, while in cancer, molecules are developed to abrogate Treg function. To develop these molecules, Treg suppression assays are commonly used to assess whether a potential therapeutic can inhibit or promote Treg function. In these assays, Tregs are co-cultured with responder cells, such as CD8 T cells or PBMCs, and functional readouts of the responder cells are performed at the end of the culture period. Proliferation and cytokine production are often used to assess function.

iQ Biosciences has made it simple to perform these assays with our Human Treg Suppression Assay kit. The kit includes human CD4+CD25+FOXP3+ Tregs validated for suppressive function and your choice of CD8 T cells or PBMCs. The kit contains plenty of cells to evaluate multiple Treg:responder cell ratios to demonstrate functional consistency and provide insights into the potency of Treg modulation if therapeutic molecules are included in the co-cultures.

The responder cells can be labeled with CFSE (or a proliferation dye of your choosing) so downstream interrogation of proliferation is made simple. After the culture period, the cells can be harvested and analyzed with a flow cytometer to examine dilution of CFSE in responder cells, which reflects proliferative function. Molecules that promote Treg function will demonstrate less proliferation while those that inhibit Treg function will demonstrate more proliferation relative to untreated control.

To test the effect of your molecule with the Human Treg Suppression Assay Kit, simply thaw the cells and co-culture the Tregs and responders at the desired ratios in the presence of the molecule. After the culture period, CFSE dilution can be analyzed on a flow cytometer.

Tregs and Responder Cells in the Treg Suppression Assay Kit

Tregs in the Treg Suppression Assay Kit are analyzed for the expression of CD4, CD25, FOXP3, and CD127. Characterization of Treg cells, as defined by CD4, CD25, FOXP3, and CD127 expression, was shown to be greater than 85% pure.

The Treg Suppression Assay Kit also comes with your choice of either pure CD8 T cells or PBMCs as responder cells. PBMCs are isolated from blood obtained from a local world-renowned collection facility using a sterile isolation method that permits T cells to retain function. In contrast, CD8 T cells are obtained from isolated PBMCs using magnetic cell sorting. Typical purities of the isolated CD8 cells are greater than 90%.

The Tregs included in the kit have been validated to have suppressive characteristics (Figure 1 and 2) so researchers can have confidence using these cells. Investigators simply need to follow the included protocol and add the therapeutic molecule they are assessing.

Figure 1. Human Tregs suppress proliferation of CD8 T cells. PBMCs were labeled with CFSE and co-cultured at different ratios of Tregs in the presence or absence of stimulation. After 4 days of co-culture, cells were stained with CD8 antibody and flow cytometric analysis was performed. (Left) Histograms of CFSE staining for unstimulated (gray) and stimulated (purple) CD8 T cells. Marker for proliferation is set based on unstimulated cells. (Right) Histograms of CFSE staining for CD8 T cells co-cultured with different ratios of Tregs.

Figure 2. Human Tregs suppress proliferation of CD8 T cells. PBMCs were labeled with CFSE and co-cultured at different ratios of Tregs in the presence or absence of stimulation. After 4 days of co-culture, cells were stained with CD8 antibody and flow cytometric analysis was performed. For analysis, dilution of CFSE was assessed on CD8 positive gate. Marker for CFSE dilution was set using unstimulated cells. (Left, Right) Graphs show the percent of divided CD8 cells based on CFSE dilution analysis from 2 different donors. At lower responder:Treg ratio, more suppression is observed.

Cryopreservation and Storage

Human Tregs, PBMCs, and CD8 T cells were cryopreserved carefully using iQ Biosciences’ cryopreservation protocol that ensures high viability (> 90%) after thawing.

Cells should be stored at < -120°C once they are received, such as within a liquid nitrogen tank (vapor phase).

Quality Control Process

We implement random sampling per lot to test for viability and cell counts to ensure they meet specifications, which are recorded on the Certificate of Analysis that is included with each shipment. Cell counts are obtained using a manual hemocytometer and then cross-referenced with an automated cell counter. Each lot is also characterized for unique cell populations by immunophenotyping, in which the results are also recorded on the Certificate of Analysis.