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Ataxia telangiectasia (AT) and Aicardi–Goutières syndrome (AGS) are inherited disorders of immunity with prevalent neurological phenotype. Available treatments are only partially effective, and the prognosis is poor. Induced pluripotent stem cells (iPSCs) are obtained by reprogramming patient somatic cells, preserving the donor individual genetic heritage and creating patient-specific disease models, useful to investigate pathogenesis and drug effects and to develop precision therapies. The aim is to investigate the cytotoxicity of a panel of immunomodulators using iPSCs of patients with AT or different forms of AGS (AGS1, AGS2, and AGS7). iPSCs were obtained by reprogramming AT and AGS patients’ cells and, as a control, the BJ normal human fibroblast line, using Sendai virus. Cytotoxic effects of two drugs proposed to treat respectively AT and AGS (dexamethasone and mepacrine) were tested by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay after 72 hours' exposure. Data were obtained also for other immunomodulatory drugs (thioguanine, mercaptopurine, thalidomide, and lenalidomide). Relative expression of genes involved in the tested drug pathways was analyzed. AGS7-derived iPSCs displayed altered viability when treated with a low dose of mepacrine and higher expression of cyclic guanosine monophosphate–adenosine monophosphate synthase, which is the main target for mepacrine action. AGS7-derived iPSCs were also more sensitive to thioguanine, while AGS2 and AT iPSCs were less sensitive to this medication than the BJ-iPSC. All iPSCs were equally sensitive to mercaptopurine and resistant to dexamethasone, thalidomide, and lenalidomide. This work establishes an innovative in vitro model that is useful to investigate the mechanisms of drugs potentially effective in AT and AGS.

C-X-C motif chemokine 13 (CXCL-13), which is expressed by synovial follicular dendritic cells and activated mature antigen-experienced T-helper cells, has been described as a surrogate marker of lymphoid phenotype of synovitis in rheumatoid arthritis (RA). A preferential response to anti-interleukin-6 receptor (IL-6R) as compared to anti-tumour necrosis factor alpha (TNFα) monotherapy has been described in patients with increased levels of CXCL-13. We hypothesised that serum levels of CXCL-13 could be used as a biomarker of response to treatment with abatacept (ABA), a T-cell co-stimulation blocker.Serum levels of CXCL-13 and of soluble intracellular adhesion molecule 1 (sICAM-1) (a putative marker of the myeloid subtype of synovitis) were measured by indirect solid-phase enzyme immunoassays, before (T0) and after 6 months of therapy with ABA (T6) in 63 patients with RA. Circulating T follicular helper cells and B cell subpopulations were identified by flow-cytometry.At T0, CXCL-13 serum levels were higher in RA patients than in healthy controls (p=0.0001) and correlated with disease activity, while no difference between the two groups was observed as far as sICAM-1 levels. Serum levels of CXCL-13 levels decreased after therapy with ABA both in patients who achieved a clinical response (p0.01) and in non-responders (p=0.01), whereas sICAM-1 levels did not significantly change. When comparing RA patients who responded to ABA with non-responders no significant difference of baseline serum levels of CXCL-13 was observed.CXCL-13 serum levels are raised in RA patients and decrease after therapy with ABA. We were not able to demonstrate that serum CXCL-13 levels predict the clinical response to ABA in RA patients.

