Antibodies recognizing Mycobacterium avium paratuberculosis epitopes cross-react with the beta-cell antigen ZnT8 in Sardinian type 1 diabetic patients

The environmental factors at play in the pathogenesis of type 1 diabetes (T1D) remain enigmatic. Mycobacterium avium subspecies paratuberculosis (MAP) is transmitted from dairy herds to humans through food contamination. MAP causes an asymptomatic infection that is highly prevalent in Sardinian T1D patients compared with type 2 diabetes (T2D) and healthy controls. Moreover, MAP elicits humoral responses against several mycobacterial proteins. We asked whether antibodies (Abs) against one of these proteins, namely MAP3865c, which displays a sequence homology with the β-cell protein zinc transporter 8 (ZnT8) could be cross-reactive with ZnT8 epitopes. To this end, Ab responses against MAP3865c were analyzed in Sardinian T1D, T2D and healthy subjects using an enzymatic immunoassay. Abs against MAP3865c recognized two immunodominant transmembrane epitopes in 52-65% of T1D patients, but only in 5-7% of T2D and 3-5% of healthy controls. There was a linear correlation between titers of anti-MAP3865c and anti-ZnT8 Abs targeting these two homologous epitopes, and pre-incubation of sera with ZnT8 epitope peptides blocked binding to the corresponding MAP3865c peptides. These results demonstrate that Abs recognizing MAP3865c epitopes cross-react with ZnT8, possibly underlying a molecular mimicry mechanism, which may precipitate T1D in MAP-infected individuals.     

Extrapancreatic Autoantibody Profiles in Type I Diabetes

Type I diabetes (T1D) is an autoimmune disease characterized by destruction of insulin-producing β-cells in the pancreas. Although several islet cell autoantigens are known, the breadth and spectrum of autoantibody targets has not been fully explored. Here the luciferase immunoprecipitation systems (LIPS) antibody profiling technology was used to study islet and other organ-specific autoantibody responses in parallel. Examination of an initial cohort of 93 controls and 50 T1D subjects revealed that 16% of the diabetic subjects showed anti-gastric ATPase autoantibodies which did not correlate with autoantibodies against GAD65, IA2, or IA2-β. A more detailed study of a second cohort with 18 potential autoantibody targets revealed marked heterogeneity in autoantibody responses against islet cell autoantigens including two polymorphic variants of ZnT8. A subset of T1D subjects exhibited autoantibodies against several organ-specific targets including gastric ATPase (11%), thyroid peroxidase (14%), and anti-IgA autoantibodies against tissue transglutaminase (12%). Although a few T1D subjects showed autoantibodies against a lung-associated protein KCNRG (6%) and S100-β (8%), no statistically significant autoantibodies were detected against several cytokines. Analysis of the overall autoantibody profiles using a heatmap revealed two major subgroups of approximately similar numbers, consisting of T1D subjects with and without organ-specific autoantibodies. Within the organ-specific subgroup, there was minimal overlap among anti-gastric ATPase, anti-thyroid peroxidase, and anti-transglutaminase seropositivity, and these autoantibodies did not correlate with islet cell autoantibodies. Examination of a third cohort, comprising prospectively collected longitudinal samples from high-risk individuals, revealed that anti-gastric ATPase autoantibodies were present in several individuals prior to detection of islet autoantibodies and before clinical onset of T1D. Taken together, these results suggest that autoantibody portraits derived from islet and organ-specific targets will likely be useful for enhancing the clinical management of T1D.     

