PTPN22 R620W gene editing in T cells enhances low-avidity TCR responses
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Evaluation Summary:
PTPN22 is a protein tyrosine phosphatase which negatively regulates antigen receptor signaling. It has been proposed that several genetic variants of PTPN22 might be loss of function (LOF) variants, leading to hyper-responsive T cell proliferative and effector responses. The authors investigate how the PTPN22 R620W variant, associated with multiple autoimmune diseases, might contribute to breech of peripheral T cell tolerance. This work greatly advances and clarifies ongoing confusion of whether PTPN22 SNP(620W) is a LOF mutant.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 and Reviewer #3 agreed to share their name with the authors.)
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Abstract
A genetic variant in the gene PTPN22 (R620W, rs2476601) is strongly associated with increased risk for multiple autoimmune diseases and linked to altered TCR regulation and T cell activation. Here, we utilize Crispr/Cas9 gene editing with donor DNA repair templates in human cord blood-derived, naive T cells to generate PTPN22 risk edited (620W), non-risk edited (620R), or knockout T cells from the same donor. PTPN22 risk edited cells exhibited increased activation marker expression following non-specific TCR engagement, findings that mimicked PTPN22 KO cells. Next, using lentiviral delivery of T1D patient-derived TCRs against the pancreatic autoantigen, islet-specific glucose-6 phosphatase catalytic subunit-related protein (IGRP), we demonstrate that loss of PTPN22 function led to enhanced signaling in T cells expressing a lower avidity self-reactive TCR, but not a high-avidity TCR. In this setting, loss of PTPN22 mediated enhanced proliferation and Th1 skewing. Importantly, expression of the risk variant in association with a lower avidity TCR also increased proliferation relative to PTPN22 non-risk T cells. Together, these findings suggest that, in primary human T cells, PTPN22 rs2476601 contributes to autoimmunity risk by permitting increased TCR signaling and activation in mildly self-reactive T cells, thereby potentially expanding the self-reactive T cell pool and skewing this population toward an inflammatory phenotype.
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Evaluation Summary:
PTPN22 is a protein tyrosine phosphatase which negatively regulates antigen receptor signaling. It has been proposed that several genetic variants of PTPN22 might be loss of function (LOF) variants, leading to hyper-responsive T cell proliferative and effector responses. The authors investigate how the PTPN22 R620W variant, associated with multiple autoimmune diseases, might contribute to breech of peripheral T cell tolerance. This work greatly advances and clarifies ongoing confusion of whether PTPN22 SNP(620W) is a LOF mutant.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 and Reviewer #3 agreed to share their name with the authors.)
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Reviewer #1 (Public Review):
This study has some neat technological features that go a long way to reconcile contradictory data regarding functions of disease associated PTPN22 variants. These include:
• Crispr/Cas9 gene editing of exon 14 of PTPN22 in primary human T cells to generate HDR for WT, and gene editing for risk and KO sequences
• Use of cord blood T cells, mitigating against any variability in T cell responses that could be influenced by activation or differentiation state
• Lentiviral infection of these T cells with high and low avidity TCRs that recognise the same peptide from the islet cell autoantigen IGRP, presented by HLA-DRB1*0401; the TCRs are chimeric, allowing detection of LV transgene and detection of TCRs that have not cross-paired with endogenous TCR chains
• Cis-linked GFP to detect those T cells expressing TCR …Reviewer #1 (Public Review):
This study has some neat technological features that go a long way to reconcile contradictory data regarding functions of disease associated PTPN22 variants. These include:
• Crispr/Cas9 gene editing of exon 14 of PTPN22 in primary human T cells to generate HDR for WT, and gene editing for risk and KO sequences
• Use of cord blood T cells, mitigating against any variability in T cell responses that could be influenced by activation or differentiation state
• Lentiviral infection of these T cells with high and low avidity TCRs that recognise the same peptide from the islet cell autoantigen IGRP, presented by HLA-DRB1*0401; the TCRs are chimeric, allowing detection of LV transgene and detection of TCRs that have not cross-paired with endogenous TCR chains
• Cis-linked GFP to detect those T cells expressing TCR transgenes. Infection is undertaken using titres of virus likely to avoid high copy number TCRs and therefore variable TCR expression
• Repeat experiments using multiple donors
• TCR stimulations using a range of different readoutsThe main findings and things to look out for are:
• The HDR editing process leads to reduced expression of PTPN22 when compared to unedited/mock edited wild type T cells; thresholds of signalling are therefore different. But this is ok because expression of phosphatase in edited wild type and risk variants is equivalent, albeit at lower levels (Fig 1).
