Identification of DNA methylation episignature for the intellectual developmental disorder, autosomal dominant 21 syndrome, caused by variants in the CTCF gene.

PURPOSE: The main objective of this study was to assess clinical features and genome-wide DNA methylation profiles in individuals affected by intellectual developmental disorder, autosomal dominant 21 (IDD21) syndrome, caused by variants in the CCCTC-binding factor (CTCF) gene. METHODS: DNA samples were extracted from peripheral blood of 16 individuals with clinical features and genetic findings consistent with IDD21. DNA methylation analysis was performed using the Illumina Infinium Methylation EPIC Bead Chip microarrays. The methylation levels were fitted in a multivariate linear regression model to identify the differentially methylated probes. A binary support vector machine classification model was constructed to differentiate IDD21 samples from controls. RESULTS: We identified a highly specific, reproducible, and sensitive episignature associated with CTCF variants. Six variants of uncertain significance were tested, of which 2 mapped to the IDD21 episignature and clustered alongside IDD21 cases in both heatmap and multidimensional scaling plots. Comparison of the genomic DNA methylation profile of IDD21 with that of 56 other neurodevelopmental disorders provided insights into the underlying molecular pathophysiology of this disorder. CONCLUSION: The robust and specific CTCF/IDD21 episignature expands the growing list of neurodevelopmental disorders with distinct DNA methylation profiles, which can be applied as supporting evidence in variant classification.

Epilepsy is an important feature of KBG syndrome associated with poorer developmental outcome.

OBJECTIVE: The aim of this study was to describe the epilepsy phenotype in a large international cohort of patients with KBG syndrome and to study a possible genotype-phenotype correlation. METHODS: We collected data on patients with ANKRD11 variants by contacting University Medical Centers in the Netherlands, an international network of collaborating clinicians, and study groups who previously published about KBG syndrome. All patients with a likely pathogenic or pathogenic ANKRD11 variant were included in our patient cohort and categorized into an "epilepsy group" or "non-epilepsy group". Additionally, we included previously reported patients with (likely) pathogenic ANKRD11 variants and epilepsy from the literature. RESULTS: We included 75 patients with KBG syndrome of whom 26 had epilepsy. Those with epilepsy more often had moderate to severe intellectual disability (42.3% vs 9.1%, RR 4.6 [95% CI 1.7-13.1]). Seizure onset in patients with KBG syndrome occurred at a median age of 4 years (range 12 months - 20 years), and the majority had generalized onset seizures (57.7%) with tonic-clonic seizures being most common (23.1%). The epilepsy type was mostly classified as generalized (42.9%) or combined generalized and focal (42.9%), not fulfilling the criteria of an electroclinical syndrome diagnosis. Half of the epilepsy patients (50.0%) were seizure free on anti-seizure medication (ASM) for at least 1 year at the time of last assessment, but 26.9% of patients had drug-resistant epilepsy (failure of ≥2 ASM). No genotype-phenotype correlation could be identified for the presence of epilepsy or epilepsy characteristics. SIGNIFICANCE: Epilepsy in KBG syndrome most often presents as a generalized or combined focal and generalized type. No distinctive epilepsy syndrome could be identified. Patients with KBG syndrome and epilepsy had a significantly poorer neurodevelopmental outcome compared with those without epilepsy. Clinicians should consider KBG syndrome as a causal etiology of epilepsy and be aware of the poorer neurodevelopmental outcome in individuals with epilepsy.

POU3F3-related disorder: Defining the phenotype and expanding the molecular spectrum.

