In the setting of hematopoietic stem cell transplantation (HSCT), Rituximab (RTX) is used for the treatment and prevention of EBV-associated post-transplantation lymphoproliferative disease or... Show moreIn the setting of hematopoietic stem cell transplantation (HSCT), Rituximab (RTX) is used for the treatment and prevention of EBV-associated post-transplantation lymphoproliferative disease or autoimmune phenomena such as autoimmune hemolytic anemia (AIHA). Persistent hypogammaglobulinemia and immunoglobulin substitution dependence has been observed in several patients after RTX treatment despite the normalization of total B cell numbers. We aimed to study whether this is a B cell intrinsic phenomenon. We analyzed four patients with different primary diseases who were treated with myeloablative conditioning and matched unrelated donor HSCT who developed persistent hypogammaglobulinemia after receiving RTX treatment. They all received RTX early after HSCT to treat EBV infection or AIHA post-HSCT. All patients showed normalized total B cell numbers but absent to very low IgG positive memory B cells, and three lacked IgA positive memory B cells. All of the patients had full donor chimerism, and none had encountered graft-versus-host disease. Sorted peripheral blood naive B cells from these patients, when stimulated with CD40L, IL21, IL10 and anti-IgM, demonstrated intact B cell differentiation including the formation of class-switched memory B cells and IgA and IgG production. Peripheral blood T cell numbers including CD4 follicular T-helper (Tfh) cells were all within the normal reference range. In conclusion, in these four HSCT patients, the persistent hypogammaglobulinemia observed after RTX cannot be attributed to an acquired intrinsic B cell problem nor to a reduction in Tfh cell numbers. Show less
Kaiser, F.M.P.; Janowska, I.; Menafra, R.; Gier, M. de; Korzhenevich, J.; Pico-Knijnenburg, I.; ... ; Burg, M. van der 2023
Although absence of interleukin-7 (IL-7) signaling completely abrogates T and B lymphopoiesis in mice, patients with severe combined immunodeficiency caused by mutations in the IL-7 receptor alpha... Show moreAlthough absence of interleukin-7 (IL-7) signaling completely abrogates T and B lymphopoiesis in mice, patients with severe combined immunodeficiency caused by mutations in the IL-7 receptor alpha chain (IL-7R alpha) still generate peripheral blood B cells. Consequently, human B lymphopoiesis has been thought to be independent of IL-7 signaling. Using flow cytometric analysis and single-cell RNA sequencing of bone marrow samples from healthy controls and patients who are IL-7R alpha deficient, in combination with in vitro modeling of human B-cell differentiation, we demonstrate that IL-7R signaling plays a crucial role in human B lymphopoiesis. IL-7 drives proliferation and expansion of early B-cell progenitors but not of pre-BII large cells and has a limited role in the prevention of cell death. Furthermore, IL-7 guides cell fate decisions by enhancing the expression of BACH2, EBF1, and PAX5, which jointly orchestrate the specification and commitment of early B-cell progenitors. In line with this observation, early B-cell progenitors of patients with IL-7R alpha deficiency still expressed myeloid-specific genes. Collectively, our results unveil a previously unknown role for IL-7 signaling in promoting the B-lymphoid fate and expanding early human B-cell progenitors while defining important differences between mice and humans. Our results have implications for hematopoietic stem cell transplantation strategies in patients with T- B+ severe combined immunodeficiency and provide insights into the role of IL-7R signaling in leukemogenesis. Show less
Recombination activating genes (RAG)1 and RAG2 deficiency leads to combined T/B-cell deficiency with varying clinical presentations. This study aimed to define the clinical/laboratory spectrum of... Show moreRecombination activating genes (RAG)1 and RAG2 deficiency leads to combined T/B-cell deficiency with varying clinical presentations. This study aimed to define the clinical/laboratory spectrum of RAG1 and RAG2 deficiency. We retrospectively reviewed the clinical/laboratory data of 35 patients, grouped them as severe combined immunodeficiency (SCID), Omenn syndrome (OS), and delayed-onset combined immunodeficiency (CID) and reported nine novel mutations. The male/female ratio was 23/12. Median age of clinical manifestations was 1 months (mo) (0.5-2), 2 mo (1.25-5), and 