Evidence for in vivo primed and expanded autoreactive T cells as a specific feature of patients with type 1 diabetes

Identifying beta cell autoantigen-reactive T cells that are involved in the pathogenesis of type 1 diabetes has been troublesome for many laboratories. Disease-relevant autoreactive T cells should be in vivo Ag experienced. The aim of this study was to test this hypothesis and then use this principle as a strategy for identifying diabetes-relevant autoreactive T cells. In this study, a CSFE dilution assay was used to detect glutamic acid decarboxylase 65 (GAD65)- and insulin-responsive T cells and HLA-0201*-GAD65(114-122) pentamers were used to detect CD8(+) GAD-responsive T cells in memory CD45RO(+) and naive CD45RO(-) cell populations from patients with type 1 diabetes and healthy control subjects. T cell proliferative history was evaluated by flow cytometry telomere length measurement. CD4(+) and CD8(+) T cells specific for GAD65 and insulin were present in patients with type 1 diabetes and control subjects. Within the naive CD45RO(-) cells, CD4(+) and CD8(+) T cell responses were similar between patients and controls. Within the memory CD45RO(+) cells, CD4(+) T cell responses against whole GAD65 and insulin and HLA-0201*-GAD65(114-122) pentamer-positive CD8(+) T cells were found in patients with type 1 diabetes, but not in control subjects (p < 0.05 for all). Responding cells from the CD45RO(+) T cell population had substantially shorter telomere lengths than responding cells from the CD45RO(-) cell population. Diabetes-specific autoreactive T cells in the circulation have uniquely undergone sustained in vivo proliferation and differentiation into memory T cells. Prior selection of these cells is possible and is a way to identify diabetes-relevant target Ags and epitopes.     

A mimic of p21WAF1/CIP1 ameliorates murine lupus

Systemic lupus erythematosus (SLE) is a progressive autoimmune disease characterized by the production of high levels of affinity-matured IgG autoantibodies to dsDNA and, possibly, visceral involvement. Pathogenic autoantibodies result from the activation and proliferation of autoreactive T and B lymphocytes stimulated by epitopes borne by nucleosomal histones. To inhibit the proliferation of autoreactive cells and abrogate the development of SLE, a novel tool, cell cycle inhibiting peptide therapy, was used. Thus, a peptidyl mimic of p21WAF1/CIP1 that inhibits the interaction between cyclin-dependent kinase 4 and type D cyclins abrogated the in vitro proliferative response of T cells to histones and T-independent and T-dependent proliferative responses of B cells. The WAF1/CIP1 mimic also abrogated the in vitro production of total and anti-dsDNA IgG Abs by B cells. Similarly, the p21WAF1/CIP1 construct inhibited the ex vivo T and B cell proliferative responses to histones and decreased the numbers of activated/memory B and T spleen cells. The alterations in the balance of spleen cell subsets resulted from proapoptotic effects of the p21WAF1/CIP1)construct on activated splenocytes. Finally, in vivo, four i.v. injections of the p21WAF1/CIP1 mimic were sufficient to inhibit the progression of the lupus-like syndrome in (NZB x NZW)F1 mice. The levels of anti-dsDNA IgG autoantibodies and the incidence and severity of renal involvement were lower in treated mice than in nontreated mice. Those observations open new avenues for the treatment of SLE and prompt us to evaluate the potential interest of peptidic therapy in human SLE.     

T Cell Repertoire Diversity Is Decreased in Type 1 Diabetes Patients

Type 1 diabetes mellitus (T1D) is an immune-mediated disease. The autoreactive T cells in T1D patients attack and destroy their own pancreatic cells. In order to systematically investigate the potential autoreactive T cell receptors (TCRs), we used a high-throughput immune repertoire sequencing technique to profile the spectrum of TCRs in individual T1D patients and controls. We sequenced the T cell repertoire of nine T1D patients, four type 2 diabetes (T2D) patients and six nondiabetic controls. The diversity of the T cell repertoire in T1D patients was significantly decreased in comparison with T2D patients (P = 7.0E
08 for CD4+ T cells, P = 1.4E
04 for CD8+ T cells) and nondiabetic controls (P = 2.7E
09 for CD4+ T cells, P = 7.6E
06 for CD8+ T cells). Moreover, T1D patients had significantly more highly-expanded T cell clones than T2D patients (P = 5.2E
06 for CD4+ T cells, P = 1.9E
07 for CD8+ T cells) and nondiabetic controls (P =1.7E
07 for CD4+ T cells, P= 3.3E
03 for CD8+ T cells). Furthermore, we identified a group of highly-expanded T cell receptor clones that are shared by more than two T1D patients. Although further validation in larger cohorts is needed, our data suggest that T cell receptor diversity measurements may become a valuable tool in investigating diabetes, such as using the diversity as an index to distinguish different types of diabetes.     

