Analysis of genetically defined immunodeficient patients allows study of the effect of the absence of specific proteins on human immune function in real‐world conditions. Here we have addressed the ...importance of type I interferon signalling for human NK cell development by studying the phenotype and function of circulating NK cells isolated from patients suffering primary immunodeficiency disease due to mutation of either the human interferon regulatory factor 9 (IRF9) or the signal transducer and activator of transcription 2 (STAT2) genes. IRF9, together with phosphorylated STAT1 and STAT2, form a heterotrimer called interferon stimulated gene factor 3 (ISGF3) which promotes the expression of hundreds of IFN‐stimulated genes that mediate antiviral function triggered by exposure to type I interferons. IRF9‐ and STAT2‐deficient patients are unable to respond efficiently to stimulation by type I interferons and so our experiments provide insights into the importance of type I interferon signalling and the consequences of its impairment on human NK cell biology. Surprisingly, the NK cells of these patients display essentially normal phenotype and function.
Mutations in IRF9 and STAT2 that render an individual unable to respond to Type I interferons have no discernible effect on human natural killer cell development and function.
Humoral immunocompetence develops stepwise throughout life and contributes to individual susceptibility to infection, immunodeficiency, autoimmunity, and neoplasia. Immunoglobulin heavy chain (IgH) ...isotype serum levels can partly explain such age-related differences, but their relationship with the IgH isotype distribution within memory B-cell (MBC) and plasma cell (PCs) compartments remains to be investigated.
We studied the age-related distribution of MBCs and PCs expressing different IgH isotypes in addition to the immature/transitional and naive B-cell compartments.
B-cell and PC subsets and plasma IgH isotype levels were studied in cord blood (n = 19) and peripheral blood (n = 215) from healthy donors aged 0 to 90 years by using flow cytometry and nephelometry, respectively.
IgH-switched MBCs expressing IgG1, IgG2, IgG3, IgA1, and IgA2 were already detected in cord blood and newborns at very low counts, whereas CD27+IgM++IgD+ MBCs only became detectable at 1 to 5 months and remained stable until 2 to 4 years, and IgD MBCs peaked at 2 to 4 years, with both populations decreasing thereafter. MBCs expressing IgH isotypes of the second immunoglobulin heavy chain constant region (IGHC) gene block (IgG1, IgG3, and IgA1) peaked later during childhood (2-4 years), whereas MBCs expressing third IGHC gene block immunoglobulin isotypes (IgG2, IgG4, and IgA2) reached maximum values during adulthood. PCs were already detected in newborns, increasing in number until 6 to 11 months for IgM, IgG1, IgG2, IgG3, IgA1, and IgA2; until 2 to 4 years for IgD; and until 5 to 9 years for IgG4 and decreasing thereafter. For most IgH isotypes (except IgD and IgG4), maximum plasma levels were reached after PC and MBC counts peaked.
PC counts reach maximum values early in life, followed by MBC counts and plasma IgH isotypes. Importantly, IgH isotypes from different IGHC gene blocks show different patterns, probably reflecting consecutive cycles of IgH isotype switch recombination through life.
Display omitted
Chimeric antigen receptor (CAR) T‐cell therapy is a breakthrough in hematologic malignancies, such as acute B lymphoblastic leukemia (B‐ALL). Monitoring this treatment is recommended, although ...standardized protocols have not been developed yet. This work compares two flow cytometry monitoring strategies and correlates this technique with qPCR method. CAR‐T cells were detected by two different flow‐cytometry protocols (A and B) in nine blood samples from one healthy donor and five B‐ALL patients treated with Tisagenlecleucel (Kymriah®, USA). HIV‐1 viral load allowed CAR detection by qPCR, using samples from seven healthy donors and nine B‐ALL patients. CAR detection by protocol A and B did not yield statistically significant differences (1.9% vs. 11.8% CD3 + CAR+, p = 0.07). However, protocol B showed a better discrimination of the CD3 + CAR+ population. A strong correlation was observed between protocol B and qPCR (r = 0.7, p < 0.0001). CD3 + CAR+ cells were detected by flow cytometry only when HIV‐1 viral load was above 104 copies/mL. In conclusion, protocol B was the most specific flow‐cytometry procedure for the identification of CAR‐T cells and showed a high correlation with qPCR. Further efforts are needed to achieve a standardized monitoring approach.
