Abstract 1213: RNA-based screens in primary human immune cells

• Published in: Cancer research (Chicago, Ill.) Ill.), 2019-07, Vol.79 (13_Supplement), p.1213-1213
• Main Authors: Joubert, Bronwyn, Ghirelli, Cristina, Nett, Isabelle, Prime, John, Martin, Glynn, Moore, Jonathan, Cross, Benedict, McCarthy, Nicola J.
• Format: Article
• Language: English
• ISSN: 0008-5472; 1538-7445
• DOI: 10.1158/1538-7445.AM2019-1213

Genetic screens have long been used as an approach to identify and validate new targets for drug discovery. The vast majority of these have been carried out in cell lines: mostly cancer cell lines. However, with improvements in tissue culture techniques, the increasing interest in using the immune system to tackle disease and the discovery of CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-Cas9 mediated genome editing, screening primary cells that have not been subverted by transformation into immortal lines, is both appealing and feasible. We have successfully carried out a CRISPR-Cas9 screen in primary T cells using a combined lentivirus and electroporation protocol. Freshly isolated primary T cells are stimulated with anti-CD3 and anti-CD28 antibodies and then transduced with a pooled sgRNA library. After antibiotic selection, T cells successfully transduced with sgRNAs are electroporated to introduce Cas9 mRNA. We chose to validate this approach by carrying out a screen similar to that published by Birsoy et al., 2015, in which they ran a CRISPR-Cas9 screen in Jurkat T cells in the presence and absence of the electron transport chain inhibitor phenformin. In our screen, we exposed the CRISPR-Cas9 edited pool of primary T cells to a dose of phenformin that resulted in growth inhibition to a similar degree to that used by Birsoy and colleagues. Our data are in agreement with the published screen, showing that loss of the cytosolic aspartate aminotransferase GOT1 sensitises primary T cells to phenformin. We took multiple time points in our primary T cell screen and used T cells isolated from three different donors, allowing for the analysis of guide drop-out kinetics and reproducibility between donors. We anticipate that these data will be useful in building more complex screens that assess T cell biology in the presence of additional cells, such as myeloid derived suppressor cells (MDSCs). With a view to this, we have also carried out an arrayed siRNA screen in MDSCs to look for genes that when knocked down reduce the capacity of MDSCs to inhibit T cell proliferation. The endpoint for this screen is based on co-culture of siRNA transfected MDSCs with proliferating primary T cells. Using this complex data set, we have identified several potential targets, which when validated could provide new therapeutic targets through which the immunosuppressive nature of MDSCs in the tumour microenvironment can be mitigated. Birsoy, K., et al. (2015