Screening and identification of human ZnT8-specific single-chain variable fragment (scFv) from type 1 diabetes phage display library

Zinc transporter 8(ZnT8) is a major autoantigen and a predictive marker in type 1 diabetes(T1D). To investigate ZnT8-specific antibodies, a phage display library from T1 D was constructed and single-chain antibodies against ZnT 8 were screened and identified. Human T1 D single-chain variable fragment(sc Fv) phage display library consists of approximately 1í10~8 clones. After four rounds of bio-panning, seven unique clones were positive by phage ELISA. Among them, C27 and C22, which demonstrated the highest affinity to ZnT8, were expressed in Escherichia coli Top10F' and then purified by affinity chromatography. C27 and C22 specifically bound ZnT8 N/C fusion protein and ZnT8 C terminal dimer with one Arg325 Trp mutation. The specificity to human islet cells of these sc Fvs were further confirmed by immunohistochemistry. In conclusion, we have successfully constructed a T1 D phage display antibody library and identified two ZnT8-specific sc Fv clones, C27 and C22. These ZnT8-specific sc Fvs are potential agents in immunodiagnostic and immunotherapy of T1 D.     

Low frequency of CD8+CD25+FOXP3(BRIGHT) T cells and FOXP3 mRNA expression in the peripheral blood of allergic asthma patients

The aim of this study is to determine whether frequencies of CD8+CD25+ T cells and FoxP3 messenger RNA (mRNA) expression levels in CD8+ T cells isolated from peripheral blood are related to allergic asthma and disease severity. We enrolled 50 patients with allergic asthma (AA) and 25 healthy control subjects (NC) in our study. The frequencies of CD8+CD25+FoxP3 -/+ T cells were assessed with flow cytometry, and mRNA FoxP3 level in CD8+ T cells was determined with real time polymerase chain reaction (RT-PCR). Asthma patients had fewer CD8+CD25+FoxP3bright T cells [SA (median = 3.4%, IQR = 3.1) vs MA (median = 7.5%, IQR = 4.7)] than controls NC [median = 12.1 %, IQR = 8, P < 0.0001] but more CD8+CD25+FoxP3- T cells [SA (median = 96 %, IQR = 3.1) vs MA (median = 92.5%, IQR = 4.7)] than controls NC [median = 87.9%, IQR = 9.2, P < 0.0001]. FoxP3 mRNA level was significantly decreased in CD8+ T cells of severe asthma patients (median = 0.82, IQR = 0.54) than that of patients with mild to moderate asthma and controls [(median = 2.29, IQR = 4.40) vs (median = 2.11, IQR = 3.2)]. The percentage of FoxP3+ T cells was correlated positively with the percentage of forced expiratory volume in 1 second (FEV1) (r = 0.71, p< 0.01) in patients with severe asthma. The proportion of CD8+CD25+FoxP3bright T cells and the level of FOXP3 gene expression in CD8+ T cells are relevant to allergic asthma and disease severity. The manipulation of FoxP3+CD25+CD8+ T cells may prevent chronic allergic inflammation and improve lung function during an acute allergic asthma exacerbation.     

Investigating the role of T-cell avidity and killing efficacy in relation to type 1 diabetes prediction

