Periodic Reporting for period 1 - CD4CRISPR (A quorum sensing mechanism regulating CD4 T cell proliferation)
Período documentado: 2017-04-01 hasta 2019-03-31
Knowledge about the physiology of the immune response is important for vaccine development. At the core of the adaptive immune response, CD4 T cells are key players of the cellular immunity against pathogens and cancer. The regulation of their proliferation, which is altered during viral infection or in immunosuppressive tumor microenvironments, is a complex and crucial phenomenon underlying the efficiency of the cellular immune response. We have developed an unbiased approach to identify memory CD4 intrinsic inhibitors of proliferation in pathophysiological contexts like chronic infections and cancer. We have restored the mutated CD4 T cells survival and proliferation under an in vivo selective pressure. Identifying mechanisms involved in the inhibition of memory CD4 T cells proliferation is key to the development of long lasting vaccines against pathogens, cancer and also to a better understanding of the mechanisms of autoimmunity.
We first reproduced the inhibition of Ag-specific CD4 T cells proliferation in the presence of a first cohort with in vitro generated memory Marilyn cells, allowing us to perform the genome wide inactivation of these cells transduced with the lentiviral gRNA libraries. To overcome the delivery challenges of Cas9, we have crossed Marilyn CD45.1 mice to Cas9-expressing mice. In the hypothesis that genes involved in the activation or survival of memory CD4 T cells are also involved in their proliferation as well as their retention in the lymph node, we will also use a Tamoxifen-inducible strategy (Rosa26-LSL-Cas9 mice crossed to Tam inductible-Cre-ERT2 Marilyn mice, allowing for temporally controlled gene inactivation. We have already optimized Marilyn CD4 T cells transduction. We have also validated the coverage of sgRNAs in both amplified libraries by deep sequencing. We will perform the pool screening several times and 10 recipient mice will be used to generate sufficient numbers of T cells for DNA isolation and deep sequencing, as well as to reduce potential variability. Likewise, a large number of control sgRNAs within each pool are present to assess the frequency of off-target effects. Model-based Analysis of Genome-wide CRISPR/Cas9 Knockout (MAGeCK) method will be performed to robustely identify the targets of the in vivo positive screen. New gRNAs corresponding to the identified hits will be designed and validated in OT2 model prior to a depth functional annotation of immunosuppressive pathways study by RNAseq with Nextera protocol and HiSeq sequencer.
The balance between protective and regulatory CD4 T cells and the governing rules of CD4 antitumor immunity will be the subject of active investigation over the coming years. Here, we present a functional genetic approach combining genome wide screening and primary T cells engineering in vivo to rapidly identify factors limiting memory CD4 T cells expansion. We propose a highly competitive research program, combining all the latest advances and technologies developed in the field of cells engineering, pre-clinical cancer models and high throughput cells processing. Understanding the precise regulation of various immune modulatory pathways will enable improved diagnostics and the development of more appropriate therapies for specific patient populations. A major application is the discovery of sgRNAs that enhance the therapeutic activity of adoptively transferred human T cells. To this end, we are initiating such efforts and experiments are underway using human T cells in a xenotransplantation model. Our approach may also impact the field by guiding the pursuit of selected targets for the development of antibody or small molecule based inhibitors, depending on their cellular localization and function.