Androgens contribute to sex bias of autoimmunity in mice by T cell-intrinsic regulation of Ptpn22 phosphatase expression.

Autoimmune diseases such as systemic lupus erythematosus (SLE) display a strong female bias. Although sex hormones have been associated with protecting males from autoimmunity, the molecular mechanisms are incompletely understood. Here we report that androgen receptor (AR) expressed in T cells regulates genes involved in T cell activation directly, or indirectly via controlling other transcription factors. T cell-specific deletion of AR in mice leads to T cell activation and enhanced autoimmunity in male mice. Mechanistically, Ptpn22, a phosphatase and negative regulator of T cell receptor signaling, is downregulated in AR-deficient T cells. Moreover, a conserved androgen-response element is found in the regulatory region of Ptpn22 gene, and the mutation of this transcription element in non-obese diabetic mice increases the incidence of spontaneous and inducible diabetes in male mice. Lastly, Ptpn22 deficiency increases the disease severity of male mice in a mouse model of SLE. Our results thus implicate AR-regulated genes such as PTPN22 as potential therapeutic targets for autoimmune diseases.

PTPN22 through the regulation of Th17/Treg balance acts as a potential target for the treatment of Graves' disease.

Graves' disease (GD) is an autoimmune disease and the most common cause of hyperthyroidism. While the phosphotyrosine phosphatase non-receptor type 22 (PTPN22) variant is associated with GD susceptibility, its precise role and mechanism in GD remain unclear. To investigate this, we induced GD in mice using Ad-TSHR289 and isolated CD4+ T cells from spleen tissues. We conducted a series of experiments, including hematoxylin-eosin staining, enzyme-linked immunosorbent assay (ELISA), immunohistochemistry, flow cytometry, immunofluorescence (IF), reverse transcription quantitative PCR (RT-qPCR), and western blotting. PTPN22 expression was found to be downregulated in GD mice. Overexpression of PTPN22 ameliorated pathological damage and increased serum levels of T4 and thyroid stimulating hormone receptor antibody (TRAb), as well as the ratio of thyroid weight to body weight in GD mice. Furthermore, GD mice exhibited elevated levels of CD4+ and IL-17+ T cells, an increased Th17/Treg ratio, and upregulation of IL-17A mRNA expression. Conversely, there was a decrease in Foxp3+ T cells and transcriptional levels of Foxp3, which were reversed by PTPN22 overexpression. In vitro experiments showed that PTPN22 overexpression in CD4+ T cells from spleen tissues of GD mice enhanced Foxp3 expression while reducing IL-17A expression. Mechanistically, PTPN22 overexpression led to decreased levels of phosphorylated Lck (p-Lck), Lck, phosphorylated Fyn (p-Fyn), Fyn, phosphorylated Zap70 (p-Zap70), and Zap70 in both in vivo and in vitro GD models. In summary, PTPN22 can alleviate thyroid dysfunction in GD by modulating Th17/Treg balance through the downregulation of the Lck/Zap70 signaling axis.

A novel gain-of-function phosphorylation site modulates PTPN22 inhibition of TCR signaling.

Protein tyrosine phosphatase nonreceptor type 22 (PTPN22) is encoded by a major autoimmunity gene and is a known inhibitor of T cell receptor (TCR) signaling and drug target for cancer immunotherapy. However, little is known about PTPN22 posttranslational regulation. Here, we characterize a phosphorylation site at Ser325 situated C terminal to the catalytic domain of PTPN22 and its roles in altering protein function. In human T cells, Ser325 is phosphorylated by glycogen synthase kinase-3 (GSK3) following TCR stimulation, which promotes its TCR-inhibitory activity. Signaling through the major TCR-dependent pathway under PTPN22 control was enhanced by CRISPR/Cas9-mediated suppression of Ser325 phosphorylation and inhibited by mimicking it via glutamic acid substitution. Global phospho-mass spectrometry showed Ser325 phosphorylation state alters downstream transcriptional activity through enrichment of Swi3p, Rsc8p, and Moira domain binding proteins, and next-generation sequencing revealed it differentially regulates the expression of chemokines and T cell activation pathways. Moreover, in vitro kinetic data suggest the modulation of activity depends on a cellular context. Finally, we begin to address the structural and mechanistic basis for the influence of Ser325 phosphorylation on the protein's properties by deuterium exchange mass spectrometry and NMR spectroscopy. In conclusion, this study explores the function of a novel phosphorylation site of PTPN22 that is involved in complex regulation of TCR signaling and provides details that might inform the future development of allosteric modulators of PTPN22.