During the progression of the clinical onset of Type 1 Diabetes (T1D), high-risk individuals exhibit multiple islet autoantibodies and high-avidity T cells which progressively destroy beta cells causing overt T1D. In particular, novel autoantibodies, such as those against IA-2 epitopes (aa1-577), had a predictive rate of 100% in a 10-year follow up (rapid progressors), unlike conventional autoantibodies that required 15 years of follow up for a 74% predictive rate (slow progressors). The discrepancy between these two groups is thought to be associated with T-cell avidity, including CD8 and/or CD4 T cells. For this purpose, we build a series of mathematical models incorporating first one clone then multiple clones of islet-specific and pathogenic CD8 and/or CD4 T cells, together with B lymphocytes, to investigate the interaction of T-cell avidity with autoantibodies in predicting disease onset. These models are instrumental in examining several experimental observations associated with T-cell avidity, including the phenomenon of avidity maturation (increased average T-cell avidity over time), based on intra- and cross-clonal competition between T cells in high-risk human subjects. The model shows that the level and persistence of autoantibodies depends not only on the avidity of T cells, but also on the killing efficacy of these cells. Quantification and modeling of autoreactive T-cell avidities can thus determine the level of risk associated with each type of autoantibodies and the timing of T1D disease onset in individuals that have been tested positive for these autoantibodies. Such studies may lead to early diagnosis of the disease in high-risk individuals and thus potentially serve as a means of staging patients for clinical trials of preventive or interventional therapies far before disease onset.     

Polyfunctional Type-1, -2, and -17 CD8+ T Cell Responses to Apoptotic Self-Antigens Correlate with the Chronic Evolution of Hepatitis C Virus Infection

Caspase-dependent cleavage of antigens associated with apoptotic cells plays a prominent role in the generation of CD8⁺ T cell responses in various infectious diseases. We found that the emergence of a large population of autoreactive CD8⁺ T effector cells specific for apoptotic T cell-associated self-epitopes exceeds the antiviral responses in patients with acute hepatitis C virus infection. Importantly, they endow mixed polyfunctional type-1, type-2 and type-17 responses and correlate with the chronic progression of infection. This evolution is related to the selection of autoreactive CD8⁺ T cells with higher T cell receptor avidity, whereas those with lower avidity undergo prompt contraction in patients who clear infection. These findings demonstrate a previously undescribed strict link between the emergence of high frequencies of mixed autoreactive CD8⁺ T cells producing a broad array of cytokines (IFN-γ, IL-17, IL-4, IL-2…) and the progression toward chronic disease in a human model of acute infection.     

Recognition of Zinc Transporter 8 and MAP3865c Homologous Epitopes by Hashimoto's Thyroiditis Subjects from Sardinia: A Common Target with Type 1 Diabetes?

Mycobacterium avium subspecies paratuberculosis (MAP) asymptomatic infection has been previously linked to Type 1 diabetes (T1D) and Multiple Sclerosis. An association between MAP infection and Hashimoto''s thyroiditis (HT) was also proposed only in a case report. This study aimed to investigate the robustness of the latter association, testing a large cohort of HT and healthy control (HCs) subjects, all from Sardinia. Prevalence of anti-MAP3865c Abs was assessed by indirect enzyme-linked immunosorbent assay (ELISA). Moreover, given that human ZnT8 is specifically expressed in the pancreatic β-cells, in the follicle epithelial cells and in the parafollicular cells of the thyroid gland, we also tested ZnT8 epitopes homologues to the MAP3865c immunodominant peptides previously identified. Indeed, Abs targeting MAP3865c and ZnT8 homologous regions display similar frequencies in patients and controls, thus suggesting that Abs recognizing these epitopes could be cross-reactive. A statistically significant difference was found between HT patients and HCs when analyzing the humoral response mounted against MAP3865c/ZnT8 homologues epitopes. To our knowledge, this is the first report, which provides statistically significant evidence sustaining the existence of an association between MAP sero-reactivity and HT. Further studies are required to investigate the relevance of MAP to HT, aimed at deciphering if this pathogen can be at play in triggering this autoimmune disease. Likewise, genetic polymorphism of the host, and other environmental factors need to be investigated.     

Inhibition of Increased Circulating Tfh Cell by Anti-CD20 Monoclonal Antibody in Patients with Type 1 Diabetes

Objectives

Follicular helper T (Tfh) cells exert an important role in autoimmune diseases. Whether it might be involved in type 1 diabetes (T1D) is unknown. Our aim was to investigate the role of Tfh cells in patients with T1D and the effect of anti-CD20 monoclonal antibody (rituximab) on Tfh cells from T1D patients.

