CD45 pre-exclusion from the tips of T cell microvilli prior to antigen recognition.

The tyrosine phosphatase CD45 is a major gatekeeper for restraining T cell activation. Its exclusion from the immunological synapse (IS) is crucial for T cell receptor (TCR) signal transduction. Here, we use expansion super-resolution microscopy to reveal that CD45 is mostly pre-excluded from the tips of microvilli (MV) on primary T cells prior to antigen encounter. This pre-exclusion is diminished by depleting cholesterol or by engineering the transmembrane domain of CD45 to increase its membrane integration length, but is independent of the CD45 extracellular domain. We further show that brief MV-mediated contacts can induce Ca2+ influx in mouse antigen-specific T cells engaged by antigen-pulsed antigen presenting cells (APC). We propose that the scarcity of CD45 phosphatase activity at the tips of MV enables or facilitates TCR triggering from brief T cell-APC contacts before formation of a stable IS, and that these MV-mediated contacts represent the earliest step in the initiation of a T cell adaptive immune response.

Genetic bioengineering of overexpressed guanylate binding protein family BmAtlastin-n enhances silkworm resistance to Nosema bombycis.

Microsporidia are obligate single-celled eukaryote parasites. Microsporidian infection can cause large economic losses to beneficial insects such as silkworms and honey bees. Identification of resistance biomacromolecules and breeding of transgenic lines resistant to the microsporidian Nosema bombycis are important for disease management. We previously used transcriptome analysis to identify a guanylate binding protein family BmAtlastin-n gene that was significantly upregulated after Nosema bombycis infection, and we determined that the molecule was highly expressed in resistance-related tissues such as the midgut, fat body and the epidermis. The transgenic silkworm line overexpressing BmAtlastin-n biomolecules had economic characters similar to those of non-transgenic lines. The transgenic OE-BmAtlastin-n lines had significantly improved survival after microspore infection. We used RT-PCR and H&E staining to show that the number of spores in the transgenic lines was significantly lower than in the control lines. In this study, we identified a BmAtlastin-n macromolecule with resistance to N. bombycis and developed a transgenic line. The results improved understanding of the GBP protein family and provided biomacromolecule material for the treatment and prevention of microsporidia.

Yersinia pseudotuberculosis YopH targets SKAP2-dependent and independent signaling pathways to block neutrophil antimicrobial mechanisms during infection.

Yersinia suppress neutrophil responses by using a type 3 secretion system (T3SS) to inject 6-7 Yersinia effector proteins (Yops) effectors into their cytoplasm. YopH is a tyrosine phosphatase that causes dephosphorylation of the adaptor protein SKAP2, among other targets in neutrophils. SKAP2 functions in reactive oxygen species (ROS) production, phagocytosis, and integrin-mediated migration by neutrophils. Here we identify essential neutrophil functions targeted by YopH, and investigate how the interaction between YopH and SKAP2 influence Yersinia pseudotuberculosis (Yptb) survival in tissues. The growth defect of a ΔyopH mutant was restored in mice defective in the NADPH oxidase complex, demonstrating that YopH is critical for protecting Yptb from ROS during infection. The growth of a ΔyopH mutant was partially restored in Skap2-deficient (Skap2KO) mice compared to wild-type (WT) mice, while induction of neutropenia further enhanced the growth of the ΔyopH mutant in both WT and Skap2KO mice. YopH inhibited both ROS production and degranulation triggered via integrin receptor, G-protein coupled receptor (GPCR), and Fcγ receptor (FcγR) stimulation. SKAP2 was required for integrin receptor and GPCR-mediated ROS production, but dispensable for degranulation under all conditions tested. YopH blocked SKAP2-independent FcγR-stimulated phosphorylation of the proximal signaling proteins Syk, SLP-76, and PLCγ2, and the more distal signaling protein ERK1/2, while only ERK1/2 phosphorylation was dependent on SKAP2 following integrin receptor activation. These findings reveal that YopH prevents activation of both SKAP2-dependent and -independent neutrophilic defenses, uncouple integrin- and GPCR-dependent ROS production from FcγR responses based on their SKAP2 dependency, and show that SKAP2 is not required for degranulation.

