Identification of gene combinations to target in dendritic cells to enhance T cell activation using HMPCITE-seq platform.
2Core Research Facility, The Hebrew University of Jerusalem
Introduction
Dendritic cells (DCs) are innate immune cells that mediate the priming and activation of adaptive immune cells. In the absence of DCs, the adaptive immune system is not functional. This is crucial in the context of cancer since the immune system can eliminate malignant cells. However, tumor microenvironment attenuates the immune response. Suppressive signals produced by malignant cells were shown to affect DCs, reducing their ability to prime T cells and inhibiting a proper adaptive immune response against cancer. CD86 (B7-2) is one of the critical molecules that are expressed on the surface of DCs, and it interacts with CD28 on T cells to deliver a signal that is essential for T cell priming. Expression of CD86 was shown to depend on environmental signals and, in many cases, was found to be reduced in the immunosuppressive environment. We found that CD86 is a rate-limiting factor in T cell priming and that co-incubation of bone marrow-derived dendritic cells (BMDCs) with pancreatic cancer cell line restricts the expression of CD86. To find how the expression of CD86 is regulated and reveal targeted genes that will increase the expression of CD86 and improve the activation of DCs even in the presence of immunosuppressive conditions, we introduced the High Multiplicity of Perturbations and Cellular Indexing of Transcriptomes and Epitopes platform (HMPCITE-seq).
Materials and methods
The HMPCITE-seq platform is based on two parts: genome-wide CRISPR screen and high-order perturb-seq screen. The CRISPR screen aims to identify genes that increase CD86 expression, while the perturb-seq aims to find gene combinations that improve the overall DC program.
Results and discussion
We found that a combination of targeting Med12 and Cebpb together enhances the increase of CD86 level. In addition, functional validation demonstrated that this combination of perturbations results in improved CD8 T cell priming and enhanced expression and secretion of cytotoxic cytokines.
Conclusions
HMPCITE-seq platform reveals a set of targeted genes that enhances CD86 expression and DC activation. This methodology allows us to find the effect of each target on the gene expression program and improve the desired phenotype by targeting two genes simultaneously.