There is considerable variation in vaccination practices between pediatric transplant centers. This study aims to evaluate active immunization attitudes and practices among ERN-TransplantChild ...centers and identify potential areas of improvement that could be addressed by shared evidence-based protocols.
A cross-sectional questionnaire of attitudes and practices toward immunization of pediatric SOT and HSCT candidates and recipients was sent to a representative member of multidisciplinary teams from 27 European centers belonging to the ERN-TransplantChild.
A total of 28/62 SOT programs and 6/12 HSCT programs across 21 European centers participated. A quarter of centers did not have an on-site protocol for the immunizations. At the time of transplantation, pediatric candidates were fully immunized (80%-100%) in 57% and 33% of the SOT and HSCT programs. Variations in the time between vaccine administration and admission to the waiting list were reported between the centers, with 2 weeks for inactivated vaccines and variable time (2-4 weeks) for live-attenuated vaccines (LAVs). Almost all sites recommended immunization in the post-transplant period, with a time window of 4-8 months for the inactivated vaccines and 16-24 months for MMR and Varicella vaccines. Only five sites administer LAVs after transplantation, with seroconversion evaluated in 80% of cases.
The immunization coverage of European pediatric transplant recipients is still inconsistent and far from adequate. This survey is a starting point for developing shared evidence-based immunization protocols for safe vaccination among pediatric transplant centers and generating new research studies.
“An International Meeting on Wolf‐Hirschhorn Syndrome (WHS)” was held at The University Hospital La Paz in Madrid, Spain (October 13–14, 2017). One hundred and twenty‐five people, including ...physicians, scientists and affected families, attended the meeting. Parent and patient advocates from the Spanish Association of WHS opened the meeting with a panel discussion to set the stage regarding their hopes and expectations for therapeutic advances. In keeping with the theme on therapeutic development, the sessions followed a progression from description of the phenotype and definition of therapeutic endpoints, to definition of genomic changes. These proceedings will review the major points of discussion.
Background
Genetic diagnosis of inborn errors of immunity (IEI) is complex due to the large number of genes involved and their molecular features. Missense variants have been reported as the most ...common cause of IEI. However, the frequency of copy number variants (CNVs) may be underestimated since their detection requires specific quantitative techniques. At this point, the use of Next Generation Sequencing (NGS) is acquiring relevance.
Methods
In this article, we present our experience in the genetic diagnosis of IEI based on three diagnostic algorithms that allowed the detection of single nucleotide variants (SNVs) and CNVs. Following this approximation, 703 index cases were evaluated between 2014 and 2021. Sanger sequencing, MLPA, CGH array, breakpoint spanning PCR or a customized NGS‐based multigene‐targeted panel were performed.
Results
A genetic diagnosis was reached in 142 of the 703 index cases (20%), 19 of them presented deletions as causal variants. Deletions were also detected in 5 affected relatives and 16 healthy carriers during the family studies. Additionally, we compile, characterize and present all the CNVs detected by our diagnostic algorithms, representing the largest cohort of deletions related to IEI to date. Furthermore, three bioinformatic tools (LACONv, XHMM, VarSeq™) based on NGS data were evaluated. VarSeq™ was the most sensitive and specific bioinformatic tool; detecting 21/23 (91%) deletions located in captured regions.
Conclusion
Based on our results, we propose a strategy to guide the molecular diagnosis that can be followed by expert and non‐expert centres in the field of IEI.
The diagnosis of IEI is complex due to the clinical overlap, the presence of pseudogenes and the difficult detection of CNVs that requires quantitative techniques. In our centre, 703 patients with an IEI suspicion were evaluated: 142 were diagnosed, of whom 19 had a causative CNV. We characterized these CNVs and compared the sensitivity and specificity of MLPA, CHG array and NGS for the detection of CNVS. Finally, we propose a genetic algorithm for the evaluation of IEI suspicions.