Research of anti-GAD and anti-IA2 autoantibodies by ELISA test in a series of Moroccan pediatric patients with diabetes type 1.

INTRODUCTION: Type I diabetes (T1D) is an autoimmune disease with a prediabetic, asymptomatic period characterized by the selective destruction of insulin-producing ß cells. During the pre-clinical phase, various auto-antibodies are generated against several beta cell antigens such as anti glutamate acid decarboxylase (Anti-GAD), anti tyrosine phosphatase (Anti-IA2). Today, the coupled detection of Anti-IA2 with that of Anti-GAD proves its great importance in the diagnosis and prediction of type 1 diabetes. The combined positivity for both antibodies has a specificity and a positive predictive value of 100%. OBJECTIVES: In this work, we evaluate the diagnostic value of anti-GAD and anti-IA2 antibodies in a series based on 78 Moroccan subjects initially under 16, suspected T1D. RESULTS AND DISCUSSION: Our series consists mainly of 74% of newly diagnosed patients for T1D and 26% of confirmed diagnostic patients, of whom 52% are females. The mean age of diagnosis is 7 ± 4 years, the mean of HbA1c at the time of diagnosis is 11.63 ± 2.16%, and the percentage of family history in our series is 69%. The proportion of positive results for anti-IA2 antibodies and anti-GAD antibodies are, respectively, 76.92% and 62.82%, and 52.56% of patients are positive for both auto-antibodies. This study confirms that anti-GAD and anti-IA2 auto-antibodies assays can detect patients early and the autoantibodies can persist several years after diagnosis of type 1 diabetes. CONCLUSION: This study confirmed the diagnosis and classification of T1D (type 1A) in 87.18% of patients, and we reported that the prevalence of anti-GAD and anti-IA2 is higher in girls than in boys.

Identification and Characterization of Post-activated B Cells in Systemic Autoimmune Diseases.

Autoimmune diseases (AID) such as systemic lupus erythematosus (SLE), primary Sjögren's syndrome (pSS), and rheumatoid arthritis (RA) are chronic inflammatory diseases in which abnormalities of B cell function play a central role. Although it is widely accepted that autoimmune B cells are hyperactive in vivo, a full understanding of their functional status in AID has not been delineated. Here, we present a detailed analysis of the functional capabilities of AID B cells and dissect the mechanisms underlying altered B cell function. Upon BCR activation, decreased spleen tyrosine kinase (Syk) and Bruton's tyrosine kinase (Btk) phosphorylation was noted in AID memory B cells combined with constitutive co-localization of CD22 and protein tyrosine phosphatase (PTP) non-receptor type 6 (SHP-1) along with hyporesponsiveness to TLR9 signaling, a Syk-dependent response. Similar BCR hyporesponsiveness was also noted specifically in SLE CD27- B cells together with increased PTP activities and increased transcripts for PTPN2, PTPN11, PTPN22, PTPRC, and PTPRO in SLE B cells. Additional studies revealed that repetitive BCR stimulation of normal B cells can induce BCR hyporesponsiveness and that tissue-resident memory B cells from AID patients also exhibited decreased responsiveness immediately ex vivo, suggesting that the hyporesponsive status can be acquired by repeated exposure to autoantigen(s) in vivo. Functional studies to overcome B cell hyporesponsiveness revealed that CD40 co-stimulation increased BCR signaling, induced proliferation, and downregulated PTP expression (PTPN2, PTPN22, and receptor-type PTPs). The data support the conclusion that hyporesponsiveness of AID and especially SLE B cells results from chronic in vivo stimulation through the BCR without T cell help mediated by CD40-CD154 interaction and is manifested by decreased phosphorylation of BCR-related proximal signaling molecules and increased PTPs. The hyporesponsiveness of AID B cells is similar to a form of functional anergy.