Targeted inhibition of PTPN22 is a novel approach to alleviate osteogenic responses in aortic valve interstitial cells and aortic valve lesions in mice.

BACKGROUND: Calcific aortic valve disease (CAVD) is the most prevalent valvular disease and has high morbidity and mortality. CAVD is characterized by complex pathophysiological processes, including inflammation-induced osteoblastic differentiation in aortic valve interstitial cells (AVICs). Novel anti-CAVD agents are urgently needed. Protein tyrosine phosphatase nonreceptor type 22 (PTPN22), an intracellular nonreceptor-like protein tyrosine phosphatase, is involved in several chronic inflammatory diseases, including rheumatoid arthritis and diabetes. However, it is unclear whether PTPN22 is involved in the pathogenesis of CAVD. METHODS: We obtained the aortic valve tissue from human and cultured AVICs from aortic valve. We established CAVD mice model by wire injury. Transcriptome sequencing, western bolt, qPCR, and immunofluorescence were performed to elucidate the molecular mechanisms. RESULTS: Here, we determined that PTPN22 expression was upregulated in calcific aortic valve tissue, AVICs treated with osteogenic medium, and a mouse model of CAVD. In vitro, overexpression of PTPN22 induced osteogenic responses, whereas siRNA-mediated PTPN22 knockdown abolished osteogenic responses and mitochondrial stress in the presence of osteogenic medium. In vivo, PTPN22 ablation ameliorated aortic valve lesions in a wire injury-induced CAVD mouse model, validating the pathogenic role of PTPN22 in CAVD. Additionally, we discovered a novel compound, 13-hydroxypiericidin A 10-O-α-D-glucose (1 → 6)-ß-D-glucoside (S18), in a marine-derived Streptomyces strain that bound to PTPN22 with high affinity and acted as a novel inhibitor. Incubation with S18 suppressed osteogenic responses and mitochondrial stress in human AVICs induced by osteogenic medium. In mice with aortic valve injury, S18 administration markedly alleviated aortic valve lesions. CONCLUSION: PTPN22 plays an essential role in the progression of CAVD, and inhibition of PTPN22 with S18 is a novel option for the further development of potent anti-CAVD drugs. Therapeutic inhibition of PTPN22 retards aortic valve calcification through modulating mitochondrial dysfunction in AVICs.

Protein tyrosine phosphatase PTPN22 negatively modulates platelet function and thrombus formation.

Protein tyrosine phosphatase nonreceptor type 22 (PTPN22) is a protein tyrosine phosphatase that negatively regulates T-cell signaling. However, whether it is expressed and functions in platelets remains unknown. Here we investigated the expression and role of PTPN22 in platelet function. We reported PTPN22 expression in both human and mouse platelets. Using PTPN22-/- mice, we showed that PTPN22 deficiency significantly shortened tail-bleeding time and accelerated arterial thrombus formation without affecting venous thrombosis and the coagulation factors VIII and IX. Consistently, PTPN22-deficient platelets exhibited enhanced platelet aggregation, granule secretion, calcium mobilization, lamellipodia formation, spreading, and clot retraction. Quantitative phosphoproteomic analysis revealed the significant difference of phosphodiesterase 5A (PDE5A) phosphorylation in PTPN22-deficient platelets compared with wild-type platelets after collagen-related peptide stimulation, which was confirmed by increased PDE5A phosphorylation (Ser92) in collagen-related peptide-treated PTPN22-deficient platelets, concomitant with reduced level and vasodilator-stimulated phosphoprotein phosphorylation (Ser157/239). In addition, PTPN22 interacted with phosphorylated PDE5A (Ser92) and dephosphorylated it in activated platelets. Moreover, purified PTPN22 but not the mutant form (C227S) possesses intrinsic serine phosphatase activity. Furthermore, inhibition of PTPN22 enhanced human platelet aggregation, spreading, clot retraction, and increased PDE5A phosphorylation (Ser92). In conclusion, our study shows a novel role of PTPN22 in platelet function and arterial thrombosis, identifying new potential targets for future prevention of thrombotic or cardiovascular diseases.

