Modeling autoimmune associated genetics in primary human T cells using CRISPR/Cas9 gene editing

Abstract

Genome wide association studies have identified genetic risk variants associated with multiple autoimmune diseases, thereby impacting large numbers of patients. Prominent examples are found within the phosphatase encoding genes PTPN22 and PTPN2. Studies have shown that risk variants in these genes impact a variety of cell types, but their expression or ablation in lymphocytes alone can be sufficient to drive autoimmunity in certain mouse models, due in part, to hyper-active lymphocyte signaling. Notably, cross-sectional studies of human carriers of the PTPN22 or PTPN2 risk variants have shown these donors to possess T cells that are hypo-responsive to TCR or cytokine stimuli. To investigate this discrepancy and understand the functional impact of these variants, new methods of studying these genes are required. The purpose of this study is to establish methods of using gene editing approaches in primary human T cells to modify expression the genes PTPN22 and PTPN2 and assess the impact of altered gene expression. Using this approach we found that ablation of PTPN22 and PTPN2 in primary human CD4+ T cells results in T cell responses which mirror current mouse models, with increased responses to TCR stimulation upon disruption of either gene, and enhanced responsiveness to IFNγ and IL-2 in PTPN2 disrupted cells. Interestingly, in the case of PTPN2 disruption, we found responses to IL-2 to be dynamic, eventually resulting in loss of responsiveness to IL-2, mirroring current human data from carriers of a reduced expression variant. Furthermore, we have explored several efficient methods to alter the coding sequence of PTPN22 to reflect its risk variant in non-risk donor T cells. Collectively our data shows that gene editing is a powerful tool for investigating how gene variants can contribute to disease, and that the effects of genetic risk variants may impart contextual and dynamic phenotypes on human lymphocytes. Finally, the methods we have established in this study are applicable to many other gene variants, and potentially could be utilized in multiple primary cell types.