14 mo (3.63-27), age at diagnosis was 4 mo (3-6), 4.5 mo (2.5-9.75), and 27 mo (14.5-70) in SCID (n = 25; 71.4%), OS (n = 5; 14.3%), and CID (n = 5; 14.3%) patients, respectively. Common clinical manifestations were recurrent sinopulmonary infections 82.9%, oral moniliasis 62.9%, diarrhea 51.4%, and eczema/dermatitis 42.9%. Autoimmune features were present in 31.4% of the patients; 80% were in CID patients. Lymphopenia was present in 92% of SCID, 80% of OS, and 80% of CID patients. All SCID and CID patients had low T (CD3, CD4, and CD8), low B, and increased NK cell numbers. Twenty-eight patients underwent hematopoietic stem cell transplantation (HSCT), whereas seven patients died before HSCT. Median age at HSCT was 7 mo (4-13.5). Survival differed in groups; maximum in SCID patients who had an HLA-matched family donor, minimum in OS. Totally 19 (54.3%) patients survived. Early molecular genetic studies will give both individualized therapy options, and a survival advantage because of timely diagnosis and treatment. Further improvement in therapeutic outcomes will be possible if clinicians gain time for HSCT.The recombination activating genes (RAG) are key players for T- and B-cell development and functions. Multifarious clinical presentations have been defined as patients with RAG deficiency. Early diagnosis, effective treatment, and early hematopoietic stem cell transplantation will increase the patients' chances of survival, especially for late-onset forms.Graphical Abstract Show less
Weitering, T.J.; Willemsen, M.A.A.P.; Taylor, A.M.R.; Weemaes, C.M.R.; Burg, M. van der; Berghuis, D. 2023
Early B cell development in the bone marrow ensures the replenishment of the peripheral B cell pool. Immature B cells continuously develop from hematopoietic stem cells, in a process guided by an... Show moreEarly B cell development in the bone marrow ensures the replenishment of the peripheral B cell pool. Immature B cells continuously develop from hematopoietic stem cells, in a process guided by an intricate network of transcription factors as well as chemokine and cytokine signals. Humans and mice possess somewhat similar regulatory mechanisms of B lymphopoiesis. The continuous discovery of monogenetic defects that impact early B cell development in humans substantiates the similarities and differences with B cell development in mice. These differences become relevant when targeted therapeutic approaches are used in patients; therefore, predicting potential immunological adverse events is crucial. In this review, we have provided a phenotypical classification of human and murine early progenitors and B cell stages, based on surface and intracellular protein expression. Further, we have critically compared the role of key transcription factors (Ikaros, E2A, EBF1, PAX5, and Aiolos) and chemo- or cytokine signals (FLT3, c-kit, IL-7R, and CXCR4) during homeostatic and aberrant B lymphopoiesis in both humans and mice. Show less
Vissers, L.; Burg, M. van der; Lankester, A.; Smiers, F.; Mohseny, A. 2023
Peripheral blood cytopenia, a frequent presenting symptom in pediatric patients, can be caused by bone marrow failure (BMF). Timely identification of patients with non-reversible BMF is of crucial... Show morePeripheral blood cytopenia, a frequent presenting symptom in pediatric patients, can be caused by bone marrow failure (BMF). Timely identification of patients with non-reversible BMF is of crucial importance to reduce the risks of invasive infections and bleeding complications. Most pediatric patients with severe persistent cytopenia, independent of the underlying cause, are offered allogeneic hematopoietic stem cell transplantation (HSCT) as curative therapy. Here we report on our management guidelines and HSCT outcomes of pediatric BMF patients to pinpoint improvements and future challenges. We formulated recommendations based on this 50 years' experience, which were implemented at our center in 2017. By analysis of the HSCT cohort of 2017-2023, the 5-year outcome data is presented and compared to historical outcome data. In addition, outcomes of patients transplanted for identified inherited bone marrow failure syndromes (IBMFS) are compared to severe aplastic anemia (SAA) outcomes to underline the often multiorgan disease in IBMFS with implications for long-term survival. Survival of pediatric patients with irreversible BMF has improved tremendously. SAA patients transplanted after 2017 had a superior 5-year overall (OS) and event-free