PTPN22: structure, function, and developments in inhibitor discovery with applications for immunotherapy.

INTRODUCTION: While immunotherapy strategies such as immune checkpoint inhibition and adoptive T cell therapy have become commonplace in cancer therapy, they suffer from limitations, including lack of patient response and toxicity. To wield the maximum potential of the immune system, cancer immunotherapy must integrate novel targets and therapeutic strategies with potential to augment clinical efficacy of currently utilized immunotherapies. PTPN22, a member of the protein tyrosine phosphatase (PTP) superfamily that downregulates T cell signaling and proliferation, has recently emerged as a systemically druggable and novel immunotherapy target. AREAS COVERED: This review describes the basics of PTPN22 structure and function and provides comprehensive insight into recent advances in small molecule PTPN22 inhibitor development and the immense potential of PTPN22 inhibition to synergize with current immunotherapies. EXPERT OPINION: It is apparent that small molecule PTPN22 inhibitors have enormous potential to augment efficacy of current immunotherapy strategies such as checkpoint inhibition and adoptive cell transfer. Nevertheless, several constraints must be overcome before these inhibitors can be applied as useful therapeutics, namely selectivity, potency, and in vivo efficacy.

SH3-domain mutations selectively disrupt Csk homodimerization or PTPN22 binding.

The kinase Csk is the primary negative regulator of the Src-family kinases (SFKs, e.g., Lck, Fyn, Lyn, Hck, Fgr, Blk, Yes), phosphorylating a tyrosine on the SFK C-terminal tail that mediates autoinhibition. Csk also binds phosphatases, including PTPN12 (PTP-PEST) and immune-cell PTPN22 (LYP/Pep), which dephosphorylate the SFK activation loop to promote autoinhibition. Csk-binding proteins (e.g., CBP/PAG1) oligomerize within membrane microdomains, and high local concentration promotes Csk function. Purified Csk homodimerizes in solution through an interface that overlaps the phosphatase binding footprint. Here we demonstrate that Csk can homodimerize in Jurkat T cells, in competition with PTPN22 binding. We designed SH3-domain mutations in Csk that selectively impair homodimerization (H21I) or PTPN22 binding (K43D) and verified their kinase activity in solution. Disruption of either interaction in cells, however, decreased the negative-regulatory function of Csk. Csk W47A, a substitution previously reported to block PTPN22 binding, had a secondary effect of impairing homodimerization. Csk H21I and K43D will be useful tools for dissecting the protein-specific drivers of autoimmunity mediated by the human polymorphism PTPN22 R620W, which impairs interaction with Csk and with the E3 ubiquitin ligase TRAF3. Future investigations of Csk homodimer activity and phosphatase interactions may reveal new facets of SFK regulation in hematopoietic and non-hematopoietic cells.

Improvement of Lipoplexes With a Sialic Acid Mimetic to Target the C1858T PTPN22 Variant for Immunotherapy in Endocrine Autoimmunity.

The C1858T variant of the protein tyrosine phosphatase N22 (PTPN22) gene is associated with pathophysiological phenotypes in several autoimmune conditions, namely, Type 1 diabetes and autoimmune thyroiditis. The R620W variant protein, encoded by C1858T, leads to a gain of function mutation with paradoxical reduced T cell activation. We previously exploited a novel personalized immunotherapeutic approach based on siRNA delivered by liposomes (lipoplexes, LiposiRNA) that selectively inhibit variant allele expression. In this manuscript, we functionalize lipoplexes carrying siRNA for variant C1858T with a high affinity ligand of Siglec-10 (Sig10L) coupled to lipids resulting in lipoplexes (LiposiRNA-Sig10L) that enhance delivery to Siglec-10 expressing immunocytes. LiposiRNA-Sig10L lipoplexes more efficiently downregulated variant C1858T PTPN22 mRNA in PBMC of heterozygous patients than LiposiRNA without Sig10L. Following TCR engagement, LiposiRNA-Sig10L more significantly restored IL-2 secretion, known to be paradoxically reduced than in wild type patients, than unfunctionalized LiposiRNA in PBMC of heterozygous T1D patients.