POU3F3 variants cause developmental delay, behavioral problems, hypotonia and dysmorphic features. We investigated the phenotypic and genetic landscape, and genotype-phenotype correlations in individuals with POU3F3-related disorders. We recruited unpublished individuals with POU3F3 variants through international collaborations and obtained updated clinical data on previously published individuals. Trio exome sequencing or single exome sequencing followed by segregation analysis were performed in the novel cohort. Functional effects of missense variants were investigated with 3D protein modeling. We included 28 individuals (5 previously published) from 26 families carrying POU3F3 variants; 23 de novo and one inherited from an affected parent. Median age at study inclusion was 7.4 years. All had developmental delay mainly affecting speech, behavioral difficulties, psychiatric comorbidities and dysmorphisms. Additional features included gastrointestinal comorbidities, hearing loss, ophthalmological anomalies, epilepsy, sleep disturbances and joint hypermobility. Autism, hearing and eye comorbidities, dysmorphisms were more common in individuals with truncating variants, whereas epilepsy was only associated with missense variants. In silico structural modeling predicted that all (likely) pathogenic variants destabilize the DNA-binding region of POU3F3. Our study refined the phenotypic and genetic landscape of POU3F3-related disorders, it reports the functional properties of the identified pathogenic variants, and delineates some genotype-phenotype correlations.

Trio-based whole exome sequencing in patients with suspected sporadic inborn errors of immunity: A retrospective cohort study.

Background: De novo variants (DNVs) are currently not routinely evaluated as part of diagnostic whole exome sequencing (WES) analysis in patients with suspected inborn errors of immunity (IEI). Methods: This study explored the potential added value of systematic assessment of DNVs in a retrospective cohort of 123 patients with a suspected sporadic IEI that underwent patient-parent trio-based WES. Results: A (likely) molecular diagnosis for (part) of the immunological phenotype was achieved in 12 patients with the diagnostic in silico IEI WES gene panel. Systematic evaluation of rare, non-synonymous DNVs in coding or splice site regions led to the identification of 14 candidate DNVs in genes with an annotated immune function. DNVs were found in IEI genes (NLRP3 and RELA) and in potentially novel candidate genes, including PSMB10, DDX1, KMT2C, and FBXW11. The FBXW11 canonical splice site DNV was shown to lead to defective RNA splicing, increased NF-κB p65 signalling, and elevated IL-1ß production in primary immune cells extracted from the patient with autoinflammatory disease. Conclusions: Our findings in this retrospective cohort study advocate the implementation of trio-based sequencing in routine diagnostics of patients with sporadic IEI. Furthermore, we provide functional evidence supporting a causal role for FBXW11 loss-of-function mutations in autoinflammatory disease. Funding: This research was supported by grants from the European Union, ZonMW and the Radboud Institute for Molecular Life Sciences.

Implementation of Early Next-Generation Sequencing for Inborn Errors of Immunity: A Prospective Observational Cohort Study of Diagnostic Yield and Clinical Implications in Dutch Genome Diagnostic Centers.

Objective: Inborn errors of immunity (IEI) are a heterogeneous group of disorders, affecting different components of the immune system. Over 450 IEI related genes have been identified, with new genes continually being recognized. This makes the early application of next-generation sequencing (NGS) as a diagnostic method in the evaluation of IEI a promising development. We aimed to provide an overview of the diagnostic yield and time to diagnosis in a cohort of patients suspected of IEI and evaluated by an NGS based IEI panel early in the diagnostic trajectory in a multicenter setting in the Netherlands. Study Design: We performed a prospective observational cohort study. We collected data of 165 patients with a clinical suspicion of IEI without prior NGS based panel evaluation that were referred for early NGS using a uniform IEI gene panel. The diagnostic yield was assessed in terms of definitive genetic diagnoses, inconclusive diagnoses and patients without abnormalities in the IEI gene panel. We also assessed time to diagnosis and clinical implications. Results: For children, the median time from first consultation to diagnosis was 119 days versus 124 days for adult patients (U=2323; p=0.644). The median turn-around time (TAT) of genetic testing was 56 days in pediatric patients and 60 days in adult patients (U=1892; p=0.191). A definitive molecular diagnosis was made in 25/65 (24.6%) of pediatric patients and 9/100 (9%) of adults. Most diagnosed disorders were identified in the categories of immune dysregulation (n=10/25; 40%), antibody deficiencies (n=5/25; 20%), and phagocyte diseases (n=5/25; 20%). Inconclusive outcomes were found in 76/165 (46.1%) patients. Within the patient group with a genetic diagnosis, a change in disease management occurred in 76% of patients. Conclusion: In this cohort, the highest yields of NGS based evaluation for IEI early in the diagnostic trajectory were found in pediatric patients, and in the disease categories immune dysregulation and phagocyte diseases. In cases where a definitive diagnosis was made, this led to important disease management implications in a large majority of patients. More research is needed to establish a uniform diagnostic pathway for cases with inconclusive diagnoses, including variants of unknown significance.

