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Summary: Common variable immunodeficiency (CVID) is a primary immunodeficiency characterized by hypogammaglobulinemia and different degrees of B cell compartment alteration. Memory B cell differentiation requires the orchestrated activation of several intracellular signaling pathways that lead to the activation of a number of factors, such as nuclear factor kappa B (NF‐κB) which, in turn, promote transcriptional programs required for long‐term survival. The aim of this study was to determine if disrupted B cell differentiation, survival and activation in B cells in CVID patients could be related to defects in intracellular signaling pathways. For this purpose, we selected intracellular readouts that reflected the strength of homeostatic signaling pathways in resting cells, as the protein expression levels of the Bcl‐2 family which transcription is promoted by NF‐κB. We found reduced Bcl‐2 protein levels in memory B cells from CVID patients. We further explored the possible alteration of this crucial prosurvival signaling pathway in CVID patients by analysing the expression levels of mRNAs from anti‐apoptotic proteins in naive B cells, mimicking T cell‐dependent activation in vitro with CD40L and interleukin (IL)‐21. BCL‐XL mRNA levels were decreased, together with reduced levels of AICDA, after naive B‐cell activation in CVID patients. The data suggested a molecular mechanism for this tendency towards apoptosis in B cells from CVID patients. Lower Bcl‐2 protein levels in memory B cells could compromise their long‐term survival, and a possible less activity of NF‐κB in naive B cells, may condition an inabilityto increase BCL‐XL mRNA levels, thus not promoting survival in the germinal centers. [ABSTRACT FROM AUTHOR]

An increasing number of AB0-incompatible (AB0i) liver transplantations (LT) are being undertaken internationally in recent years due to organ shortages and the need for urgent transplantation. The aim of our study was establish the value of ABOi LT from available retrospective results of AB0i pediatric liver transplantations performed in European reference centers now belonging to the TransplantChild, European Reference Network (ERN). Data from medical records were analyzed, including demographic data, diagnosis, urgency of transplantation, time on the waiting list, PELD/MELD score, desensitization procedures, immunosuppression, selected post-transplant complications, and patient and graft survival. A total of 142 patients (pts) with transplants between 1986 and 2018 in 8 European transplant centers were included in the study. The indications for liver transplantation were: cholestatic diseases in 62 pts, acute liver failure in 42 pts, and other conditions in the remaining 38 pts. Sixty-six patients received grafts from living donors, and seventy-six received grafts from deceased donors. Both patient and graft survival were significantly affected by deceased donor type, urgent transplantation, and the development of vascular complications. In the multivariate analysis, vascular complications had a negative impact on patient and graft survival, while a longer time from the first AB0i LT in the study showed better results, suggesting an international learning experience. In conclusion, we believe that AB0i LT in children is now a safe procedure that may be adopted more readily in children.

(1) Background: Post-transplant lymphoproliferative disease (PTLD) is a significant complication of solid organ transplantation (SOT). However, there is lack of consensus in PTLD management. Our aim was to establish a present benchmark for comparison between international centers and between various organ transplant systems and modalities; (2) Methods: A cross-sectional questionnaire of relevant PTLD practices in pediatric transplantation was sent to multidisciplinary teams from 17 European center members of ERN TransplantChild to evaluate the centers' approach strategies for diagnosis and treatment and how current practices impact a cross-sectional series of PTLD cases; (3) Results: A total of 34 SOT programs from 13 European centers participated. The decision to start preemptive treatment and its guidance was based on both EBV viremia monitoring plus additional laboratory methods and clinical assessment (61%). Among treatment modalities the most common initial practice at diagnosis was to reduce the immunosuppression (61%). A total of 126 PTLD cases were reported during the period 2012-2016. According to their histopathological classification, monomorphic lesions were the most frequent (46%). Graft rejection after PTLD remission was 33%. Of the total cases diagnosed with PTLD, 88% survived; (4) Conclusions: There is still no consensus on prevention and treatment of PTLD, which implies the need to generate evidence. This might successively allow the development of clinical guidelines.