Regulation of CD4+ T Cell Signaling and Immunological Synapse by Protein Tyrosine Phosphatases: Molecular Mechanisms in Autoimmunity.

T cell activation and effector function is mediated by the formation of a long-lasting interaction established between T cells and antigen-presenting cells (APCs) called immunological synapse (IS). During T cell activation, different signaling molecules as well as the cytoskeleton and the endosomal compartment are polarized to the IS. This molecular dynamics is tightly regulated by phosphorylation networks, which are controlled by protein tyrosine phosphatases (PTPs). While some PTPs are known to be important regulators of adhesion, ligand discrimination or the stimulation threshold, there is still little information about the regulatory role of PTPs in cytoskeleton rearrangements and endosomal compartment dynamics. Besides, spatial and temporal regulation of PTPs and substrates at the IS is only barely known. Consistent with an important role of PTPs in T cell activation, multiple mutations as well as altered expression levels or dynamic behaviors have been associated with autoimmune diseases. However, the precise mechanism for the regulation of T cell activation and effector function by PTPs in health and autoimmunity is not fully understood. Herein, we review the current knowledge about the regulatory role of PTPs in CD4+ T cell activation, IS assembly and effector function. The potential molecular mechanisms mediating the action of these enzymes in autoimmune disorders are discussed.

Interaction with tumor­associated macrophages promotes PRL­3­induced invasion of colorectal cancer cells via MAPK pathway­induced EMT and NF­κB signaling­induced angiogenesis.

Protein phosphatase of regenerating liver­3 (PRL­3) is considered to be metastasis­associated phosphatase and is associated with a poor prognosis. Additionally, tumor­associated macrophages (TAMs) participate in cancer progression. A previous study demonstrated that PRL­3 promotes invasion and metastasis by inducing TAM infiltration. However, the underlying mechanism has not been elucidated. In the present study, western blot analysis, polymerase chain reaction, immunohistochemistry, ELISA, mouse model experiments and functional experiments were performed to confirm that the interaction between TAMs and colorectal cancer (CRC) cells induced epithelial­mesenchymal transition (EMT)­associated features in CRC cells by activating mitogen­activated protein kinase (MAPK) pathways in TAMs and upregulating the expression of interleukin (IL)­6 and IL­8. The neutralization of IL­6 and IL­8 reduced EMT and the invasive and migratory abilities of CRC cells. Therefore, IL­6 and IL­8 were considered important factors in EMT, and in CRC invasion and metastasis. In addition, increased angiogenesis was observed after TAMs were co­cultured with CRC cells that overexpress PRL­3. Vascular endothelial growth factor­A was significantly upregulated, and the nuclear factor­κB (NF­κB) signaling pathway was activated in CRC cells after co­culture. Moreover, nude mice injected with CRC cells with high PRL­3 expression levels tended to generate larger xenografts. Immunohistochemistry results from xenografted CRC cells overexpressing PRL­3 also confirmed the activation of MAPK pathways in xenografts. Overall, the findings indicate that PRL­3 promotes CRC cell invasion and metastasis by activating MAPK pathways in TAMs to initiate the EMT, and PRL­3 promotes angiogenesis by activating the NF­κB pathway in CRC cells.

Alteration of gut microbiota-associated epitopes in children with autism spectrum disorders.

