Immune suppression 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.
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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
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
Although cells within a tumor express immune checkpoint inhibitor molecules, such as PD-L1, which suppress T cell activity, the immune-inhibitory tumor microenvironment can also lead to the differentiation of T cells into inhibitory regulatory T cells (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)
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.
Resting CD8+ T cells cultured alone (blue line) show low rates of suppression and this is taken as a baseline for the assay, 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.