• The technology inevitably leads to hemizygosity with biallelic editing events, and this needs to be born in mind when considering the homogeneity of T cell populations
• The impact of the PTPN22 risk variant or phosphatase deficiency is uncovered under conditions of lower avidity/low signal strength, where loss of negative regulation leads to increased proliferation and cytokine production (IFN or IL-2)
• Consistent with this PTPN22 regulates responses of T cells expressing low avidity L-TCR, but not high avidity H-TCR
• Thus, the risk variant mimics the knockout, to a large extentAdditional things/experiments that might strengthen the study:
• The claims of the authors might be further substantiated if they extended the range of T cell stimulatory readouts eg different cell surface markers such PD-1, OX-40, 41BB, ICOS, GPR56, whose expression is linked to TCR signalling thresholds
• Additional signalling experiments such as phospho-flow using phospho-Erk specific antibodies would be a bonus; I worry a bit about only showing pS6 data
• Repeat the experiments comparing wild type and ko T cells and study cytokine expression eg IFNg in non-risk edited and risk edited T cells. As it stands the only data we see comparing these genotypes is proliferation. -
Reviewer #2 (Public Review):
I appreciated reading this manuscript, which is well written and contains very interesting data based on well controlled experiments.
As I understood it the aim was to solve the controversy that human T cells with the disease associated PTPN SNP (associated with several autoimmune disease (T1D, RA) give a poor antigen response in vitro whereas mouse models with the same SNP (or knockouts) give a higher T cell responsiveness. This discrepancy has led to the conclusion that mouse models may not be useful for solving the functional consequence of the PTPN22 polymorphism. The question is basically what to believe, to have a solid point to navigate from.
The authors argue that they need to test naïve T cell which in a controlled experimental setting. They, therefore, isolated human cord blood T cells and CRISP …Reviewer #2 (Public Review):
I appreciated reading this manuscript, which is well written and contains very interesting data based on well controlled experiments.
As I understood it the aim was to solve the controversy that human T cells with the disease associated PTPN SNP (associated with several autoimmune disease (T1D, RA) give a poor antigen response in vitro whereas mouse models with the same SNP (or knockouts) give a higher T cell responsiveness. This discrepancy has led to the conclusion that mouse models may not be useful for solving the functional consequence of the PTPN22 polymorphism. The question is basically what to believe, to have a solid point to navigate from.
The authors argue that they need to test naïve T cell which in a controlled experimental setting. They, therefore, isolated human cord blood T cells and CRISP mutated them, resulting in T cells that have the disease SNP allele, T cells with a deleted PTPN22 and wild type T cells. They also transfected them with a mouse TCR recognizing a specific peptide in the context of the human MHC II molecule *0401. They tested two different TCRs with different affinity. They found that, in similarity with published mouse data, the PTPN22 disease allele and the deleted PTPN22 led to a higher response using the low affinity TCR. -
Reviewer #3 (Public Review):
This work is important for understanding both how immune cells are regulated and how alterations in receptor signaling can affect the balance of health and development of autoimmune diseases. The work uses CRISPR-based genetic manipulation of the autoimmunity associated PTPN22 gene in single donor human cord-derived naïve T cells to analyze T-cell receptor functions. The authors conclude that the autoimmunity associated PTPN22 variant PTPN22(620W) is a loss-of-function mutant as T cells expressing PTPN22(620W) phenocopies PTPN22 deficient T cells. The use of a single donor minimizes potential other effects that would be observed when comparison cellular functions from multiple donors.
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