Background/Purpose Anti-carbamylated protein antibodies (anti-CarP) were detected in a large cohort of patients with Systemic Lupus Erythematosus (SLE) in correlation with erosive arthritis but not with disease activity indexes. In animal models, T-cells may be activated by carbamylated epitopes playing a role in the development of arthritis. The imbalance between circulating regulatory (Treg) and CD28- effector T-cells was described in active SLE patients, explaining its involvement in disease’s pathogenesis. Actually, no data are available about the possible correlation with these T-cell subpopulations and anti-CarP levels in SLE. Methods Eight SLE patients with a median (10th-90th percentile) SLEDAI-2K=0 (0–4), anti-dsDNA levels=34.1 (15.6–427.4) UI/ml (nv Results Enrolled patients showed levels of anti-CarP=189.38 (93.5–341.1) AU/ml, Treg=2.2 (% of CD4+), CD4+CD28-=7.7 (4.8–22.5) (% of CD4+) and CD8+CD28-=30.3 (17.2–35.6) (% of CD8+). Analyzing possible correlations among different T-cell subtypes and anti-CarP levels, a significant inverse correlation was found between these autoantibodies and CD4+CD28- T cells (r=-0.8, p Conclusions In a small cohort of patients with serologically active SLE, anti-CarP autoantibodies were found as negatively correlated to circulating CD4+CD28- T-cells, which were described in association with disease damage, independently of age, gender, disease duration and activity. This suggest a potential role of anti-CarP as marker of SLE with a minor extent of T-cell activation and, consequently, with a possible better prognosis.

We investigated the molecular and cellular basis of severe combined immunodeficiency (SCID) in six patients with otofaciocervical syndrome type 2 who failed to attain T cell reconstitution after allogeneic hematopoietic stem cell transplantation, despite successful engraftment in three of them. We identified rare bi-allelic PAX1 rare variants in all patients. We demonstrated that these mutant PAX1 proteins have an altered conformation and flexibility of the paired box domain and reduced transcriptional activity. We generated patient-derived induced pluripotent stem cells and differentiated them into thymic epithelial progenitor cells, and found that they have an altered transcriptional profile, including for genes involved in the development of the thymus and other tissues derived from pharyngeal pouches. These results identify bi-allelic, loss-of-function PAX1 mutations as the cause of a syndromic form of SCID due to altered thymus development.

Autosomal recessive osteopetrosis (ARO) is a rare inherited disorder leading to increased bone density with impairment in bone resorption. Among the genes responsible for ARO, the TCIRG1 gene, coding for the a3 subunit of the osteoclast proton pump, is mutated in more than 50% of the cases, increasing the importance of TCIRG1-iPSCs as disease model. We generated 3 iPSC clones derived from Peripheral Blood Mononuclear Cells (PBMCs) of a patient carrying the heterozygous mutations p.Y512X and c.2236 + 1G > A. A Sendai virus-based vector was used and the iPSCs were characterized for genetic identity to parental cells, genomic integrity, pluripotency, and differentiation ability.

Using a Sendai Virus based vector delivering Yamanaka Factors, we generated induced Pluripotent Stem Cells (iPSCs) from peripheral blood mononuclear cells of a patient affected by Ataxia Telangiectasia (AT), caused by a novel homozygous deletion in ATM, spanning exons 5–7. Three clones were fully characterized for pluripotency and capability to differentiate. These clones preserved the causative mutation of parental cells and genomic stability over time (>100 passages). Furthermore, in AT derived iPSCs we confirmed the impaired DNA damage response after ionizing radiation. All these data underline potential usefulness of our clones as in vitro AT disease model.

Aicardi-Goutières syndrome (AGS) is an early-onset monogenic encephalopathy characterized by intracranial calcification, leukodystrophy and cerebrospinal fluid lymphocytosis. To date, seven genes have been related to AGS. Among these, IFIH1 encodes for MDA5, a cytosolic double-stranded RNA receptor, and is responsible for AGS type 7. We generated three isogenic iPSC clones, using a Sendai virus-based vector, starting from fibroblasts of a patient carrying a dominant mutation in IFIH1. All lines were characterized for genomic integrity, genetic uniqueness, pluripotency, and differentiation capability. Our clones might offer a good model to investigate AGS7 pathophysiological mechanism and to discover new biomarkers for this condition treatment.

The Cri du Chat Syndrome (CdCS) is a genetic disease resulting from variable size deletion occurring on the short arm of chromosome 5. The main clinical features are a high-pitched monochromatic cry, microcephaly, severe psychomotor and mental retardation with characteristics of autism spectrum disorders such as hand flapping, obsessive attachments to objects, twirling objects, repetitive movements, and rocking. We reprogrammed to pluripotency peripheral blood mononuclear cells derived from a patient carrying large deletion on the short arm of chromosome 5, using a commercially available non-integrating expression system. The iPSCs expressed pluripotency markers and differentiated in the three embryonic germ layers.