BACKGROUND: Autism spectrum disorder (ASD) affects 1% of children and has no cure. Gastrointestinal (GI) problems are common in children with ASD, and although gut microbiota is known to play an important role in ASD through the gut-brain axis, the specific mechanism is unknown. Recent evidence suggests that gut microbiota may participate in the pathogenesis of ASD through immune- and inflammation-mediated pathways. Here, we identified potentially immunogenic epitopes derived from gut microbiota in stool samples from ASD children with and without GI problems and typically developing (TD) children. METHODS: Candidate gut microbiota-associated epitopes (MEs) were identified by blast shotgun metagenomic sequencing of fecal samples from 43 ASD children (19 with and 24 without GI involvement) and 31 sex- and age-matched typically developing (TD) children. Potentially immunogenic epitopes were screened against a predictive human Immune Epitope Database. The composition and abundance of candidate MEs were compared between the three groups of children. RESULTS: MEs identified in ASD children with GI problems were significantly more diverse than those in TD children. ME composition could discriminate between the three groups of children. We identified 34 MEs that were significantly more or less abundant in ASD children than TD children, most (29/34) of the differences in MEs were reduced in ASD and associated with abnormal gut IgA level and altered gut microbiota composition, these MEs were limited effected by clinical factors such as age, gender, and GI problems, of which eleven MEs were pathogenic microorganisms peptides with strong T or B cell response, nine MEs showed high homology to peptides from human self proteins associated with autoimmune disease occurrence, eliciting immune attack against hematopoietic stem cells and inhibition antigen binding. We also found that the abundance of five MEs were increased in ASD, including three human self proteins, gap junction alpha-1 (GJA1), paired box protein Pax-3 (PAX3) and eyes absent homolog 1 isoform 4 (EYA1) which associated with cancer, and a ME with homology to a Listeriolysin O peptide from the pathogenic bacterium Listeria monocytogenes was significantly increased in ASD children compared with TD children. CONCLUSIONS: Our findings demonstrate the abnormal of MEs composition in the gut of children with ASD, moreover, the abnormality in MEs composition was associated with abnormal gut IgA levels and altered gut microbiota composition, this abnormality also suggests that there may be abnormalities in intestinal immunity in children with ASD; In all, thirty-four MEs identified were potential biomarker of ASD, and alterations in MEs may contribute to abnormalities in gut immunity and/or homeostasis in ASD children. Further study of the MEs identified here may advance our understanding of the pathogenesis of ASD.

Prolyl Hydroxylase Domain-2 Protein Regulates Lipopolysaccharide-Induced Vascular Inflammation.

Acute lung injury and its more severe form, acute respiratory distress syndrome, are life-threatening respiratory disorders. Overwhelming pulmonary inflammation and endothelium disruption are commonly observed. Endothelial cells (ECs) are well recognized as key regulators in leukocyte adhesion and migration in response to bacterial infection. Prolyl hydroxylase domain (PHD)-2 protein, a major PHD in ECs, plays a critical role in intracellular oxygen homeostasis, angiogenesis, and pulmonary hypertension. However, its role in endothelial inflammatory response is unclear. We investigated the role of PHD2 in ECs during endotoxin-induced lung inflammatory responses with EC-specific PHD2 inducible knockout mice. On lipopolysaccharide challenge, PHD2 depletion in ECs attenuates lipopolysaccharide-induced increases of lung vascular permeability, edema, and inflammatory cell infiltration. Moreover, EC-specific PHD2 inducible knockout mice exhibit improved adherens junction integrity and endothelial barrier function. Mechanistically, PHD2 knockdown induces vascular endothelial cadherin in mouse lung microvascular primary endothelial cells. Moreover, PHD2 knockdown can increase hypoxia-inducible factor/vascular endothelial protein tyrosine phosphatase signaling and reactive oxygen species-dependent p38 activation, leading to the induction of vascular endothelial cadherin. Data indicate that PHD2 depletion prevents the formation of leaky vessels and edema by regulating endothelial barrier function. It provides direct in vivo evidence to suggest that PHD2 plays a pivotal role in vascular inflammation. The inhibition of endothelial PHD2 activity may be a new therapeutic strategy for acute inflammatory diseases.

Phosphatase of Regenerating Liver-1 (PRL-1) Regulates Actin Dynamics During Immunological Synapse Assembly and T Cell Effector Function.