Patients and Methods

Fifty-four patients with T1D and 37 healthy controls were enrolled in the current study. 20 of those patients were treated with rituximab. The frequencies of circulating CD4⁺CXCR5⁺ICOS⁺T cells were analyzed by flow cytometry. The serum autoantibodies were detected by radioligand assay. The levels of IL-21, IL-6 and BCL-6 were assessed using ELISA and/or real-time PCR.

Results

Increased frequencies of circulating Tfh cells together with enhanced expression of IL-21 were detected in patients. The correlation between the frequencies of circulating Tfh cells and the serum autoantibodies or C-peptide level was comfirmed. After rituximab therapy, follow-up analysis demonstrated that the frequencies of circulating Tfh cell and serum IA2A were decreased. The levels of IL-21, IL-6 and Bcl-6 mRNA were decreased after treatment. Furthermore, beta cell function in 10 of 20 patients was improved.

Conclusions

These data indicate Tfh cells may participate in the T1D-relatede immune responses and B cells might play a role in the development of Tfh responses in the disease progression.     

MHC class II molecules play a role in the selection of autoreactive class I-restricted CD8 T cells that are essential contributors to type 1 diabetes development in nonobese diabetic mice

Development of autoreactive CD4 T cells contributing to type 1 diabetes (T1D) in both humans and nonobese diabetic (NOD) mice is either promoted or dominantly inhibited by particular MHC class II variants. In addition, it is now clear that when co-expressed with other susceptibility genes, some common MHC class I variants aberrantly mediate autoreactive CD8 T cell responses also essential to T1D development. However, it was unknown whether the development of diabetogenic CD8 T cells could also be dominantly inhibited by particular MHC variants. We addressed this issue by crossing NOD mice transgenically expressing the TCR from the diabetogenic CD8 T cell clone AI4 with NOD stocks congenic for MHC haplotypes that dominantly inhibit T1D. High numbers of functional AI4 T cells only developed in controls homozygously expressing NOD-derived H2(g7) molecules. In contrast, heterozygous expression of some MHC haplotypes conferring T1D resistance anergized AI4 T cells through decreased TCR (H2(b)) or CD8 expression (H2(q)). Most interestingly, while AI4 T cells exert a class I-restricted effector function, H2(nb1) MHC class II molecules can contribute to their negative selection. These findings provide insights to how particular MHC class I and class II variants interactively regulate the development of diabetogenic T cells and the TCR promiscuity of such autoreactive effectors.     

Islet-Specific CTL Cloned from a Type 1 Diabetes Patient Cause Beta-Cell Destruction after Engraftment into HLA-A2 Transgenic NOD/SCID/IL2RG Null Mice

Despite increasing evidence that autoreactive CD8 T-cells are involved in both the initiation of type 1 diabetes (T1D) and the destruction of beta-cells, direct evidence for their destructive role in-vivo is lacking. To address a destructive role for autoreactive CD8 T-cells in human disease, we assessed the pathogenicity of a CD8 T-cell clone derived from a T1D donor and specific for an HLA-A2-restricted epitope of islet-specific glucose-6-phosphatase catalytic-subunit related protein (IGRP). HLA-A2/IGRP tetramer staining revealed a higher frequency of IGRP-specific CD8 T-cells in the peripheral blood of recent onset human individuals than of healthy donors. IGRP_265–273-specific CD8 T-cells that were cloned from the peripheral blood of a recent onset T1D individual were shown to secrete IFNγ and Granzyme B after antigen-specific activation and lyse HLA-A2-expressing murine islets in-vitro. Lytic capacity was also demonstrated in-vivo by specific killing of peptide-pulsed target cells. Using the HLA-A2 NOD-scid IL2rγ^null mouse model, HLA-A2-restricted IGRP-specific CD8 T-cells induced a destructive insulitis. Together, this is the first evidence that human HLA-restricted autoreactive CD8 T-cells target HLA-expressing beta-cells in-vivo, demonstrating the translational value of humanized mice to study mechanisms of disease and therapeutic intervention strategies.     