Background Common variable immunodeficiency disorders (CVIDs) are the most common forms of symptomatic primary antibody failure in adults and children. Replacement immunoglobulin is the standard ...treatment, although there are few consistent data on optimal dosages and target trough IgG levels required for infection prevention. Objective To provide data to support the hypothesis that each patient requires an individual dose of therapeutic immunoglobulin to prevent breakthrough infections and that efficacious trough IgG levels vary between patients. Methods Data, collected prospectively from a cohort of 90 patients with confirmed CVIDs from 1 center over a follow-up period of 22 years, was validated and analyzed. Immunoglobulin doses had been adjusted in accordance with infections rather than to achieve a particular trough IgG level. Doses to achieve infection-free periods were determined and resultant trough levels analyzed. A smaller group of patients with X-linked agammaglobulinemia was analyzed for comparison. Results Patients with a CVID had a range of trough IgG levels that prevented breakthrough bacterial infections (5-17 g/L); viral and fungal infections were rare. Doses of replacement immunoglobulin to prevent breakthrough infections ranged from 0.2 to 1.2 g/kg/mo. Those with proven bronchiectasis or particular clinical phenotypes required higher replacement doses. Patients with X-linked agammaglobulinemia showed a similar range of IgG levels to stay infection-free (8-13 g/L). Conclusion These data offer guidance regarding optimal doses and target trough IgG levels in individual patients with CVIDs with or without bronchiectasis and for particular clinical phenotypes. The goal of replacement therapy should be to improve clinical outcome and not to reach a particular IgG trough level.
There is a need to optimise the management of atopic dermatitis (AD), improving the efficacy of treatments and reducing the toxicity associated with them. Although the efficacy of ciclosporine (CsA) ...in the treatment of AD has been thoroughly documented in the literature, the optimal dose has not been yet established. The use of multiomic predictive models of treatment response could optimise CsA therapy in AD.
The study is a low-intervention phase 4 trial to optimise the treatment of patients with moderate-severe AD requiring systemic treatment. The primary objectives are to identify biomarkers that could allow for the selection of responders and non-responders to first-line treatment with CsA and to develop a response prediction model to optimise the CsA dose and treatment regimen in responding patients based on these biomarkers. The study is divided into two cohorts: the first comprised of patients starting treatment with CsA (cohort 1), and the second, of patients already receiving or who have received CsA therapy (cohort 2).
The study activities began following authorisation by the Spanish Regulatory Agency (AEMPS) and the Clinical Research Ethics Committee of La Paz University Hospital approval. Trial results will be submitted for publication in an open access peer-reviewed medical speciality-specific publication.Trial registration of this study can be located at the EU Clinical Trials Register, available from https://euclinicaltrials.eu/search-for-clinical-trials/?lang=en. Our clinical trial was registered in the website before the enrolment of the first patient complying with European regulations. EU Clinical Trials Register is a primary registry according the WHO. Once our trial was included in a primary and official registry, in order to extend the accessibility to our research, we also registered it retrospectively in clinicaltrials.gov; however, this is not mandatory as per our regulation.
NCT05692843.
BACKGROUND: Epstein-Barr virus (EBV) infection is a well characterized etiopathogenic factor for a variety of immune-related conditions, including lymphomas, lymphoproliferative disorders and ...autoimmune diseases. EBV-mediated transformation of resting B cells to proliferating lymphoblastoid cells occurs in early stages of infection and is an excellent model for investigating the mechanisms associated with acquisition of unlimited growth. RESULTS: We investigated the effects of experimental EBV infection of B cells on DNA methylation profiles by using high-throughput analysis. Remarkably, we observed hypomethylation of around 250 genes, but no hypermethylation. Hypomethylation did not occur at repetitive sequences, consistent with the absence of genomic instability in lymphoproliferative cells. Changes in methylation only occurred after cell divisions started, without the participation of the active demethylation machinery, and were concomitant with acquisition by B cells of the ability to proliferate. Gene Ontology analysis, expression profiling, and high-throughput analysis of the presence of transcription factor binding motifs and occupancy revealed that most genes undergoing hypomethylation are active and display the presence of NF-κB p65 and other B cell-specific transcription factors. Promoter hypomethylation was associated with upregulation of genes relevant for the phenotype of proliferating lymphoblasts. Interestingly, pharmacologically induced demethylation increased the efficiency of transformation of resting B cells to lymphoblastoid cells, consistent with productive cooperation between hypomethylation and lymphocyte proliferation. CONCLUSIONS: Our data provide novel clues on the role of the B cell transcription program leading to DNA methylation changes, which we find to be key to the EBV-associated conversion of resting B cells to proliferating lymphoblasts.
PDF