The regulatory role of most dual specific phosphatases during T cell activation remains unknown. Here, we have studied the expression and function of phosphatases of regenerating liver (PRLs: PRL-1, PRL-2, and PRL-3) during T cell activation, as well as, the dynamic delivery of PRL-1 to the Immunological Synapse (IS). We found that T cell activation downregulates the expression of PRL-2, resulting in an increased PRL-1/PRL-2 ratio. PRL-1 redistributed at the IS in two stages: Initially, it was transiently accumulated at scanning membranes enriched in CD3 and actin, and at later times, it was delivered at the contact site from pericentriolar, CD3ζ-containing, vesicles. Once at the established IS, PRL-1 distributed to LFA-1 and CD3ε sites. Remarkably, PRL-1 was found to regulate actin dynamics during IS assembly and the secretion of IL-2. Moreover, pharmacological inhibition of the catalytic activity of the three PRLs reduced the secretion of IL-2. These results provide evidence indicating a regulatory role of PRL-1 during IS assembly and highlight the involvement of PRLs in immune responses by mature T cells.

Eya3 promotes breast tumor-associated immune suppression via threonine phosphatase-mediated PD-L1 upregulation.

Eya proteins are critical developmental regulators that are highly expressed in embryogenesis but downregulated after development. Amplification and/or re-expression of Eyas occurs in many tumor types. In breast cancer, Eyas regulate tumor progression by acting as transcriptional cofactors and tyrosine phosphatases. Intriguingly, Eyas harbor a separate threonine (Thr) phosphatase activity, which was previously implicated in innate immunity. Here we describe what we believe to be a novel role for Eya3 in mediating triple-negative breast cancer-associated immune suppression. Eya3 loss decreases tumor growth in immune-competent mice and is associated with increased numbers of infiltrated CD8+ T cells, which, when depleted, reverse the effects of Eya3 knockdown. Mechanistically, Eya3 utilizes its Thr phosphatase activity to dephosphorylate Myc at pT58, resulting in a stabilized form. We show that Myc is required for Eya3-mediated increases in PD-L1, and that rescue of PD-L1 in Eya3-knockdown cells restores tumor progression. Finally, we demonstrate that Eya3 significantly correlates with PD-L1 in human breast tumors, and that tumors expressing high levels of Eya3 have a decreased CD8+ T cell signature. Our data uncover a role for Eya3 in mediating tumor-associated immune suppression, and suggest that its inhibition may enhance checkpoint therapies.

Immunogenicity of PtpA secreted during Mycobacterium avium ssp. paratuberculosis infection in cattle.

AIMS: Mycobacterium avium subsp. paratuberculosis (MAP) is the etiological agent of Johne's disease. To survive within host macrophages, the pathogen secretes a battery of proteins to interfere with the immunological response of the host. One of these proteins is tyrosine phosphate A (PtpA), which has been identified as a secreted protein critical for survival of its close relative M. tuberculosis within infected macrophages. METHODS AND RESULTS: In this study, the immune response to recombinant PtpA used as an antigen was investigated in a cohort of ∼1000 cows infected with MAP compared to negative control animals using ELISA. The sera from MAP-infected cows had significantly higher levels of antibodies against PtpA when compared to uninfected cows. CONCLUSIONS: The data presented here indicate that the antibodies produced against PtpA are sensitive enough to detect infected animals before the appearance of the disease symptoms. SIGNIFICANCE AND IMPACT OF STUDY: The use of PtpA as an antigen can be developed as an early diagnostic test. Moreover, PtpA is a candidate antigen for detection of humoral immune responses in cows infected with MAP.

CCL26 Participates in the PRL-3-Induced Promotion of Colorectal Cancer Invasion by Stimulating Tumor-Associated Macrophage Infiltration.