Modulation of PTPN2/22 Function by Spermidine in CRISPR-Cas9-Edited T-Cells Associated with Crohn's Disease and Rheumatoid Arthritis.

Crohn's Disease (CD) and Rheumatoid Arthritis (RA) share some single nucleotide polymorphisms (SNPs) in protein tyrosine phosphatase non-receptor types 2 and 22 (PTPN2/22). Recently, we reported that clinical samples from CD and RA patients associated with PTPN2:rs478582 or PTPN22:rs2476601 genotypes were linked to overactive immune response and exacerbation of inflammation. Here, we investigated in vitro the effects of these SNPs in Jurkat T-cells using CRISPR-Cas9. All cells were evaluated for PTPN22/22 loss of function and effects on cell response. We measured gene expression via RT-qPCR and cytokines by ELISA. We also measured cell proliferation using a BrdU labeling proliferation ELISA, and T-cell activation using CD-25 fluorescent immunostaining. In PTPN2 SNP-edited cells, PTPN2 expression decreased by 3.2-fold, and proliferation increased by 10.2-fold compared to control. Likewise, expression of PTPN22 decreased by 2.4-fold and proliferation increased by 8.4-fold in PTPN22 SNP-edited cells. IFN-γ and TNF-α secretions increased in both edited cell lines. CD25 expression (cell activation) was 80.32% in PTPN2 SNP-edited cells and 85.82% in PTPN22 SNP-edited cells compared to 70.48% in unedited Jurkat T-cells. Treatment of PTPN2 and PTPN22-edited cells with a maximum 20 µM spermidine restored PTPN2/22 expression and cell response including cell proliferation, activation, and cytokines secretion. Most importantly, the effect of spermidine on edited cells restored normal expression and secretion of IFN-γ and TNF-α. The data clearly demonstrated that edited SNPs in PTPN2 or PTPN22 were associated with reduced gene expression, which resulted in an increase in cell proliferation and activation and overactive immune response. The data validated our earlier observations in CD and RA clinical samples. Surprisingly, spermidine restored PTPN2/22 expression in edited Jurkat T-cells and the consequent beneficial effect on cell response and inflammation. The study supports the use of polyamines dietary supplements for management of CD and in RA patients.

Overexpression of the PTPN22 Autoimmune Risk Variant LYP-620W Fails to Restrain Human CD4+ T Cell Activation.

A missense mutation (R620W) of protein tyrosine phosphatase nonreceptor type 22 (PTPN22), which encodes lymphoid-tyrosine phosphatase (LYP), confers genetic risk for multiple autoimmune diseases including type 1 diabetes. LYP has been putatively demonstrated to attenuate proximal T and BCR signaling. However, limited data exist regarding PTPN22 expression within primary T cell subsets and the impact of the type 1 diabetes risk variant on human T cell activity. In this study, we demonstrate endogenous PTPN22 is differentially expressed and dynamically controlled following activation. From control subjects homozygous for the nonrisk allele, we observed 2.1- (p < 0.05) and 3.6-fold (p < 0.001) more PTPN22 transcripts in resting CD4+ memory and regulatory T cells (Tregs), respectively, over naive CD4+ T cells, with expression peaking 24 h postactivation. When LYP was overexpressed in conventional CD4+ T cells, TCR signaling and activation were blunted by LYP-620R (p < 0.001) but only modestly affected by the LYP-620W risk variant versus mock-transfected control, with similar results observed in Tregs. LYP overexpression only impacted proliferation following activation by APCs but not anti-CD3- and anti-CD28-coated microbeads, suggesting LYP modulation of pathways other than TCR. Notably, proliferation was significantly lower with LYP-620R than with LYP-620W overexpression in conventional CD4+ T cells but was similar in Treg. These data indicate that the LYP-620W variant is hypomorphic in the context of human CD4+ T cell activation and may have important implications for therapies seeking to restore immunological tolerance in autoimmune disorders.