survival (EFS) of 97% and 85% compared to 68% and 59% in the cohort transplanted before 2017 (p = 0.0011 and p = 0.017). A similar trend was seen for BMF, with an OS and EFS of 89% for those transplanted after 2017 compared to 62% and 59% (p > 0.05). This improvement is mainly related to better survival in the first months after HSCT. The long-term survival after HSCT is lower in IBMFS patients as compared to SAA patients due to secondary malignancies and multiorgan toxicity.Conclusion: Unbiased protocolized in-depth diagnostic strategies are crucial to increase the frequency of identifiable causes within the heterogeneous group of pediatric BMF. A comprehensive approach to identify the cause of BMF can prevent treatment delay and be useful to tailor treatment and follow-up protocols. Show less
IntroductionInborn errors of immunity (IEI) are an expanding group of rare diseases whose field has been boosted by next-generation sequencing (NGS), revealing several new entities, accelerating... Show moreIntroductionInborn errors of immunity (IEI) are an expanding group of rare diseases whose field has been boosted by next-generation sequencing (NGS), revealing several new entities, accelerating routine diagnoses, expanding the number of atypical presentations and generating uncertainties regarding the pathogenic relevance of several novel variants. MethodsResearch laboratories that diagnose and provide support for IEI require accurate, reproducible and sustainable phenotypic, cellular and molecular functional assays to explore the pathogenic consequences of human leukocyte gene variants and contribute to their assessment. We have implemented a set of advanced flow cytometry-based assays to better dissect human B-cell biology in a translational research laboratory. We illustrate the utility of these techniques for the in-depth characterization of a novel (c.1685G>A, p.R562Q) de novo gene variant predicted as probably pathogenic but with no previous insights into the protein and cellular effects, located in the tyrosine kinase domain of the Bruton's tyrosine kinase (BTK) gene, in an apparently healthy 14-year-old male patient referred to our clinic for an incidental finding of low immunoglobulin (Ig) M levels with no history of recurrent infections. Results and discussionA phenotypic analysis of bone marrow (BM) revealed a slightly high percentage of pre-B-I subset in BM, with no blockage at this stage, as typically observed in classical X-linked agammaglobulinemia (XLA) patients. The phenotypic analysis in peripheral blood also revealed reduced absolute numbers of B cells, all pre-germinal center maturation stages, together with reduced but detectable numbers of different memory and plasma cell isotypes. The R562Q variant allows Btk expression and normal activation of anti-IgM-induced phosphorylation of Y551 but diminished autophosphorylation at Y223 after anti IgM and CXCL12 stimulation. Lastly, we explored the potential impact of the variant protein for downstream Btk signaling in B cells. Within the canonical nuclear factor kappa B (NF-kappa B) activation pathway, normal I kappa B alpha degradation occurs after CD40L stimulation in patient and control cells. In contrast, disturbed I kappa B alpha degradation and reduced calcium ion (Ca2(+)) influx occurs on anti-IgM stimulation in the patient's B cells, suggesting an enzymatic impairment of the mutated tyrosine kinase domain. Show less
Epigenetic immune cell counting is a DNA (de)methylation-based technique which can be used to quantify lymphocyte subsets on dried blood spots (DBS). The foregoing techniques allow for a... Show moreEpigenetic immune cell counting is a DNA (de)methylation-based technique which can be used to quantify lymphocyte subsets on dried blood spots (DBS). The foregoing techniques allow for a retrospective investigation of immune cell profiles in newborns. In this study, we used this technique for determining lymphocyte subcounts as a potential biomarker for necrotizing enterocolitis (NEC). We investigated whether this technique can be implemented in the field of neonatology, by testing whether regulatory T cell (Treg) levels are pre-existently low in preterms with NEC. Newborn screening (NBS) cards from 32 preterms with NEC and 32 age- and weight-matched preterm controls, and 60 healthy term newborns, were analyzed. Relative and absolute cell counts were determined for CD3+, CD4+, CD8+, Th17, and Treg T cells. For both relative and absolute cell counts of CD3+, CD4+, CD8+, and Th17 T cells, significant differences were found between healthy term controls and both preterm groups, but not between preterm groups. For Tregs, no significant differences were found in either relative or absolute counts between any of the newborn groups. This study demonstrates the principle of epigenetic immune cell counting to analyze lymphocyte subsets in preterm neonates. Show less