Both phosphatase of regenerating liver-3 (PRL-3) and tumor-associated macrophages (TAM) influence cancer progression. Whether PRL-3 plays a critical role in colorectal cancer invasion and metastasis by inducing TAM infiltration remains unclear. In the current study, we investigated the effects of chemokine ligand 26 (CCL26) on TAM infiltration and colorectal cancer invasion and the underlying mechanism in colorectal cancer cells by overexpressing or silencing PRL-3. We found that PRL-3 upregulated CCL26 expression correlatively and participated in cell migration, according to the results of gene ontology analysis. In addition, IHC analysis results indicated that the PRL-3 and CCL26 levels were positively correlated and elevated in stage III and IV colorectal cancer tissues and were associated with a worse prognosis in colorectal cancer patients. Furthermore, we demonstrated that CCL26 induced TAM infiltration by CCL26 binding to the CCR3 receptor. When LoVo-P and HT29-C cells were cocultured with TAMs, CCL26 binding to the CCR3 receptor enhanced the invasiveness of LoVo-P and HT29-C cells by mobilizing intracellular Ca2+of TAMs to increase the expression of IL6 and IL8. In addition, IHC results indicated that protein levels of CCR3 and TAMs counts were higher in stage III and IV colorectal cancer tissues and correlated with CCL26. Moreover, similar results were observed in vivo using mice injected with LoVo-P and HT29-C cells. These data indicate that PRL-3 may represent a potential prognostic marker that promotes colorectal cancer invasion and metastasis by upregulating CCL26 to induce TAM infiltration. Mol Cancer Ther; 17(1); 276-89. ©2017 AACR.

Prevalence of pancreatic autoantibodies in non-diabetic patients with autoimmune thyroid disease and its relation to insulin secretion and glucose tolerance

ABSTRACT Objective We evaluated the prevalence of glutamic acid decarboxylase (GADA) and tyrosine phosphatase-protein antibodies (IA2A), their titers and their relation to first phase insulin response (FPIR) and glucose tolerance in autoimmune thyroid diseases (ATDs) patients. Subjects and methods Graves' disease (GD; n = 181) and Hashimoto's thyroiditis (HT; n = 143) patients in addition to healthy controls (n = 93) were studied. Secondly, FPIR and oral glucose tolerance tests (OGTT) were performed in 11 anti-pancreatic islet-cell (+) and in 20 anti-pancreatic-cell (-) patients. Results There was a non significant trend for higher prevalence of GADA positivity in GD vs HT (7.2% vs 2% p = 0.06), but the GADA titers were higher in HT. We also did not find a significant difference in IA2 prevalence (0.7% vs 0.0%) between these two groups or compared to the control group. In the subsequent analysis, low FPIR was found in 10% of these patients but without statistical difference for OGTT between pancreatic antibody-positive and -negative patients. Conclusion A trend for greater prevalence of GADA was observed for GD patients than for HT or control. However, the titers of these autoantibodies were higher in HT patients, but there was no significant relation to insulin secretion and glucose tolerance at that moment and stage of autoimmune diseases.

Prevalence of pancreatic autoantibodies in non-diabetic patients with autoimmune thyroid disease and its relation to insulin secretion and glucose tolerance.

OBJECTIVE: We evaluated the prevalence of glutamic acid decarboxylase (GADA) and tyrosine phosphatase-protein antibodies (IA2A), their titers and their relation to first phase insulin response (FPIR) and glucose tolerance in autoimmune thyroid diseases (ATDs) patients. SUBJECTS AND METHODS: Graves' disease (GD; n = 181) and Hashimoto's thyroiditis (HT; n = 143) patients in addition to healthy controls (n = 93) were studied. Secondly, FPIR and oral glucose tolerance tests (OGTT) were performed in 11 anti-pancreatic islet-cell (+) and in 20 anti-pancreatic-cell (-) patients. RESULTS: There was a non significant trend for higher prevalence of GADA positivity in GD vs HT (7.2% vs 2% p = 0.06), but the GADA titers were higher in HT. We also did not find a significant difference in IA2 prevalence (0.7% vs 0.0%) between these two groups or compared to the control group. In the subsequent analysis, low FPIR was found in 10% of these patients but without statistical difference for OGTT between pancreatic antibody-positive and -negative patients. CONCLUSION: A trend for greater prevalence of GADA was observed for GD patients than for HT or control. However, the titers of these autoantibodies were higher in HT patients, but there was no significant relation to insulin secretion and glucose tolerance at that moment and stage of autoimmune diseases.