Mesenchymal Stem Cells Derived Extracellular Vesicles Alleviate Traumatic Hemorrhagic Shock Induced Hepatic Injury via IL-10/PTPN22-Mediated M2 Kupffer Cell Polarization.

Traumatic hemorrhagic shock (THS) is a major cause of mortality and morbidity worldwide in severely injured patients. Mesenchymal stem cells (MSCs) possess immunomodulatory properties and tissue repair potential mainly through a paracrine pathway mediated by MSC-derived extracellular vesicles (MSC-EVs). Interleukin 10 (IL-10) is a potent anti-inflammatory cytokine that plays a crucial role during the inflammatory response, with a broad range of effects on innate and adaptive immunity, preventing damage to the host and maintaining normal tissue homeostasis. However, the function and mechanism of IL-10 in MSC-mediated protective effect in THS remain obscure. Here, we show that MSCs significantly attenuate hepatic injury and inflammation from THS in mice. Notably, these beneficial effects of MSCs disappeared when IL-10 was knocked out in EVs or when recombinant IL-10 was administered to mice. Mechanistically, MSC-EVs function to carry and deliver IL-10 as cargo. WT MSC-EVs restored the function of IL-10 KO MSCs during THS injury. We further demonstrated that EVs containing IL-10 mainly accumulated in the liver during THS, where they were captured by Kupffer cells and induced the expression of PTPN22. These effects subsequently shifted Kupffer cells to an anti-inflammatory phenotype and mitigated liver inflammation and injury. Therefore, our study indicates that MSC-EVs containing IL-10 alleviate THS-induced hepatic injury and may serve as a cell-free therapeutic approach for THS.

Autoimmunity linked protein phosphatase PTPN22 as a target for cancer immunotherapy.

BACKGROUND: Cancer immunotherapy has evolved from interferon-alpha (IFNα) and interleukin-2 in the 1980s to CTLA-4 and PD-1/PD-L1 checkpoint inhibitors (CPIs), the latter highlighting the importance of enhancing T-cell functions. While the search for novel immunomodulatory pathways continues, combination therapies augmenting multiple pathways can also increase efficacy. The association of autoimmune-related adverse events with clinical efficacy following CPI treatment has been inferred and suggests that breaking tolerance thresholds associated with autoimmunity may affect host immune responses for effective cancer immunotherapy. RESULTS: Here, we show that loss of autoimmune associated PTPN22, a key desensitization node for multiple signaling pathways, including IFNα receptor (IFNAR) and T-cell receptor, can augment tumor responses. Implantation of syngeneic tumors in Ptpn22-/- mice led to expansion and activation of peripheral and intratumoral T cells and, in turn, spontaneous tumor regression as well as enhanced responses in combination with anti-PD-L1 treatment. Using genetically modified mice expressing a catalytically inactive PTPN22 or the autoimmunity-associated human single-nucleotide polymorphism variant, augmentation of antitumor immunity was dependent on PTPN22 phosphatase activity and partially on its adaptor functions. Further, antitumor responses were dependent on both CD4+ and CD8+T cells and, in part, IFNAR function. Finally, we demonstrate that the autoimmune susceptibility Ptpn22(C1858T) variant is associated with lower risk of developing non-melanoma skin cancers, improved overall survival and increased risk for development of hyperthyroidism or hypothyroidism following atezolizumab (anti-PD-L1) treatment. CONCLUSIONS: Together, these data suggest that inhibition of PTPN22 phosphatase activity may provide an effective therapeutic option for cancer immunotherapy and that exploring genetic variants that shift immune tolerance thresholds may serve as a paradigm for finding new cancer immunotherapy targets.

Design, synthesis and biological evaluation of imidazolidine-2,4-dione and 2-thioxothiazolidin-4-one derivatives as lymphoid-specific tyrosine phosphatase inhibitors.