Common variable immunodeficiency (CVID) is a primary immunodeficiency characterized by hypogammaglobulinemia and different degrees of B cell compartment alteration. Memory B cell differentiation requires the orchestrated activation of several intracellular signaling pathways that lead to the activation of a number of factors, such as nuclear factor kappa B (NF-κB) which, in turn, promote transcriptional programs required for long-term survival. The aim of this study was to determine if disrupted B cell differentiation, survival and activation in B cells in CVID patients could be related to defects in intracellular signaling pathways. For this purpose, we selected intracellular readouts that reflected the strength of homeostatic signaling pathways in resting cells, as the protein expression levels of the Bcl-2 family which transcription is promoted by NF-κB. We found reduced Bcl-2 protein levels in memory B cells from CVID patients. We further explored the possible alteration of this crucial prosurvival signaling pathway in CVID patients by analysing the expression levels of mRNAs from anti-apoptotic proteins in naive B cells, mimicking T cell-dependent activation in vitro with CD40L and interleukin (IL)-21. BCL-XL mRNA levels were decreased, together with reduced levels of AICDA, after naive B-cell activation in CVID patients. The data suggested a molecular mechanism for this tendency towards apoptosis in B cells from CVID patients. Lower Bcl-2 protein levels in memory B cells could compromise their long-term survival, and a possible less activity of NF-κB in naive B cells, may condition an inabilityto increase BCL-XL mRNA levels, thus not promoting survival in the germinal centers., (© 2020 British Society for Immunology.)

Introduction: Common Variable Immunodeficiency (CVID) is characterized by defective antibody production and hypogammaglobulinemia. Flow cytometry immunophenotyping of blood lymphocytes has become of great relevance for the diagnosis and classification of CVID, due to an impaired differentiation of mature post-germinal-center (GC) class-switched memory B-cells (MBC) and severely decreased plasmablast/plasma cell (Pb) counts. Here, we investigated in detail the pre-GC B-cell maturation compartment in blood of CVID patients., Methods: In this collaborative multicentric study the EuroFlow PID 8-color Pre-GC B-cell tube , standardized sample preparation procedures (SOPs) and innovative data analysis tools, were used to characterize the maturation profile of pre-GC B-cells in 100 CVID patients, vs 62 age-matched healthy donors (HD)., Results: The Pre-GC B-cell tube allowed identification within pre-GC B-cells of three subsets of maturation associated immature B-cells and three subpopulations of mature naïve B-lymphocytes. CVID patients showed overall reduced median absolute counts (vs HD) of the two more advanced stages of maturation of both CD5 + CD38 +/++ CD21 het CD24 ++ (2.7 vs 5.6 cells/µl, p=0.0004) and CD5 + CD38 het CD21 + CD24 + (6.5 vs 17 cells/µl, p<0.0001) immature B cells (below normal HD levels in 22% and 37% of CVID patients). This was associated with an expansion of CD21 - CD24 - (6.1 vs 0.74 cells/µl, p<0.0001) and CD21 - CD24 ++ (1.8 vs 0.4 cells/µl, p<0.0001) naïve B-cell counts above normal values in 73% and 94% cases, respectively. Additionally, reduced IgMD + (21 vs 32 cells/µl, p=0.03) and IgMD - (4 vs 35 cells/µl, p<0.0001) MBC counts were found to be below normal values in 25% and 77% of CVID patients, respectively, always together with severely reduced/undetectable circulating blood pb. Comparison of the maturation pathway profile of pre-GC B cells in blood of CVID patients vs HD using EuroFlow software tools showed systematically altered patterns in CVID. These consisted of: i) a normally-appearing maturation pathway with altered levels of expression of >1 (CD38, CD5, CD19, CD21, CD24, and/or smIgM) phenotypic marker (57/88 patients; 65%) for a total of 3 distinct CVID patient profiles (group 1: 42/88 patients, 48%; group 2: 8/88, 9%; and group 3: 7/88, 8%) and ii) CVID patients with a clearly altered pre-GC B cell maturation pathway in blood (group 4: 31/88 cases, 35%)., Conclusion: Our results show that maturation of pre-GC B-cells in blood of CVID is systematically altered with up to four distinctly altered maturation profiles. Further studies, are necessary to better understand the impact of such alterations on the post-GC defects and the clinical heterogeneity of CVID., Competing Interests: JD, MB, TK, MP-A, EL-G, A-KK, EB, and AO each report being one of the inventors on the EuroFlow-owned patent PCT/NL 2015/050762 (Diagnosis of primary immunodeficiencies), which is licensed to Cytognos, a company that pays royalties to the EuroFlow Consortium., (Copyright © 2021 del Pino-Molina, López-Granados, Lecrevisse, Torres Canizales, Pérez-Andrés, Blanco, Wentink, Bonroy, Nechvatalova, Milota, Kienzler, Philippé, Sousa, van der Burg, Kalina, van Dongen and Orfao.)

