Immune suppression assays

MDSC assays

The tumor microenvironment is well-characterized as highly immunosuppressive, and there are many mechanisms responsible. Myeloid-derived suppressor cells (MDSC) are macrophage-like cells that have attained an immunosuppressive phenotype as a result of the tumor microenvironment, and are known to suppress the activity of tumor-directed T cells.

We have developed an assay that synthetically derives MDSCs and measures their ability to suppress T cell proliferation. Test your compound’s ability to mitigate this suppression and boost T cell activity.

How does the MDSC suppression assay work?

  • PBMC are stimulated with a cytokine cocktail including GM-CSF and IL-6 to induce MDSC differentiation
  • MDSC are recovered using CD33+ isolation
  • Autologous T cells are incubated with the CD33+ MDSCs and stimulated with anti-CD3/CD28
  • Endpoints measured: T cell proliferation and activation surface markers

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What can we observe?

Blood-derived monocytes can be efficiently differentiated to myeloid derived suppressor cells using a cytokine cocktail including GM-CSF and IL-6, and are enriched using the marker CD33 (see diagram).

CD33+ MDSC can be cocultured with autologous CD8+ T cells to measure their suppressive effects.

We have demonstrated that cytokine-derived MDSCs dose-dependently inhibit autologous CD8+ T cell proliferation compared to CD8+ T cells alone (green vs red or blue bubbles).

In a culture of CD8+ T cells alone, 20% of T cells do not proliferate, whereas the addition of MDSC to the culture dose-dependently increases the percentage of undivided cells.

We can assess your compound for its ability to relieve suppression on T cells.

Treg suppression assays

While tumors upregulate immune checkpoint molecules such as PD-L1 on their surface to inhibit T cell activity, the generalized immune-inhibitory tumor microenvironment can also polarize T cells to a suppressive phenotype known as regulatory T cells, or Tregs. Tregs are CD4+ T cells characterized by the secretion of the regulatory cytokine IL-10, which is inhibitory to both macrophages and T cells, and can suppress the activity of tumor-directed T cells.

We have developed an assay that robustly generates Tregs and measures their ability to suppress T cell proliferation. Test your compound’s ability to modulate Treg polarization or to reverse suppression of T cell proliferation, thereby boosting anti-tumor T cell activity.

How does the Treg suppression assay work?

  • CD4+ T cells are stimulated with a cytokine cocktail including IL-2 and TGF-β1 to induce Treg polarization
  • Tregs are incubated with effector T cells (Teff) and stimulated with anti-CD3/CD28
  • Endpoints measured: Treg polarization and T cell proliferation
  • Add your compounds in either Step 1 (to measure effects on Treg polarization) or Step 2 (to measure effects on T cell suppression)
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What can we observe?

CD4+ T cells can be efficiently polarized to Tregs using a cytokine cocktail containing IL-2 and TGF-β1. Efficient polarization to the Treg phenotype is measured by the up-regulation of the transcription factor Foxp3 using flow cytometry (see diagram).

We have demonstrated that co-culture of naive CD8+ T cells (effector T cells, or Teff) with polarized Tregs dose-dependently inhibits CD8+ T cell proliferation compared to incubation of CD8+ T cells alone. Results are expressed as % suppression.

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There is a baseline lack of proliferation in CD8+ T cells alone (blue line), with a dose-dependent increase in suppression following the addition of Tregs (orange line), leading to 90% suppression of CD8+ proliferation at the highest dose of Tregs.

We can assess your compound for its ability to relieve Treg suppression on T cells, as well as its ability to prevent T cell polarization to the Treg state.

For more information on services or to discuss your requirements with our team.

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