Dichotomy of autoreactive Th1 and Th2 cell responses to desmoglein 3 in patients with pemphigus vulgaris (PV) and healthy carriers of PV-associated HLA class II alleles

Pemphigus vulgaris (PV) is the most severe autoimmune bullous skin disorder and is primarily associated with circulating autoantibodies (autoAb) against desmoglein 3 (Dsg3). In light of recent evidence that autoreactive T cells are critical for the induction and regulation of Ab production, the goal of this study was to characterize and quantitate autoreactive T cells in patients with PV and healthy controls. Peripheral Dsg3-reactive Th cells from 28 patients with acute-onset, chronic active, and remittent PV were quantitated by MACS secretion assay. Dsg3-reactive Th2 cells were detected at similar frequencies in all studied PV patients, while the number of autoreactive Th1 cells exceeded those of Th2 cells in chronic active PV. In contrast, healthy carriers of the PV-associated HLA class II alleles, DRB1*0402 and DQB1*0503, exhibited exclusively Dsg3-reactive Th1 cell responses, while healthy carriers of other HLA class II alleles did not. Moreover, the presence of IgG1 and IgG4 against Dsg3 was directly related to the ratio of Dsg3-reactive Th1/Th2 cells. T cell recognition of Dsg3 was restricted by HLA-DRB1*0402 and DQB1*0503 in PV patients and Dsg3-responsive healthy donors. These observations strongly suggest 1) that the appearance of Dsg3-reactive Th2 cells is restricted to patients with PV; 2) that specific HLA class II alleles that are prevalent in PV are critical for T cell recognition of Dsg3 in PV patients and Dsg3-responsive healthy donors; and 3) that autoAb production is associated with both Th1 and Th2 cells.     

Targeting Insulin-Like Growth Factor 1 Leads to Amelioration of Inflammatory Demyelinating Disease

In patients with multiple sclerosis (MS) and in mice with experimental autoimmune encephalomyelitis (EAE), proliferating autoreactive T cells play an important role in the pathogenesis of the disease. Due to the importance of these myelin-specific T cells, these cells have been therapeutic targets in a variety of treatments. Previously we found that Lenaldekar (LDK), a novel small molecule, could inhibit exacerbations in a preclinical model of MS when given at the start of an EAE exacerbation. In those studies, we found that LDK could inhibit human T cell recall responses and murine myelin responses in vitro. In these new studies, we found that LDK could inhibit myelin specific T cell responses through the insulin-like growth factor-1 receptor (IGF-1R) pathway. Alteration of this pathway led to marked reduction in T cell proliferation and expansion. Blocking this pathway could account for the observed decreases in clinical signs and inflammatory demyelinating disease, which was accompanied by axonal preservation. Our data indicate that IGF-1R could be a potential target for new therapies for the treatment of autoimmune diseases where autoreactive T cell expansion is a requisite for disease.     

Seroreactivity against Specific L5P Antigen from Mycobacterium avium subsp. paratuberculosis in Children at Risk for T1D

Aims/HypothesisAlthough numerous environmental agents have been investigated over the years as possible triggers of type 1 diabetes (T1D), its causes remain unclear. We have already demonstrated an increased prevalence of antibodies against peptides derived from Mycobacterium avuim subsp. paratuberculosis (MAP) homologous to human zinc transporter 8 protein (ZnT8) and proinsulin in Italian subjects at risk for or affected by T1D. In this study, we compared titers of the previously detected antibodies with seroreactivity to MAP lipopentapetide (L5P) that recently emerged as a strong immunogenic component able to specifically distinguish MAP from other mycobacteria.MethodsPlasma of 32 children and youth at risk for T1D including follow-up samples and 42 age-matched healthy controls (HC) recruited at the Tor Vergata University Hospital in Rome was analyzed by indirect ELISA for the presence of antibodies against MAP-derived epitopes MAP3865c_133–141, MAP3865c_125-133, MAP2404c_70-85 and MAP1,4αgbp_157-173 along with their ZnT8 and proinsulin homologs. The data were analyzed through two-tailed Mann-Whitney U test and relation between variables was determined by principal component analysis.ResultsResponses to L5P were not detectable in subjects whose initial seroreactivity to MAP peptides and their human homologs was lost in follow-up samples, whereas anti-L5P antibodies appeared constantly in individuals with a stable immunity against MAP antigens. The overall coincidence in positivity to L5P and the four MAP epitopes both in children at risk for T1D and HC exceeded 90%.ConclusionsMAP-derived homologs may cross-react with ZnT8 and proinsulin peptides inducing immune responses at a young age in subjects predisposed for T1D. Thus, L5P may have a diagnostic value to immediately indicate the presence of anti-MAP seroreactivity when evaluation of a more complex antibody status is not required. Almost complete coincidence in responses to both types of antigens lends support to the involvement of MAP in T1D.     

