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Getting to know an immunosuppressive microenvironment



Clinically relevant tumors have largely evolved in an immune-suppressive microenvironment that makes them mostly invisible to the immune system. At Horizon, we have mimicked aspects of this immune suppressive state by building screening assays using regulatory T cells (Tregs), myeloid derived suppressor cells (MDSCs) and regulatory B cells (Bregs). 

The science and applicability of Treg assays
Outside of a diseased state, Tregs are not abundant in the peripheral blood of healthy individuals, so to generate enough Tregs to run an assay with, the Treg phenotype needs to be induced in vitro. At Horizon we develop iTreg cells by culturing naïve CD4+ T cells isolated from different blood donors using media supplemented with interleukin-2 (IL-2) and transforming growth factor beta (TGF-β). By FACS we confirm the polarization to iTregs by staining for intranuclear Foxp3 expression, and cell surface expression of CD25 and CD127. To verify that these cells are suppressive, we co-culture these iTregs with proliferating CD3+ T cells and use FACS analyses to determine levels of proliferation using the Celltrace stain present in the proliferating T cells. The iTreg differentiation protocol and the co-culture of iTregs with proliferating T cells can be used to examine the impact of compounds, biologics or small molecules on iTreg polarization and/or changes in the suppressive nature of iTregs on T cell proliferation. 
Taking a closer look at myeloid derived suppressor cells
Similar to Tregs, MDSCs are not common in the peripheral blood from healthy donors, so need to be generated in the lab for use in suppression assays. At Horizon we generate MDSC by stimulating peripheral blood mononuclear cells (PBMCs) with a cytokine cocktail that includes granulocyte-macrophage colony stimulating factor (GM-CSF) and IL-6, followed by isolation of the resulting CD33+ cells to recover the MDSC phenotype. To validate the MDSC phenotype, these cells are co-cultured with proliferating T cells to verify inhibition of cell proliferation using Celltrace detection via FACS (Fig 1). Our MDSC: T cell co-culture assay can be used to examine the capacity of compounds, biologics or small molecules to either inhibit or enhance the suppression to T cell proliferation by MDSCs. In addition to proliferation, we can assess cytokine production and T cell activation markers, such as CD25, to add further biological context to these assays. 

 

Figure 1

Welcoming the new kid on the block: Bregs

The more recently described regulatory B cell (Breg) also contributes to immune suppressive environments. Again, Bregs are not common in PBMCs and so we generate these in vitro using media supplemented with CD40L, CpG and IL-21. One prominent characteristic of Bregs is their production of the immune suppressive cytokine IL10.We used this to determine both the best method for the generation of Bregs in vitro, and to check that our in vitro generated iBregs produce this cytokine in detectable quantities (Figure 2).iBregs can be used in co-culture assays with proliferating T cells, again labelled with Celltrace, to examine the effect of compounds, biologics or small molecules on iBreg suppression of T cell proliferation.

 

Horizon's primary immune cell CRISPR screening platform

Horizon has been performing CRISPR screens for more that 12 years and have expanded our services to include pooled and arrayed CRISPR screening in primary immune cells. We can run pooled CRISPR screens in primary T cells and arrayed CRISPR screens in primary T cells and Breg cells. Using Bregs as an example, we have developed and tested an arrayed CRISPR screen, which uses co-culture with proliferating T cells as an end-point assay.In this assay we introduce a small arrayed CRISPR library consisting of crRNA targeting genes of interest and tracrRNA along with Cas9 protein via electroporation. Two days after electroporation, we analysed both levels of IL-10 production and whether the edited Bregs were still able to suppress T cell proliferation. In this screen we identified genes that impact the capacity of Bregs to produce IL-10 and identified genes that when knocked out inhibit the capacity of Bregs to suppress T cell proliferation (Figure 3).

Fig 3

Make Horizon your partner of choice

Horizon’s capabilities help researchers address timely biological questions, like the underlying genes that enable Bregs to suppress T cell proliferation. Such assays can aid the development of new therapeutics to treat auto-immune related diseases or enhance patient immune response in the treatment of cancer. We are ready to work with you on your next immune-based therapeutic.