Background:T-cells play a pivotal role in the pathogenesis of rheumatoid arthritis (RA) and in its cardiovascular (CV) comorbidities, acting at microvascular level [1]. Since small artery remodeling is the earliest form of target organ damage in hypertension, the evaluation of microvascular alterations might provide clinically useful information. The evaluation of retinal arterioles is a non-invasive technique to identify a precocious microvascular damage, which is related to an increase of the wall-to-lumen ratio (WLR) [2]. CD3+CD31+CXCR4+ T-cells may be involved in damaged endothelium repair and are increased in patients with morphological microvascular alterations [3]. In addition to its effect on disease activity, abatacept (ABA), a co-stimulator blocker which is approved for the treatment of RA, may have specific CV protective action, modulating the numbers of certain subtypes of lymphocytes [4].Objectives:To non-invasively investigate morphological characteristics of retinal arterioles and to evaluate CD3+CD31+CXCR4+T-cells in a cohort of RA patients treated with ABA.Methods:Eleven RA patients [median (25th-75thpercentile) age=58 (50-65) years, baseline C-reactive protein (CRP)-DAS28=4.4 (3.8-4.6), body mass index (BMI)=23.4 (21.6-25.6) kg/m2, rheumatoid factor (RF) positive:45%, anti-citrullinated peptide autoantibodies (ACPA) positive:73%] without known CV risk factors (arterial hypertension, diabetes, hypercholesterolemia, previous CV events, smoking) were evaluated by adaptive optics, a validated technique quantifying microvascular damage [5], before and every 6 months of therapy with ABA (T0, T6 and T12). Phenotypic analysis of peripheral blood T lymphocytes was made by flow-cytometry in 5 patients of the cohort at T0 and T6.Results:A progressive significant reduction of the WLR of retinal arterioles was observed [T0=0.28 (0.25-0.30), T6=0.27 (0.24-0.31), T12=0.23 (0.23-0.26); p T0 vs T6=0.4; p T6 vs T12=0.01; p T0 vs T12=0.01] (Figure 1), without significant variations in the other parameters [internal diameter: T0=94.4 (84.1-104.0), T6=94.8 (84.6-107.7), T12=99.2 (89.1-109.1) µm; external diameter: T0=125.8 (111.1-131.0), T6=122.4 (109.1-134.5), T12=125.6 (113.9-134.4) µm; wall thickness: T0=13.2 (12.2-14.4), T6=13.4 (11.7-14.4), T12=12.5 (11.6-13.0) µm; wall cross-sectional area: T0=4581.0 (3788.7-5263.7), T6=4563.3 (3788.5-5295.2), T12=4099.7 (3899.0-5145.7) µm2)]. In 5 patients evaluated also for T-cell immunophenotyping a negative correlation was observed between CD3+CD31+CXCR4+ T-cell number and the retinal wall thickness at baseline (R=0.871;p=0.05). After ABA therapy a trend for reduction of CD3+CD31+CXCR4+T-cells [19.0 (13.8-38.3) vs 12.4 (5.2-18.0) % of CD3+], was observed as well as of significant reduction of retinal wall cross-sectional area [5123.3 (4385.0-5470.3) vs 4852.3 (4118.3-5228.0) µm2;p=0.04].Conclusion:In a cohort of RA patients without known CV risk factors, a reduction in retinal microvascular alterations arterioles was demonstrated after treatment for 12 months with ABA. CD3+CD31+CXCR4+T-cell number was inversely related to the possible presence of subclinical CV involvement. These results may suggest the possibility of microvascular abnormalities regression induced by the immune system modulation.References:[1]Dessein PH, J Rheumatol 2005.[2]Rizzoni D, Am J Hypertens. 2018.[3]Hur J, Circulation 2007.[4]Kallikourdis M, Nat Commun 2017.[5]De Ciuceis C, J Hypertens 2018.Acknowledgements:Bristol-Myers-Squibb Italy provided an unrestricted research grant for the study conduct.Figure 1.Disclosure of Interests:None declared