The quest for personalized B-cell depletion therapy in rheumatic disease

Although B cell depletion therapy (BCDT) is now a well-accepted therapeutic option in autoimmune rheumatic disease, a significant proportion of patients remain resistant to therapy. .19pt?>A more challenging clinical problem is the high rate of relapse after B cell reconstitution, as well as the difficulty in predicting the exact timing of that relapse. In this article, we consider the immunological mechanisms that may account for the heterogeneity of clinical response to BCDT. Understanding how BCDT alters the balance between different B cell subsets, some pathogenic and some regulatory, may help us correctly target BCDT to the right patients, and thereby improve treatment responses in rheumatic disease.The identification of autoantibodies in the serum of patients with rheumatic disease was one of the landmark studies that placed B cells at the heart of research into the pathogenesis of autoimmune disease. It is now clear that B cells contribute to autoimmunity by a range of mechanisms, both directly through the secretion of inflammatory cytokines [1] and indirectly by antigen presentation and co-stimulation to activate autoreactive T cells. However, it was only at the beginning of the last decade that attention finally turned to B cells as a potential target that may ameliorate autoimmune rheumatic disease.     

Increased expression of zinc transporter ZIP4, ZIP11, ZnT1, and ZnT6 predicts poor prognosis in pancreatic cancer

IntroductionZinc homeostasis is regulated by SLC39A/ZIP, SLC30A/ZnT, and metallothionein (MT) families in human cells. Zinc dyshomeostasis may affect or be affected by the abnormal behavior of cancer cells. Although decreased serum zinc levels are observed in patients with pancreatic adenocarcinoma (PAAD), limited information is available regarding the expression pattern and prognostic roles of zinc homeostasis-related genes in PAAD.ObjectivesThe primary objective of this study was to explore the expression pattern and prognostic roles of zinc homeostasis-related genes in PAAD.MethodsThe expression pattern of 35 known zinc homeostasis-related genes in PAAD was systemically explored based on RNA-sequencing data from the Cancer Genome Atlas (TCGA) and the Genotype-Tissue Expression (GTEx) projects. The association between the expression levels of zinc homeostasis-related genes and survival of PAAD patients was evaluated using the Kaplan-Meier method and the log-rank test. Expressional correlation between zinc homeostasis-related genes with potential prognostic value in PAAD and normal pancreatic controls was evaluated using Pearson’s correlation analysis. Functional enrichment analyses were performed to elucidate possible mechanisms for the potential prognostic and therapeutic roles of these zinc homeostasis-related genes in PAAD. Effects of ZIP11, ZnT1, or ZnT6 knockdown on the proliferation and the migration of Capan-1 pancreatic cancer cells were assessed by the CCK-8 assay and the wound healing assay respectively.ResultsWe demonstrated that the expression levels of ZIP1, ZIP3, ZIP4, ZIP6, ZIP7, ZIP9, ZIP10, ZIP11, ZIP13, ZnT1, ZnT5, ZnT6, ZnT7, and ZnT9 were increased, whereas the expression levels of ZIP5, ZIP14, ZnT2, MT1 G, MT1H, and MT1X were decreased in PAAD tumors compared with normal pancreatic controls. Among these differentially-expressed genes related to zinc homeostasis, higher expression of ZIP4, ZIP11, ZnT1 or ZnT6 predicted poorer prognosis with the possible involvement of several cancer-related processes and pathways in PAAD patients. We further demonstrated that knockdown of ZIP11 attenuated Capan-1 cell proliferation with decreased activation of ERK1/2 pathway; knockdown of ZnT1 attenuated Capan-1 cell proliferation with decreased activation of ERK1/2, p38 MAPK, NF-kB, and mTOR pathways; knockdown of ZnT6 attenuated Capan-1 cell proliferation with decreased activation of ERK1/2, p38 MAPK, and NF-kB pathways.ConclusionsHigher expression of the zinc transporter ZIP4, ZIP11, ZnT1 or ZnT6 predicted poorer prognosis in patients with PAAD. These findings provide new clues for understanding the complex relationship between zinc homeostasis and pancreatic cancer.     