Lymphoid-specific tyrosine phosphatase (LYP), which exclusively exists in immune cells and down-regulates T cell receptor signaling (TCR), has becoming a potent target for various autoimmune diseases. Herein, we designed and synthesized imidazolidine-2,4-dione and 2-thioxothiazolidin-4-one derivatives as new LYP inhibitors. Among them, the cinnamic acids-based inhibitors (9p and 9r) displayed good LYP inhibitory activities (IC50 = 2.85-6.95 µM). Especially, the most potent inhibitor 9r was identified as competitive inhibitor (Ki = 1.09 µM) and bind LYP reversibly. Meanwhile, 9r exhibited better selectivity over other phosphatases than known LYP inhibitor A15. Furthermore, compound 9r could regulate TCR associated signaling pathway in Jurkat T cell.

LyP-1-Modified Oncolytic Adenoviruses Targeting Transforming Growth Factor ß Inhibit Tumor Growth and Metastases and Augment Immune Checkpoint Inhibitor Therapy in Breast Cancer Mouse Models.

We report here the development of oncolytic adenoviruses (Ads) that have reduced toxicity, enhanced tumor tropism, produce strong antitumor response, and can overcome resistance to immune checkpoint inhibitor therapy in breast cancer. We have shown that LyP-1 receptor (p32) is highly expressed on the surface of breast cancer cells and tumors from cancer patients, and that increased stromal expression of transforming growth factor ß-1 (TGFß-1) is associated with triple-negative breast cancer. Therefore, we constructed oncolytic Ads, AdLyp.sT and mHAdLyp.sT, in which the p32-binding LyP-1 peptide was genetically inserted into the adenoviral fiber protein. Both AdLyp.sT and mHAdLyp.sT express sTGFßRIIFc, a TGFß decoy that can inhibit TGFß pathways. mHAdLyp.sT is an Ad5/48 chimeric hexon virus in which hypervariable regions (HVRs 1-7) of Ad5 are replaced with the corresponding Ad48 HVRs. AdLyp.sT and mHAdLyp.sT exhibited better binding, replication, and produced higher sTGFßRIIFc protein levels in breast cancer cell lines compared with Ad.sT or mHAd.sT control viruses without LyP-1 peptide modification. Systemic delivery of mHAdLyp.sT in mice resulted in reduced hepatic/systemic toxicity compared with Ad.sT and AdLyp.sT. Intravenous delivery of AdLyp.sT and mHAdLyp.sT elicited a strong antitumor response in a human MDA-MB-231 bone metastasis model in mice, as indicated by bioluminescence imaging, radiographic tumor burden, serum TRACP 5b and calcium, and body weight analyses. Furthermore, intratumoral delivery of AdLyp.sT in 4T1 model in immunocompetent mice inhibited tumor growth and metastases, and augmented anti-PD-1 and anti-CTLA-4 therapy. Based on these studies, we believe that AdLyp.sT and mHAdLyp.sT can be developed as potential targeted immunotherapy agents for the treatment of breast cancer.

PTPN22 interacts with EB1 to regulate T-cell receptor signaling.

The PTPN22 gene encoding the Lyp/Pep protein tyrosine phosphatase is a negative regulator of T-cell receptor (TCR) signaling. Recent studies have shown that phosphorylation of end-binding protein 1 (EB1) is associated with the TCR activation. In this study, using 2-hybrid and mass spectrometry analyses, we identified EB1 as a protein associated with PTPN22. Furthermore, we discovered that EB1 specifically bound to the P1 domain of PTPN22 by competing with CSK, and the variant PTPN22-R620W does not affect the association with EB1, which is instrumental with respect to the regulation of TCR signaling. In addition, PTPN22 dephosphorylates EB1 at tyrosine-247 (Y247), which decreases the expression of the T-cell activation markers CD25 and CD69 and the phosphorylation levels of the TCR molecules ZAP-70, LAT, and Erk, leading to the eventual downregulation of the transcription factor NFAT and reduced the levels of secreted IL-2. The findings of this study provide new insights into the TCR signaling and the T-cell immune response, which are important for clarifying the mechanism of PTPN22-related autoimmune diseases.