Objective: Describe the GETH haploidentical stem cell transplantation (haplo-HSCT) activity in non-malignant disease (NMDs)., Methods: We retrospectively analyzed data from children with NMDs who underwent haplo-HSCT., Results: From January 2001 to December 2016, 26 pediatric patients underwent 31 haplo-HSCT through ex vivo T cell-depleted (TCD) graft platforms or post-transplantation cyclophosphamide (PT-Cy) at 7 Spanish centers. Five cases employed unmanipulated PT-Cy haplo-HSCT, 16 employed highly purified CD34 + cells, and 10 employed ex vivo TCD grafts manipulated either with CD3 + CD19 + depletion, TCRαβ + CD19 + selection or naive CD45RA + T-cell depletion. Peripheral blood stem cells were the sole source for patients following TCD haplo-HSCT, and bone marrow was the source for one PT-Cy haplo-HSCT. The most common indications for transplantation were primary immunodeficiency disorders (PIDs), severe aplastic anemia, osteopetrosis, and thalassemia. The 1-year cumulative incidence of graft failure was 27.4%. The 1-year III-IV acute graft-versus-host disease (GvHD) and 1-year chronic GvHD rates were 34.6% and 16.7%, respectively. The 2-year overall survival was 44.9% for PIDs, and the 2-year graft-versus-host disease-free and relapse-free survival rate was 37.6% for the other NMDs. The transplantation-related mortality at day 100 was 30.8%., Conclusion: Although these results are discouraging, improvements will come if procedures are centralized in centers of expertise., (© 2020 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

The current pandemic SARS-CoV-2 has required an unusual allocation of resources that can negatively impact chronically ill patients and high-complexity procedures. Across the European Reference Network on Pediatric Transplantation (ERN TransplantChild), we conducted a survey to investigate the impact of the COVID-19 outbreak on pediatric transplant activity and healthcare practices in both solid organ transplantation (SOT) and hematopoietic stem cell transplantation (HSCT). The replies of 30 professionals from 18 centers in Europe were collected. Twelve of 18 centers (67%) showed a reduction in their usual transplant activity. Additionally, outpatient visits have been modified and restricted to selected ones, and the use of telemedicine tools has increased. Additionally, a total of 14 COVID-19 pediatric transplanted patients were identified at the time of the survey, including eight transplant recipients and six candidates for transplantation. Only two moderate-severe cases were reported, both in HSCT setting. These survey results demonstrate the limitations in healthcare resources for pediatric transplantation patients during early stages of this pandemic. COVID-19 disease is a major worldwide challenge for the field of pediatric transplantation, where there will be a need for systematic data collection, encouraging regular discussions to address the long-term consequences for pediatric transplantation candidates, recipients, and their families., (© 2020 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

