Using CRISPR to determine the effects of mutations of PTPN22 in human T cells
The haematopoietic phosphatase PTPN22 is a key regulator in balancing immune responses between self-reactivity and tolerance. PTPN22 downregulates T cell signaling and harbors the non-HLA genetic variation most strongly associated with autoimmune disease in humans, the single nucleotide polymorphism R620W. The effect of this mutation is currently controversial due to confounding results in mouse and human models. The polymorphism is linked to increased susceptibility to autoimmunity in both human and mouse models, although the latter does depend on genetic background. However, mouse data clearly shows that the polymorphism has a loss-of-function effect on T cell signalling, whereas studies in human models largely demonstrate a gain-of-function effect for R620W. A confounding issue in human studies is that they depend on comparison of T cells from distinct individuals, on protein over-expression, or on RNA interference, techniques for which it is difficult to control for genetic and environmental variables, changes in stoichiometry, and off-target effects or incomplete knockdown, respectively. We aimed to create isogenic human cell lines with mutations in PTPN22 at the genomic level to alleviate the complications inherent in analysing human data. In addition to autoimmune pathogenesis, we are interested in the role of PTPN22 in a cancer setting. Because PTPN22 has a strong suppressive effect on T cell responses to weak affinity antigen, which encompass most tumour antigens, we postulated that knocking out PTPN22 may better enable T cells to kill tumour cells. Furthermore, we have shown that PTPN22 knockout (KO) leads to increased IL-2 expression in mouse T cells, and that this effect is protective against TGF-β mediated suppression, a common driver of T cell inhibition in the tumour microenvironment. T cell transfer experiments in mice showed that PTPN22 KO T cells are indeed more effective at reducing tumour size. Based on these findings, we aim to determine whether PTPN22 KO in human cells confers a similar effect on signaling. To investigate the effects of PTPN22 KO on human T cell signaling, we used CRISPR gene-editing to target PTPN22 in a Jurkat cell line. By combining this technique with lentiviral transduction of a specific T cell receptor, we generated human cell lines which are genetically identical, save for specific alterations to PTPN22, and which can be stimulated with strong or weak cognate antigen. We found that PTPN22 KO Jurkat cells develop an enhanced activation phenotype upon stimulation, including increased IL-2 expression. Additionally, PTPN22 KO Jurkat cells show enhanced Erk signalling following stimulation with weak affinity antigen, but this difference is lost as stimulus strength increases. CRISPR technology has presented the opportunity to create novel models of PTPN22 signalling in the context of human T cell lines. The data from these lines suggests that, unlike the R620W mutation, complete loss of PTPN22 has a comparable effect in human and mouse T cells. In conjunction with our previous findings, these results suggest that knocking out PTPN22 may lead to signalling alterations that improve adoptive T cell cancer therapy.