De novo ARHGEF9 missense variants associated with neurodevelopmental disorder in females: expanding the genotypic and phenotypic spectrum of ARHGEF9 disease in females.

Individuals harboring pathogenic variants in ARHGEF9, encoding an essential submembrane protein for gamma-aminobutyric acid (GABA)-ergic synapses named collybistin, show intellectual disability (ID), facial dysmorphism, behavioral disorders, and epilepsy. Only few affected females carrying large chromosomal rearrangements involving ARHGEF9 have been reported so far. Through next-generation sequencing (NGS)-based panels, we identified two single nucleotide variants (SNVs) in ARHGEF9 in two females with neurodevelopmental features. Sanger sequencing revealed that these variants were de novo. The X-inactivation pattern in peripheral blood cells was random. We report the first affected females harboring de novo SNVs in ARHGEF9, expanding the genotypic and phenotypic spectrum of ARHGEF9-related neurodevelopmental disorder in females.

National external quality assessment for next-generation sequencing-based diagnostics of primary immunodeficiencies.

Dutch genome diagnostic centers (GDC) use next-generation sequencing (NGS)-based diagnostic applications for the diagnosis of primary immunodeficiencies (PIDs). The interpretation of genetic variants in many PIDs is complicated because of the phenotypic and genetic heterogeneity. To analyze uniformity of variant filtering, interpretation, and reporting in NGS-based diagnostics for PID, an external quality assessment was performed. Four main Dutch GDCs participated in the quality assessment. Unannotated variant call format (VCF) files of two PID patient analyses per laboratory were distributed among the four GDCs, analyzed, and interpreted (eight analyses in total). Variants that would be reported to the clinician and/or advised for further investigation were compared between the centers. A survey measuring the experiences of clinical laboratory geneticists was part of the study. Analysis of samples with confirmed diagnoses showed that all centers reported at least the variants classified as likely pathogenic (LP) or pathogenic (P) variants in all samples, except for variants in two genes (PSTPIP1 and BTK). The absence of clinical information complicated correct classification of variants. In this external quality assessment, the final interpretation and conclusions of the genetic analyses were uniform among the four participating genetic centers. Clinical and immunological data provided by a medical specialist are required to be able to draw proper conclusions from genetic data.

Germline AGO2 mutations impair RNA interference and human neurological development.

ARGONAUTE-2 and associated miRNAs form the RNA-induced silencing complex (RISC), which targets mRNAs for translational silencing and degradation as part of the RNA interference pathway. Despite the essential nature of this process for cellular function, there is little information on the role of RISC components in human development and organ function. We identify 13 heterozygous mutations in AGO2 in 21 patients affected by disturbances in neurological development. Each of the identified single amino acid mutations result in impaired shRNA-mediated silencing. We observe either impaired RISC formation or increased binding of AGO2 to mRNA targets as mutation specific functional consequences. The latter is supported by decreased phosphorylation of a C-terminal serine cluster involved in mRNA target release, increased formation of dendritic P-bodies in neurons and global transcriptome alterations in patient-derived primary fibroblasts. Our data emphasize the importance of gene expression regulation through the dynamic AGO2-RNA association for human neuronal development.

ISSAID/EMQN Best Practice Guidelines for the Genetic Diagnosis of Monogenic Autoinflammatory Diseases in the Next-Generation Sequencing Era.

