Supression of CD80 and CD86 expression raw 264.7 cell line by using CRISPR/Cas9 gene editing system

Immunological tolerance is a condition in which the immune system does not respond to an antigen. Failure of immunological tolerance results in autoimmunity and autoimmune diseases. There has been a disturbing increase in the incidence and prevalence of autoimmune diseases in recent years. These diseases are important clinical problems due to their chronic structure, related health care costs and prevalence in young populations. In this study, it was aimed to improve tolerance mechanism by forming anergy in T cells by silencing CD80 and CD86 genes in macrophages which are one of professional antigen presenting cells that stimulate T cells. In study, the oligonucleotides specific to CD80 and CD86 genes selected by using Mouse CRISPR Knockout Pooled Library were cloned into LentiCRISPRv2 vector. Viruses of ligated CRISPR vectors were generated and Raw 264.7 cells were transduced with these viruses. These cells were then subjected to puromycin selection and the surviving cells were stimulated with IFN-γ to show whether de nova synthesis was suppressed. These cells were then threated with CD80 and CD86 monoclonal antibodies and read and analyzed by flow cytometry. In flow cytometry results following IFN-γ stimulation showed an increase in CD80 and CD86 expression in macrophages transduced with CRISPR vector similar to cells transduced with the control vector, expected CD80-CD86 silencing did not occur. Treatment attempts are being made to trigger tolerance mechanisms against autoimmune diseases and positive results are obtained. In particular studies on the suppression of CD80 and CD86 expression are new and our study is an example of these attempts. Although CD80-CD86 genes could not be silenced in our study, in further studies aimed at solving the problem, approaches to suppress the co-stimulatory signal with CRISPR/Cas9 system may be used as an alternative treatment method.