PTPN22 phosphorylation acts as a molecular rheostat for the inhibition of TCR signaling.

The hematopoietic-specific protein tyrosine phosphatase nonreceptor type 22 (PTPN22) is encoded by a major autoimmunity risk gene. PTPN22 inhibits T cell activation by dephosphorylating substrates involved in proximal T cell receptor (TCR) signaling. Here, we found by mass spectrometry that PTPN22 was phosphorylated at Ser751 by PKCα in Jurkat and primary human T cells activated with phorbol ester/ionomycin or antibodies against CD3/CD28. The phosphorylation of PTPN22 at Ser751 prolonged its half-life by inhibiting K48-linked ubiquitination and impairing recruitment of the phosphatase to the plasma membrane, which is necessary to inhibit proximal TCR signaling. Additionally, the phosphorylation of PTPN22 at Ser751 enhanced the interaction of PTPN22 with the carboxyl-terminal Src kinase (CSK), an interaction that is impaired by the PTPN22 R620W variant associated with autoimmune disease. The phosphorylation of Ser751 did not affect the recruitment of PTPN22 R620W to the plasma membrane but protected this mutant from degradation. Together, out data indicate that phosphorylation at Ser751 mediates a reciprocal regulation of PTPN22 stability versus translocation to TCR signaling complexes by CSK-dependent and CSK-independent mechanisms.

PTPN22 Acts in a Cell Intrinsic Manner to Restrict the Proliferation and Differentiation of T Cells Following Antibody Lymphodepletion.

Lymphopenic insult has been shown to precipitate the initiation of autoimmune disease in murine models such as the Non-obese diabetic mouse. Similarly, in man lymphopenia induced by mAb therapy, for instance Alemtuzumab as treatment for Multiple Sclerosis, can precipitate development of secondary autoimmune disease in up to 30 % of patients. We asked whether an identified autoimmune susceptibility locus might increase the risk of developing autoimmunity in the context of mAb-induced lymphopenia in a mouse model. A single nucleotide polymorphism (SNP) in the gene encoding the tyrosine phosphatase PTPN22 (R620W) is associated with multiple human autoimmune diseases, and PTPN22 has been shown to modulate T cell responses, particularly to weak antigens. In keeping with this, PTPN22-deficient or PTPN22 R619W mutant murine T cells adoptively transferred into immunodeficient lymphopenic hosts showed a higher lymphopenia-induced proliferation rate than WT cells. We induced lymphopenia by treating wild-type or PTPN22 knock-out mice with T cell depleting antibodies and monitored reconstitution of the T cell pool. We found that PTPN22 deficient T cells acquired a more activated effector phenotype, with significantly more IFNγ producing cells. This resulted from expansion driven by self-peptide MHC, as it was evident when the contribution of IL-7 to lymphopenic expansion was blocked with IL-7R Ab. Interestingly, Foxp3+ Tregs were also considerably expanded in PTPN22-deficient and PTPN22 R619W mice, as was the frequency of both CD25+ and CD25- CD4 T cells that produce IL-10. Using bone marrow chimeric mice, we showed that PTPN22 influenced development of both regulatory and effector T cell functions in a cell-intrinsic manner. Overall the expansion of Tregs is likely to keep the expanded T effector populations in check and sparing Treg during therapeutic mAb depletion may be a useful strategy to prevent occurrence of secondary autoimmunity.

Differential nanoscale organisation of LFA-1 modulates T-cell migration.