Background:Soluble intercellular adhesion molecule 1 (ICAM1) and C-X-C motif chemokine 13 (CXCL13) were described as differentially associated with two major subtypes of synovitis in rheumatoid arthritis (RA). Raised serum levels of ICAM1 (which is upregulated in synovial fibroblasts in response to TNFα), and of CXCL13 (which is expressed by synovial follicular dendritic cells and activated mature antigen-experienced T-helper cells), are associated with a myeloid or lymphoid synovial phenotype, respectively (1). It has been suggested that a preferential clinical response to anti-TNFα, as compared to anti-IL-6R monotherapy, can be predicted by measuring these two biomarkers (2). No information is available on the possible utility of these biomarkers in RA patients treated with abatacept (ABA), a T-cell co-stimulation blocker.Objectives:To analyze the effect of ABA on ICAM1 and CXCL13 serum levels in RA and to verify whether they predict the response to the drug.Methods:63 RA patients [F/M=51/12; median (10th-90thpercentile) age=60 (41-72) years; CRP-DAS28=4.6 (3.3-5.8); ACPA positive: 86%], before and after 6 months of treatment with ABA + methotrexate and 22 sex and age-matched healthy controls (HC) were evaluated. Serum ICAM1 and CXCL13 levels were dosed by commercial ELISA (Life Technologies and R&D). Response to treatment was defined with the EULAR criteria.Results:CXCL13 serum levels were higher in RA at baseline than in HC [136 (42-325) vs 32 (19-57) pg/ml, pConclusion:Our results confirmed that CXCL13 serum levels are directly correlated with disease activity and demonstrated that ABA therapy induces their reduction. These findings suggest that the co-stimulation blockade at central level and/or in the synovium lead to a reduced production of CXCL13. We could not demonstrate that CXCL13 levels predict the clinical response to ABA in this cohort of patients.References:[1]Rosengren S, Rheumatology 2011;2.Dennis G, Arthritis Res Ther 2014Acknowledgments:Bristol-Myers-Squibb Italy provided an unrestricted research grant for the study conduction.Disclosure of Interests:None declared

We report the generation of three isogenic iPSC clones (UNIBSi007-A, UNIBSi007-B, and UNIBSi007-C) obtained from fibroblasts of a patient with Aicardi Goutieres Syndrome (AGS) carrying a homozygous mutation in RNaseH2B. Cells were transduced using a Sendai virus based system, delivering the human OCT4, SOX2, c-MYC and KLF4 transcription factors. The resulting transgene-free iPSC lines retained the disease-causing DNA mutation, showed normal karyotype, expressed pluripotent markers and could differentiate in vitro toward cells of the three embryonic germ layers.

Fibroblasts from a patient with Aicardi Goutières Syndrome (AGS) carrying a compound heterozygous mutation in TREX1, were reprogrammed into induced pluripotent stem cells (iPSCs) to establish isogenic clonal stem cell lines: UNIBSi006-A, UNIBSi006-B, and UNIBSi006-C. Cells were transduced using the episomal Sendai viral vectors, containing human OCT4, SOX2, c-MYC and KLF4 transcription factors. The transgene-free iPSC lines showed normal karyotype, expressed pluripotent markers and displayed in vitro differentiation potential toward cells of the three embryonic germ layers.

Understanding the biological and molecular processes underlying human pathologies is fundamental in order to develop innovative approaches to treat or prevent them. Among the technologies that could provide innovative disease models, induced pluripotent stem cells (iPSCs) is one of the most promising. Indeed, one application of this technology is patient-specific disease modeling. iPSCs obtained by reprogramming patients’ cells collected from accessible tissues, have the unique capability to differentiate, under an adequate stimulus, into any human cell type. In particular, iPSCs technology can be applied to study drug adverse effects, that is a key part of the drug discovery process. Indeed, drug induced adverse effects are among the most common causes that lead to abandon the development of new candidate therapeutic molecules, increasing the cost of drug discovery. An innovative strategy that could be used in drug design to solve drug attrition rate, and to establish innovative pharmacological models, could be the application of iPSCs technology in the early stage of the drug discovery process to model druginduced adverse events. In this review, recently developed disease models based on iPSCs will be discussed, with a particular focus on available models of drugs’ adverse effect, in particular hepatic/pancreatic toxicity.