Identification of three new autoantibodies associated with systemic lupus erythematosus using two proteomic approaches

Our objective was to identify new serum autoantibodies associated with systemic lupus erythematosus (SLE), focusing on those found in patients with central nervous system (CNS) syndromes. Autoantigens in human brain proteins were screened by multiple proteomic analyses: two-dimensional polyacrylamide gel electrophoresis/Western blots followed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis and immunoprecipitation followed by liquid chromatography-tandem mass spectrometry shotgun analysis. The presence of serum IgG autoantibodies against 11 selected recombinant antigens was assessed by Western blot and enzyme-linked immunosorbent assay (ELISA) in the sera of 106 SLE patients and 100 normal healthy controls. The O.D. values in sera from SLE patients were significantly higher than those of controls for the antigens crystallin αB (p = 0.0002), esterase D (p = 0.0002), APEX nuclease 1 (p < 0.0001), ribosomal protein P0 (p < 0.0001), and PA28γ (p = 0.0005); the first three are newly reported. The anti-esterase D antibody levels were significantly higher in the CNS group than in the non-CNS group (p = 0.016). Moreover, when the SLE patients were categorized using CNS manifestations indicating neurologic or psychiatric disorders, the anti-APEX nuclease 1 antibody levels were significantly elevated in SLE patients with psychiatric disorders (p = 0.037). In conclusion, the association of SLE with several new and previously reported autoantibodies has been demonstrated. Statistically significant associations between anti-esterase D antibodies and CNS syndromes as well as between anti-APEX nuclease 1 antibodies and psychiatric disorders in SLE were also demonstrated. The combined immunoproteomic approaches used in this study are reliable and effective methods for identifying SLE autoantigens.     

Expansion of NK Cells and Reduction of NKG2D Expression in Chronic Lymphocytic Leukemia. Correlation with Progressive Disease

The immune system may mediate anti-tumor responses in chronic lymphocytic leukemia (CLL) which may affect disease progression and survival. In this study, we analyzed the immune characteristics of 99 consecutive previously diagnosed CLL patients and 50 healthy controls. The distribution of lymphocyte subsets at diagnosis was retrospectively analyzed. Compared with controls, leukemia patients showed an expansion of NK and CD8 T cells at diagnosis. The relative number of CD8 T cells at diagnosis was associated with time to treatment, suggesting that CD8 T cells may modify disease progression. The distribution of lymphocyte subsets was analyzed again when patients were enrolled in this study. The median time since these patients were diagnosed was 277 weeks. Compared with diagnosis, the absolute number of CD8 T cells significantly decreased in these patients, reaching similar values to healthy controls; however NK cells kept significantly elevated overtime. Nevertheless, NK cells showed an impaired expression of NKG2D receptor and a defective cytotoxic activity. This down-regulation of NKG2D expression was further enhanced in patients with advanced and progressive disease. Additionally, membrane NKG2D levels significantly decreased on CD8 T cells, but a significant increase of NKG2D+CD4+ T cells was observed in CLL patients. The cytotoxic activity of NK cells was diminished in CLL patients; however the treatments with IL-2, IL-15, IL-21 and lenalidomide were able to restore their activity. The effect of IL-2 and IL-15 was associated with the increase of NKG2D expression on immune cells, but the effect of IL-21 and lenalidomide was not due to NKG2D up-regulation. The expansion of NK cells and the reversibility of NK cell defects provide new opportunities for the immunotherapeutic intervention in CLL.