Immunogenicity and protective efficacy of a Salmonella Enteritidis sptP mutant as a live attenuated vaccine candidate.

BACKGROUND: Salmonella enterica serovar Enteritidis (S. Enteritidis) is a highly adaptive pathogen in both humans and animals. As a Salmonella Type III secretion system (T3SS) effector, Salmonella protein tyrosine phosphatase (SptP) is critical for virulence in this genus. To investigate the feasibility of using C50336ΔsptP as a live attenuated oral vaccine in mice, we generated the sptP gene deletion mutant C50336ΔsptP in S. Enteritidis strain C50336 by λ-Red mediated recombination and evaluated the protective ability of the S. Enteritidis sptP mutant strain C50336ΔsptP against mice salmonellosis. RESULTS: We found that C50336ΔsptP was a highly immunogenic, effective, and safe vaccine in mice. Compared to wild-type C50336, C50336ΔsptP showed reduced virulence as confirmed by the 50% lethal dose (LD50) in orally infected mice. C50336ΔsptP also showed decreased bacterial colonization both in vivo and in vitro. Immunization with C50336ΔsptP had no significant effect on body weight and did not result in obvious clinical symptoms relative to control animals treated with phosphate-buffered saline (PBS), but induced humoral and cellular immune responses at 12 and 26 days post inoculation. Immunization with 1 × 108 colony-forming units (CFU) C50336ΔsptP per mouse provided 100% protection against subsequent challenge with the wild-type C50336 strain, and immunized mice showed mild and temporary clinical symptoms as compared to those of control group. CONCLUSIONS: These results demonstrate that C50336ΔsptP can be a live attenuated oral vaccine for salmonellosis.

MAP kinase phosphatase 2 deficient mice develop attenuated experimental autoimmune encephalomyelitis through regulating dendritic cells and T cells.

Mitogen-activated protein kinase phosphatases (MKPs) play key roles in inflammation and immune mediated diseases. Here we investigated the mechanisms by which MKP-2 modulates central nervous system (CNS) inflammation in experimental autoimmune encephalomyelitis (EAE). Our results show that MKP-2 mRNA levels in the spinal cord and lymphoid organs of EAE mice were increased compared with naive controls, indicating an important role for MKP-2 in EAE development. Indeed, MKP-2-/- mice developed reduced EAE severity, associated with diminished CNS immune cell infiltration, decreased proinflammatory cytokine production and reduced frequency of CD4+ and CD8+ T cells in spleens and lymph nodes. In addition, MKP-2-/- CD11c+ dendritic cells (DCs) had reduced expression of MHC-II and CD40 compared with MKP-2+/+ mice. Subsequent experiments revealed that CD4+ T cells from naïve MKP-2-/- mice had decreased cell proliferation and IL-2 and IL-17 production relative to wild type controls. Furthermore, co-culture experiments showed that bone marrow derived DCs of MKP-2-/- mice had impaired capability in antigen presentation and T cell activation. While MKP-2 also modulates macrophage activation, our study suggests that MKP-2 is essential to the pathogenic response of EAE, and it acts mainly via regulating the important antigen presenting DC function and T cell activation.

PRL3-zumab, a first-in-class humanized antibody for cancer therapy.

Novel, tumor-specific drugs are urgently needed for a breakthrough in cancer therapy. Herein, we generated a first-in-class humanized antibody (PRL3-zumab) against PRL-3, an intracellular tumor-associated phosphatase upregulated in multiple human cancers, for unconventional cancer immunotherapies. We focused on gastric cancer (GC), wherein elevated PRL-3 mRNA levels significantly correlated with shortened overall survival of GC patients. PRL-3 protein was overexpressed in 85% of fresh-frozen clinical gastric tumor samples examined but not in patient-matched normal gastric tissues. Using human GC cell lines, we demonstrated that PRL3-zumab specifically blocked PRL-3+, but not PRL-3-, orthotopic gastric tumors. In this setting, PRL3-zumab had better therapeutic efficacy as a monotherapy, rather than simultaneous combination with 5-fluorouracil or 5-fluorouracil alone. PRL3-zumab could also prevent PRL-3+ tumor recurrence. Mechanistically, we found that intracellular PRL-3 antigens could be externalized to become "extracellular oncotargets" that serve as bait for PRL3-zumab binding to potentially bridge and recruit immunocytes into tumor microenvironments for killing effects on cancer cells. In summary, our results document a comprehensive cancer therapeutic approach to specific antibody-targeted therapy against the PRL-3 oncotarget as a case study for developing antibodies against other intracellular targets in drug discovery.