BACKGROUND: Monogenic autoinflammatory diseases are caused by pathogenic variants in genes that regulate innate immune responses, and are characterized by sterile systemic inflammatory episodes. Since symptoms can overlap within this rapidly expanding disease category, accurate genetic diagnosis is of the utmost importance to initiate early inflammation-targeted treatment and prevent clinically significant or life-threatening complications. Initial recommendations for the genetic diagnosis of autoinflammatory diseases were limited to a gene-by-gene diagnosis strategy based on the Sanger method, and restricted to the 4 prototypic recurrent fevers (MEFV, MVK, TNFRSF1A, and NLRP3 genes). The development of best practices guidelines integrating critical recent discoveries has become essential. METHODS: The preparatory steps included 2 online surveys and pathogenicity annotation of newly recommended genes. The current guidelines were drafted by European Molecular Genetics Quality Network members, then discussed by a panel of experts of the International Society for Systemic Autoinflammatory Diseases during a consensus meeting. RESULTS: In these guidelines, we combine the diagnostic strength of next-generation sequencing and recommendations to 4 more recently identified genes (ADA2, NOD2, PSTPIP1, and TNFAIP3), nonclassical pathogenic genetic alterations, and atypical phenotypes. We present a referral-based decision tree for test scope and method (Sanger versus next-generation sequencing) and recommend on complementary explorations for mosaicism, copy-number variants, and gene dose. A genotype table based on the 5-category variant pathogenicity classification provides the clinical significance of prototypic genotypes per gene and disease. CONCLUSIONS: These guidelines will orient and assist geneticists and health practitioners in providing up-to-date and appropriate diagnosis to their patients.

Ribosomal protein gene RPL9 variants can differentially impair ribosome function and cellular metabolism.

Variants in ribosomal protein (RP) genes drive Diamond-Blackfan anemia (DBA), a bone marrow failure syndrome that can also predispose individuals to cancer. Inherited and sporadic RP gene variants are also linked to a variety of phenotypes, including malignancy, in individuals with no anemia. Here we report an individual diagnosed with DBA carrying a variant in the 5'UTR of RPL9 (uL6). Additionally, we report two individuals from a family with multiple cancer incidences carrying a RPL9 missense variant. Analysis of cells from these individuals reveals that despite the variants both driving pre-rRNA processing defects and 80S monosome reduction, the downstream effects are remarkably different. Cells carrying the 5'UTR variant stabilize TP53 and impair the growth and differentiation of erythroid cells. In contrast, ribosomes incorporating the missense variant erroneously read through UAG and UGA stop codons of mRNAs. Metabolic profiles of cells carrying the 5'UTR variant reveal an increased metabolism of amino acids and a switch from glycolysis to gluconeogenesis while those of cells carrying the missense variant reveal a depletion of nucleotide pools. These findings indicate that variants in the same RP gene can drive similar ribosome biogenesis defects yet still have markedly different downstream consequences and clinical impacts.

Cohesin complex-associated holoprosencephaly.

Marked by incomplete division of the embryonic forebrain, holoprosencephaly is one of the most common human developmental disorders. Despite decades of phenotype-driven research, 80-90% of aneuploidy-negative holoprosencephaly individuals with a probable genetic aetiology do not have a genetic diagnosis. Here we report holoprosencephaly associated with variants in the two X-linked cohesin complex genes, STAG2 and SMC1A, with loss-of-function variants in 10 individuals and a missense variant in one. Additionally, we report four individuals with variants in the cohesin complex genes that are not X-linked, SMC3 and RAD21. Using whole mount in situ hybridization, we show that STAG2 and SMC1A are expressed in the prosencephalic neural folds during primary neurulation in the mouse, consistent with forebrain morphogenesis and holoprosencephaly pathogenesis. Finally, we found that shRNA knockdown of STAG2 and SMC1A causes aberrant expression of HPE-associated genes ZIC2, GLI2, SMAD3 and FGFR1 in human neural stem cells. These findings show the cohesin complex as an important regulator of median forebrain development and X-linked inheritance patterns in holoprosencephaly.