Crohn's disease is a debilitating and incurable chronic inflammatory bowel disease, affecting millions of individuals worldwide, with an increasing frequency. Surgery must be applicable in half of the cases often with a disabling course, and pharmacological treatments may have adverse complications. We generated three isogenic clones of iPSCs from peripheral blood mononuclear cells (PBMCs) of a patient with Crohn's Disease under pharmacological treatment without adverse effects. Sendai virus based vector was used and the iPSCs were characterized for genetic uniqueness, genomic integrity, pluripotency, and differentiation ability. These iPSCs will be a powerful tool to develop tailored therapies.

Introduction In AML and MDS cases, the genetic lesions inherited or acquired by the hematopoietic stem cells are considered as starting events. Familial AML and MDS, recently recognized in the revised WHO classification (2016) provide a useful model for investigation of predisposing genetic mutations. Genetic analysis of several pure familial leukemia pedigrees led to the discovery of well defined syndromes associated with inherited de novo mutations on germline DNA. Growing clinical awareness as well as a widespread use of NGS have led to an enlarged description of familial MDS/AML cases, and the number of mutations involved, suggesting they are more frequent than those previously recognized. Despite the recent discovery of well-established causative gene mutations (RUNX1, GATA2, ETV6, TERT, TERC, SRP72, ANKRD26, DDX41, CEBPA), many cases remain unexplained (about 80%), suggesting that other inherited mutations could predispose to MDS/AML. It is expected that new sequencing approaches will help to the identification of more cases, more genes as well as novel syndromes. In 2017, we started a multicentric prospective study (Clinical trial.gov NCT03058588) aiming to look for predisposing mutations in patients and relatives affected by Familial AML and MDS syndromes (FAMS) by NGS and to screen for old and new mutations potentially associated with the disease. Methods At present, 12 AML/MDS patients have been enrolled. Leukemic (bone marrow) and germline (buccal swab) DNA were analyzed by NGS gene panel approach based on a 28 genes associated to myeloid leukemogenesis, including the 9 above mentioned genes associated to FAMS. NGS libraries were performed by a Nimblegen (Roche) custom panel based on gene capture strategy and the sequencing was performed by MiSeq (Illumina). Results Ten patients did not reveal any germline mutations and the candidates are undergoing to whole exome sequencing. One presented a germline mutation on RUNX1, and the analysis of the affected relatives is on going. One revealed a new mutation. She was a 70 years old woman affected by RARS and her pedigree was characterized by 9 relatives affected by hematologic and solid neoplasia and trombocytopenia (fig 1). The NGS analysis revealed the mutation c.*514C>T in 3'UTR of ETV6 with VAF of 50% on tumor DNA. The variant has never been described before, while ETV6 has been already associated with FAMS. Sanger sequencing confirmed the mutation on the germline DNA in heterozygosis. The screening of 2 affected relatives still alive confirmed the presence of the variant in heterozygosis. In silico analysis performed on PolymiRST Database revealed that c.*514C>T in 3'UTR of ETV6 results in a gain of miRNA binding site: hsa-miR- 4717-3p and hsa-miR- 942-3p. Discussion The variant c.*514C>T in 3'UTR of ETV6 seems to repress ETV6 due to RNA interference. The new binding miRNAs have been already described as over-expressed in solid and hematologic tumors. Moreover, the down-regulation of ETV6 is associated with alteration of cell growth and hematopoiesis. Due to these evidences, c.*514C>T in 3'UTR of ETV6 could be considered as a new mutation involved in FAMS predisposition. Disclosures No relevant conflicts of interest to declare.