Prediction of type 1 diabetes in Sardinian schoolchildren using islet cell autoantibodies: 10-year follow-up of the Sardinian schoolchildren type 1 diabetes prediction study.

AIMS: Stable genetic background makes individuals from the Mediterranean island of Sardinia ideal to define the predictive power of islet-related autoantibodies (IRAs): glutamic acid decarboxylase antibodies (GADA), tyrosine phosphatase-like antibodies (IA-2A), islet cell antibodies (ICA) to identify T1DM progressors. The aims of the present study were: (1) determination of IRAs reference limits in healthy non-diabetic Sardinian schoolchildren (SSc). (2) Predictive power evaluation of IRAs as single or combined determination to identify islet to identify T1DM progressors. METHODS: Between 1986 and 1994, 8448 SSc were tested for IRAs. All were followed up for 10 years. The predictive power of single or combination of IRAs was determined as hazard ratio (HR), sensitivity, specificity, area under the ROC curve, negative and positive predictive value (NPV, PPV). RESULTS: All 43 progressors to T1DM, but three showed at least one autoantibody positivity. HR for any single-autoantibody positivity was 55.3 times greater when compared to SSc negative for all IRAs. Any single autoantibody performed at least 64.9 % sensitivity with PPV always lower than 16 %. The best performing combination was ICA, plus IA-2A (showing 52.6 % sensitivity, 99.8 % specificity, 0.76 area under the ROC curve, 51.3 % PPV and 99.8 % NPV. CONCLUSIONS: Determination of IRAs reference limits in healthy SSc by standard statistical methods is crucial to establish the power of IRAs as progression markers to T1DM. Our data offer a solid rationale for future testing of ICA and IA-2A as routine laboratory markers to identify individuals at high risk of T1DM in the general population.

Imbalanced signal transduction in regulatory T cells expressing the transcription factor FoxP3.

FoxP3(+) T regulatory (Treg) cells have a fundamental role in immunological tolerance, with transcriptional and functional phenotypes that demarcate them from conventional CD4(+) T cells (Tconv). Differences between these two lineages in the signaling downstream of T-cell receptor-triggered activation have been reported, and there are different requirements for some signaling factors. Seeking a comprehensive view, we found that Treg cells have a broadly dampened activation of several pathways and signaling nodes upon TCR-mediated activation, with low phosphorylation of CD3ζ, SLP76, Erk1/2, AKT, or S6 and lower calcium flux. In contrast, STAT phosphorylation triggered by interferons, IL2 or IL6, showed variations between Treg and Tconv in magnitude or choice of preferential STAT activation but no general Treg signaling defect. Much, but not all, of the Treg/Tconv difference in TCR-triggered responses could be attributed to lower responsiveness of antigen-experienced cells with CD44(hi) or CD62L(lo) phenotypes, which form a greater proportion of the Treg pool. Candidate regulators were tested, but the Treg/Tconv differential could not be explained by overexpression in Treg cells of the signaling modulator CD5, the coinhibitors PD-1 and CTLA4, or the regulatory phosphatase DUSP4. However, transcriptome profiling in Dusp4-deficient mice showed that DUSP4 enhances the expression of a segment of the canonical Treg transcriptional signature, which partially overlaps with the TCR-dependent Treg gene set. Thus, Treg cells, likely because of their intrinsically higher reactivity to self, tune down TCR signals but seem comparatively more attuned to cytokines or other intercellular signals.