CTCF variants in 39 individuals with a variable neurodevelopmental disorder broaden the mutational and clinical spectrum.

PURPOSE: Pathogenic variants in the chromatin organizer CTCF were previously reported in seven individuals with a neurodevelopmental disorder (NDD). METHODS: Through international collaboration we collected data from 39 subjects with variants in CTCF. We performed transcriptome analysis on RNA from blood samples and utilized Drosophila melanogaster to investigate the impact of Ctcf dosage alteration on nervous system development and function. RESULTS: The individuals in our cohort carried 2 deletions, 8 likely gene-disruptive, 2 splice-site, and 20 different missense variants, most of them de novo. Two cases were familial. The associated phenotype was of variable severity extending from mild developmental delay or normal IQ to severe intellectual disability. Feeding difficulties and behavioral abnormalities were common, and variable other findings including growth restriction and cardiac defects were observed. RNA-sequencing in five individuals identified 3828 deregulated genes enriched for known NDD genes and biological processes such as transcriptional regulation. Ctcf dosage alteration in Drosophila resulted in impaired gross neurological functioning and learning and memory deficits. CONCLUSION: We significantly broaden the mutational and clinical spectrum ofCTCF-associated NDDs. Our data shed light onto the functional role of CTCF by identifying deregulated genes and show that Ctcf alterations result in nervous system defects in Drosophila.

Improving the diagnostic yield of exome- sequencing by predicting gene-phenotype associations using large-scale gene expression analysis.

The diagnostic yield of exome and genome sequencing remains low (8-70%), due to incomplete knowledge on the genes that cause disease. To improve this, we use RNA-seq data from 31,499 samples to predict which genes cause specific disease phenotypes, and develop GeneNetwork Assisted Diagnostic Optimization (GADO). We show that this unbiased method, which does not rely upon specific knowledge on individual genes, is effective in both identifying previously unknown disease gene associations, and flagging genes that have previously been incorrectly implicated in disease. GADO can be run on www.genenetwork.nl by supplying HPO-terms and a list of genes that contain candidate variants. Finally, applying GADO to a cohort of 61 patients for whom exome-sequencing analysis had not resulted in a genetic diagnosis, yields likely causative genes for ten cases.

Exome sequencing in routine diagnostics: a generic test for 254 patients with primary immunodeficiencies.

BACKGROUND: Diagnosis of primary immunodeficiencies (PIDs) is complex and cumbersome yet important for the clinical management of the disease. Exome sequencing may provide a genetic diagnosis in a significant number of patients in a single genetic test. METHODS: In May 2013, we implemented exome sequencing in routine diagnostics for patients suffering from PIDs. This study reports the clinical utility and diagnostic yield for a heterogeneous group of 254 consecutively referred PID patients from 249 families. For the majority of patients, the clinical diagnosis was based on clinical criteria including rare and/or unusual severe bacterial, viral, or fungal infections, sometimes accompanied by autoimmune manifestations. Functional immune defects were interpreted in the context of aberrant immune cell populations, aberrant antibody levels, or combinations of these factors. RESULTS: For 62 patients (24%), exome sequencing identified pathogenic variants in well-established PID genes. An exome-wide analysis diagnosed 10 additional patients (4%), providing diagnoses for 72 patients (28%) from 68 families altogether. The genetic diagnosis directly indicated novel treatment options for 25 patients that received a diagnosis (34%). CONCLUSION: Exome sequencing as a first-tier test for PIDs granted a diagnosis for 28% of patients. Importantly, molecularly defined diagnoses indicated altered therapeutic options in 34% of cases. In addition, exome sequencing harbors advantages over gene panels as a truly generic test for all genetic diseases, including in silico extension of existing gene lists and re-analysis of existing data.

WDR26 Haploinsufficiency Causes a Recognizable Syndrome of Intellectual Disability, Seizures, Abnormal Gait, and Distinctive Facial Features.