B cell developmental defects in CVID were recently described in a limited number of cases. To date, a detailed correlation between this maturational defect and the clinical presentation of affected patients has not been reported. In this study, we correlated bone marrow B cell evaluation, peripheral B and T lymphocyte subsets and clinical findings in 15 CVID patients. Early B cell developmental defects were observed in one third of patients. Combined bone marrow and peripheral lymphocytes evaluation allowed to further subdivide CVID patients in three groups with shared clinical features at diagnosis and during follow-up. These data broaden the number of CVID patients with early B cell developmental defects and, together with the peripheral lymphocytes evaluation, offer insight into the related clinical features in affected patients.

Background Mutations in Janus kinase 3 (JAK3) are a cause of severe combined immunodeficiency, but hypomorphic JAK3 defects can result in a milder clinical phenotype, with residual development and function of autologous T cells. Maternal T-cell engraftment is a common finding in infants with severe combined immunodeficiency but is not typically observed in patients with residual T-cell development. Objective We sought to study in detail the molecular, cellular, and humoral immune phenotype and function of 3 patients with hypomorphic JAK3 mutations. Methods We analyzed the distribution and function of T and B lymphocytes in 3 patients and studied the in vitro and in vivo responses of maternal T lymphocytes in 1 patient with maternal T-cell engraftment and residual production of autologous T lymphocytes. Results B cells were present in normal numbers but with abnormal distribution of marginal zone–like and memory B cells. B-cell differentiation to plasmablasts in vitro in response to CD40 ligand and IL-21 was abolished. In 2 patients the T-cell repertoire was moderately restricted. Surprisingly, 1 patient showed coexistence of maternal and autologous T lymphocytes. By using an mAb recognizing the maternal noninherited HLA-A2 antigen, we found that autologous cells progressively accumulated in vivo but did not compete with maternal cells in vitro . Conclusion The study of 3 patients with hypomorphic JAK3 mutations suggests that terminal B-cell maturation/differentiation requires intact JAK3 function, even if partially functioning T lymphocytes are present. Maternal T-cell engraftment can occur in patients with JAK3 mutations despite the presence of autologous T cells.

A homozygous mutation that gave rise to a stop codon in the WIPF1 gene resulted in WASP protein destabilization and in symptoms resembling those of Wiskott-Aldrich syndrome, A female offspring of consanguineous parents, showed features of Wiskott-Aldrich syndrome (WAS), including recurrent infections, eczema, thrombocytopenia, defective T cell proliferation and chemotaxis, and impaired natural killer cell function. Cells from this patient had undetectable WAS protein (WASP), but normal WAS sequence and messenger RNA levels. WASP interacting protein (WIP), which stabilizes WASP, was also undetectable. A homozygous c.1301C>G stop codon mutation was found in the WIPF1 gene, which encodes WIP. Introduction of WIP into the patient’s T cells restored WASP expression. These findings indicate that WIP deficiency should be suspected in patients with features of WAS in whom WAS sequence and mRNA levels are normal.

COVID-19 is a new pandemic, caused by Severe Acute Respiratory Syndrome-CoronaVirus-2 (SARS-Cov2) infection and characterized by a broad spectrum of clinical manifestations. Inflammation and the innate immune system have been recently recognized as pivotal players in the most severe forms, characterized by significantly elevated levels of pro-inflammatory cytokines. In this setting, several studies have also reported the presence of abnormalities in coagulation parameters and platelets count, possibly identifying a subgroup of patients with poor prognosis. Some reports of full-blown thromboembolic events are emerging. Among the possible mechanisms underlying coagulation dysfunction, the so-called "cytokine storm" seems to play a pivotal role. Other candidate factors include virus-specific mechanisms, related to the virus interaction with renin angiotensin system (RAS) and the fibrinolytic pathway, but also comorbidities affecting these patients. Coagulation dysfunction is therefore a candidate risk factor for adverse outcomes in COVID-19 and should be carefully addressed in clinical practice.