The clinical value of assessing immunoglobulin (Ig)G and IgA subclasses in addition to the isotypes of soluble Igs in serum has been well established. >20years ago, the International Union of Immunological Societies and the World Health Organization performed collaborative studies in order to validate antibody (Ab) clones for the detection of IgG and IgA subclasses for a broad range of laboratory assays, except for flow cytometry. Here we analyzed the performance of commercially available Ab clones to detect IgG and IgA subclasses in memory B-cells and plasma cells (PCs) by flow cytometry. In a first step, 28 Ab clones were evaluated in peripheral blood from healthy donors. Only 17/28 clones showed reactivity against IgG and IgA subclasses expressed on the B-cell and PC surface membrane, including Ab clones for IgG 1 (SAG1, HP6188, HP6001 and HP6186), IgG 2 (SAG2, HP6014 and HP6002), IgG 3 (SAG3, HP6095 and HP6050), IgG 4 (SAG4), IgA 1 (SAA1, H69-11.4 and B3506B4) and IgA 2 (SAA2, 2E2, and A9604D2). In a second step, for each Ig subclass a single clone was selected according to its specificity and fluorescence intensity (resolution power), for further more detailed validation (SAG1, SAG2, SAG3, SAG4, SAA1 and SAA2). This validation process was carried out in 4 different laboratories by testing the selected Ab clones in human peripheral blood, bone marrow and tonsil samples, using different staining protocols (e.g. surface membrane and/or cytoplasmic staining). All selected Ab clones displayed strong positivity, high specificity and optimal resolution between negative and positive cells. Alternative Ab clones were also validated. Thus, our results show the feasibility of using the validated Ig subclass Ab clones in combination with other B cell-associated markers for detailed dissection of the memory B-cell and PC compartments that express distinct Ig subclasses in different human tissues., (Copyright © 2017. Published by Elsevier B.V.)

Common Variable Immunodeficiency (CVID) is characterized by impaired antibody production and poor terminal differentiation of the B cell compartment, yet its pathogenesis is still poorly understood. We first reported the occurrence of epigenetic alterations in CVID by high-throughput methylation analysis in CVID-discordant monozygotic twins. Data from a recent whole DNA methylome analysis throughout different stages of normal B cell differentiation allowed us to design a new experimental approach. We selected CpG sites for analysis based on two criteria: one, CpGs with potential association with the transcriptional status of relevant genes for B cell activation and differentiation; and two, CpGs that undergo significant demethylation from naïve to memory B cells in healthy individuals. DNA methylation was analyzed by bisulfite pyrosequencing of specific CpG sites in sorted naïve and memory B cell subsets from CVID patients and healthy donors. We observed impaired demethylation in two thirds of the selected CpGs in CVID memory B cells, in genes that govern B cell-specific processes or participate in B cell signaling. The degree of demethylation impairment associated with the extent of the memory B cell reduction. The impaired demethylation in such functionally relevant genes as AICDA in switched memory B cells correlated with a lower proliferative rate. Our new results reinforce the hypothesis of altered demethylation during B cell differentiation as a contributing pathogenic mechanism to the impairment of B cell function and maturation in CVID. In particular, deregulated epigenetic control of AICDA could play a role in the defective establishment of a post-germinal center B cell compartment in CVID.

Background: 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., Objective: 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., Methods: 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., Results: IgH-switched MBCs expressing IgG 1 , IgG 2 , IgG 3 , IgA 1 , and IgA 2 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 (IgG 1 , IgG 3 , and IgA 1 ) peaked later during childhood (2-4 years), whereas MBCs expressing third IGHC gene block immunoglobulin isotypes (IgG 2 , IgG 4 , and IgA 2 ) reached maximum values during adulthood. PCs were already detected in newborns, increasing in number until 6 to 11 months for IgM, IgG 1 , IgG 2 , IgG 3 , IgA 1 , and IgA 2 ; until 2 to 4 years for IgD; and until 5 to 9 years for IgG 4 and decreasing thereafter. For most IgH isotypes (except IgD and IgG 4 ), maximum plasma levels were reached after PC and MBC counts peaked., Conclusions: 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., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)