We report 15 individuals with de novo pathogenic variants in WDR26. Eleven of the individuals carry loss-of-function mutations, and four harbor missense substitutions. These 15 individuals comprise ten females and five males, and all have intellectual disability with delayed speech, a history of febrile and/or non-febrile seizures, and a wide-based, spastic, and/or stiff-legged gait. These subjects share a set of common facial features that include a prominent maxilla and upper lip that readily reveal the upper gingiva, widely spaced teeth, and a broad nasal tip. Together, these features comprise a recognizable facial phenotype. We compared these features with those of chromosome 1q41q42 microdeletion syndrome, which typically contains WDR26, and noted that clinical features are consistent between the two subsets, suggesting that haploinsufficiency of WDR26 contributes to the pathology of 1q41q42 microdeletion syndrome. Consistent with this, WDR26 loss-of-function single-nucleotide mutations identified in these subjects lead to nonsense-mediated decay with subsequent reduction of RNA expression and protein levels. We derived a structural model of WDR26 and note that missense variants identified in these individuals localize to highly conserved residues of this WD-40-repeat-containing protein. Given that WDR26 mutations have been identified in ∼1 in 2,000 of subjects in our clinical cohorts and that WDR26 might be poorly annotated in exome variant-interpretation pipelines, we would anticipate that this disorder could be more common than currently appreciated.

Punctate white matter lesions in full-term infants with neonatal seizures associated with SLC13A5 mutations.

INTRODUCTION: Early-onset epileptic encephalopathy caused by biallelic SLC13A5 mutations is characterized by seizure onset in the first days of life, refractory epilepsy and developmental delay. Little detailed information about the brain MRI features is available in these patients. METHODS: Observational study describing the neuro-imaging findings in eight patients (five families) with mutations in the SLC13A5 gene. Seven infants had an MRI in the neonatal period, two had a follow-up MRI at the age of 6 and 18 months and one only at 13 months. One patient had follow-up MRIs at 11 and 16 months and 3 and 6 years of age, but no neonatal MRI. RESULTS: All patients presented with refractory neonatal seizures on the first day of life after an uncomplicated pregnancy and term delivery. Six out of seven infants with a neonatal MRI had a characteristic MRI pattern, with punctate white matter lesions (PWML), which were no longer visible at the age of 6 months, but led to gliotic scarring visible on MRI at the age of 18 months. The same pattern of gliotic scarring was seen on the MRIs of the infant without a neonatal scan. One infant had signal abnormalities in the white matter suspected of PWML on T2WI, but these could not be confirmed on other sequences. CONCLUSION: In infants presenting with therapy resistant seizures in the first days after birth, without a clear history of hypoxic-ischemic encephalopathy, but with PWML on their neonatal MRI, a diagnosis of SCL13A5 related epileptic encephalopathy should be considered.

B56δ-related protein phosphatase 2A dysfunction identified in patients with intellectual disability.

Here we report inherited dysregulation of protein phosphatase activity as a cause of intellectual disability (ID). De novo missense mutations in 2 subunits of serine/threonine (Ser/Thr) protein phosphatase 2A (PP2A) were identified in 16 individuals with mild to severe ID, long-lasting hypotonia, epileptic susceptibility, frontal bossing, mild hypertelorism, and downslanting palpebral fissures. PP2A comprises catalytic (C), scaffolding (A), and regulatory (B) subunits that determine subcellular anchoring, substrate specificity, and physiological function. Ten patients had mutations within a highly conserved acidic loop of the PPP2R5D-encoded B56δ regulatory subunit, with the same E198K mutation present in 6 individuals. Five patients had mutations in the PPP2R1A-encoded scaffolding Aα subunit, with the same R182W mutation in 3 individuals. Some Aα cases presented with large ventricles, causing macrocephaly and hydrocephalus suspicion, and all cases exhibited partial or complete corpus callosum agenesis. Functional evaluation revealed that mutant A and B subunits were stable and uncoupled from phosphatase activity. Mutant B56δ was A and C binding-deficient, while mutant Aα subunits bound B56δ well but were unable to bind C or bound a catalytically impaired C, suggesting a dominant-negative effect where mutant subunits hinder dephosphorylation of B56δ-anchored substrates. Moreover, mutant subunit overexpression resulted in hyperphosphorylation of GSK3ß, a B56δ-regulated substrate. This effect was in line with clinical observations, supporting a correlation between the ID degree and biochemical disturbance.