Effector T-cells rely on integrins to drive adhesion and migration to facilitate their immune function. The heterodimeric transmembrane integrin LFA-1 (αLß2 integrin) regulates adhesion and migration of effector T-cells through linkage of the extracellular matrix with the intracellular actin treadmill machinery. Here, we quantified the velocity and direction of F-actin flow in migrating T-cells alongside single-molecule localisation of transmembrane and intracellular LFA-1. Results showed that actin retrograde flow positively correlated and immobile actin negatively correlated with T-cell velocity. Plasma membrane-localised LFA-1 forms unique nano-clustering patterns in the leading edge, compared to the mid-focal zone, of migrating T-cells. Deleting the cytosolic phosphatase PTPN22, loss-of-function mutations of which have been linked to autoimmune disease, increased T-cell velocity, and leading-edge co-clustering of pY397 FAK, pY416 Src family kinases and LFA-1. These data suggest that differential nanoclustering patterns of LFA-1 in migrating T-cells may instruct intracellular signalling. Our data presents a paradigm where T-cells modulate the nanoscale organisation of adhesion and signalling molecules to fine tune their migration speed, with implications for the regulation of immune and inflammatory responses.This article has an associated First Person interview with the first author of the paper.

Association of PTPN22 polymorphism and its correlation with Graves' disease susceptibility in Polish adult population-A preliminary study.

BACKGROUND: Susceptibility to Graves' disease (GD) is determined by various genetic factors; the gene encoding protein tyrosine phosphatase (PTPN22) may be one of those associated with higher risk of GD. The aim was to estimate the association of the PTPN22 gene polymorphism rs2476601:c.C>T (c.1858C>T) with the predisposition to GD within the adult north-eastern Polish population. METHODS: PTPN22 gene polymorphism was analyzed in individuals with clinical GD history (n = 166) and healthy subjects (n = 154). The presence of different variants of the investigated gene polymorphism was estimated using the DNA Sanger sequencing method. RESULTS: Patients with GD had a more frequent occurrence of the T gene allele of PTPN22 gene compared to the control group, however, it was not significant (p = 0.257). Analysis of genotype distribution showed significantly more frequent occurrence of TT homozygote in GD patients compared to control individuals (p = 0.016, OR = 9.28). Patients with ophthalmopathy had a less frequent occurrence of the T gene allele of PTPN22 gene compared to patients without ophthalmopathy, however, it was not significant (p = 0.12). Occurrence of the T gene allele of PTPN22 gene in GD manifestation in those under 40-year old was more frequent compared to individuals over 40, but the obtained difference was also not significant (p = 0.75). CONCLUSIONS: Our preliminary study suggest that PTPN22:c.1858C>T gene polymorphism may be associated with a predisposition to GD within the adult north-eastern Polish population. The studied polymorphism of the PTPN22 gene did not significantly affect the risk of ophthalmopathy developing and disease manifestation before the age of 40.

Identification and structure-function analyses of an allosteric inhibitor of the tyrosine phosphatase PTPN22.

Protein-tyrosine phosphatase nonreceptor type 22 (PTPN22) is a lymphoid-specific tyrosine phosphatase (LYP), and mutations in the PTPN22 gene are highly correlated with a spectrum of autoimmune diseases. However, compounds and mechanisms that specifically inhibit LYP enzymes to address therapeutic needs to manage these diseases remain to be discovered. Here, we conducted a similarity search of a commercial database for PTPN22 inhibitors and identified several LYP inhibitor scaffolds, which helped identify one highly active inhibitor, NC1. Using noncompetitive inhibition curve and phosphatase assays, we determined NC1's inhibition mode toward PTPN22 and its selectivity toward a panel of phosphatases. We found that NC1 is a noncompetitive LYP inhibitor and observed that it exhibits selectivity against other protein phosphatases and effectively inhibits LYP activity in lymphoid T cells and modulates T-cell receptor signaling. Results from site-directed mutagenesis, fragment-centric topographic mapping, and molecular dynamics simulation experiments suggested that NC1, unlike other known LYP inhibitors, concurrently binds to a "WPD" pocket and a second pocket surrounded by an LYP-specific insert, which contributes to its selectivity against other phosphatases. Moreover, using a newly developed method to incorporate the unnatural amino acid 2-fluorine-tyrosine and 19F NMR spectroscopy, we provide direct evidence that NC1 allosterically regulates LYP activity by restricting WPD-loop movement. In conclusion, our approach has identified a new allosteric binding site in LYP useful for selective LYP inhibitor development; we propose that the 19F NMR probe developed here may also be useful for characterizing allosteric inhibitors of other tyrosine phosphatases.