Brief report: Autologous stem cell transplantation restores immune tolerance in experimental arthritis by renewal and modulation of the Teff cell compartment.

OBJECTIVE: Autologous stem cell transplantation (ASCT) induces long-term drug-free disease remission in patients with juvenile idiopathic arthritis. This study was undertaken to further unravel the immunologic mechanisms underlying ASCT by using a mouse model of proteoglycan-induced arthritis (PGIA). METHODS: For initiation of PGIA, BALB/c mice received 2 intraperitoneal injections of human PG in a synthetic adjuvant on days 0 and 21. Five weeks after the first immunization, the mice were exposed to total body irradiation (7.5 Gy) and received (un)manipulated bone marrow (BM) grafts from mice with PGIA. Clinical scores, T cell reconstitution, (antigen-specific) T cell cytokine production, and intracellular cytokine expression were determined following autologous BM transplantation (ABMT). RESULTS: ABMT resulted in amelioration and stabilization of arthritis scores. BM grafts containing T cells and T cell-depleted grafts provided the same clinical benefit, with similar reductions in PG-induced T cell proliferation and the number of PG-specific autoantibodies. In vivo reexposure to PG did not exacerbate disease. Following ABMT, basal levels of disease-associated proinflammatory cytokines (interferon-γ [IFNγ], interleukin-17 [IL-17], and tumor necrosis factor α [TNFα]) were reduced. In addition, restimulation of T cells with PG induced a strong reduction in disease-associated proinflammatory cytokine production. Finally, although the remaining host T cells displayed a proinflammatory phenotype following ABMT, IFNγ, IL-17, and TNFα production by the newly reconstituted donor-derived T cells was significantly lower. CONCLUSION: Taken together, our data suggest that ABMT restores immune tolerance by renewal and modulation of the Teff cell compartment, leading to a strong reduction in proinflammatory (self antigen-specific) T cell cytokine production.

TLR9 agonist CpG enhances protective nasal HSP60 peptide vaccine efficacy in experimental autoimmune arthritis.

OBJECTIVES: Peptide-based immune tolerance induction is considered an attractive treatment option for autoimmune diseases. The authors have developed a novel method that can enhance the induction of protective peptide-specific T-cell responses, using a rat arthritis model. The authors focused on the Toll-like receptor 9 ligand CpG, which was shown to stimulate regulatory T-cell proliferation when added to plasmacytoid dendritic cells (pDC) using in-vitro cultures. METHODS: The peptide used is a heat shock protein 60 epitope (p1) that elicits tolerogenic peptide-specific immune responses in human arthritis patients and was recently shown to have protective capacity as a bystander antigen in the rat adjuvant arthritis model. Rats were treated with three nasal doses of p1, CpG or a combination of p1 and CpG. Antigen-presenting cells were studied in nose-draining lymph nodes (mandibular lymph nodes; MLN) after nasal treatment, and T-cell responses were analysed in joint-draining lymph nodes after arthritis induction. RESULTS: Nasal co-administration of p1/CpG significantly augmented the arthritis-protective effect of p1, while CpG treatment alone did not. Co-treatment of p1/CpG increased both the number and activation status of pDC in draining MLN, which was accompanied by amplified p1-specific T-cell proliferation and interleukin (IL)-10 production. During early arthritis, p1-specific IL-10 production was identified at the site of inflammation. P1 and p1/CpG-treated rats showed a greater amount of CD4+FoxP3+ regulatory T cells in the joint-draining lymph nodes, which correlated with lower arthritis scores. CONCLUSIONS: These clinical and immunological data suggest the use of CpG as a potent adjuvant for mucosal peptide-specific immune therapy in arthritis.