DNA ligase I deficiency is an extremely rare primary immunodeficiency with only 6 patients reported in the literature. Most common manifestations include radiosensitivity, macrocytic anemia,... Show moreDNA ligase I deficiency is an extremely rare primary immunodeficiency with only 6 patients reported in the literature. Most common manifestations include radiosensitivity, macrocytic anemia, lymphopenia with an increased percentage of gamma-delta T cells, and hypogammaglobulinemia requiring replacement therapy. Two-month-old girl with delayed development, T-B-NK+ SCID, and macrocytic anemia presented features of Omenn syndrome. Whole exome sequencing revealed two novel, heterozygous variants (c.2312 G>A, p.Arg771Gly and c.776+5G>T, p.Pro260*) in the LIG1 gene (NM_000234.1). Hematopoietic stem cell transplantation from a fully matched unrelated donor was performed at the age of 4 months using GEFA03 protocol. Mixed donor-recipient chimerism was observed, with 60-70% chimerism in the mononucleated cell compartment and over 90% in T-lymphocyte compartment, but autologous myeloid recovery. Stable CD4+ and CD8+ T-cell counts above 200/mu L were achieved after 2 months, but the patient remained transfusion-dependent. Despite satisfactory immunological reconstitution, the second transplantation due to constitutional hemolytic defect has been considered. In light of possible re-transplantation, an issue of optimal conditioning protocol with sufficient myeloid engraftment is important. For the first time Omenn syndrome is described in a compound heterozygote carrying two the novel variants p.Arg771Gly and p.Pro260* in the LIG1 gene. Patients diagnosed with SCID and Omenn syndrome showing macrocytic anemia, should be screened for DNA ligase I deficiency. Show less
Schouwenburg, P. van; Unger, S.; Payne, K.J.; Kaiser, F.M.P.; Pico-Knijnenburg, I.; Pfeiffer, J.; ... ; Warnatz, K. 2022
Common variable immunodeficiency (CVID), characterized by recurrent infections, low serum class-switched immunoglobulin isotypes, and poor antigen-specific antibody responses, comprises a... Show moreCommon variable immunodeficiency (CVID), characterized by recurrent infections, low serum class-switched immunoglobulin isotypes, and poor antigen-specific antibody responses, comprises a heterogeneous patient population in terms of clinical presentation and underlying etiology. The diagnosis is regularly associated with a severe decrease of germinal center (GC)-derived B-cell populations in peripheral blood. However, data from B-cell differentiation within GC is limited. We present a multiplex approach combining histology, flow cytometry, and B-cell receptor repertoire analysis of sorted GC B-cell populations allowing the modeling of distinct disturbances in GCs of three CVID patients. Our results reflect pathophysiological heterogeneity underlying the reduced circulating pool of post-GC memory B cells and plasmablasts in the three patients. In patient 1, quantitative and qualitative B-cell development in GCs is relatively normal. In patient 2, irregularly shaped GCs are associated with reduced somatic hypermutation (SHM), antigen selection, and class-switching, while in patient 3, high SHM, impaired antigen selection, and class-switching with large single clones imply increased re-cycling of cells within the irregularly shaped GCs. In the lymph nodes of patients 2 and 3, only limited numbers of memory B cells and plasma cells are formed. While reduced numbers of circulating post GC B cells are a general phenomenon in CVID, the integrated approach exemplified distinct defects during GC maturation ranging from near normal morphology and function to severe disturbances with different facets of impaired maturation of memory B cells and/or plasma cells. Integrated dissection of disturbed GC B-cell maturation by histology, flow cytometry, and BCR repertoire analysis contributes to unraveling defects in the essential steps during memory formation. Show less
Neirinck, J.; Emmaneel, A.; Buysse, M.; Philippe, J.; Gassen, S. van; Saeys, Y.; ... ; Bonroy, C. 2022
IntroductionMultiparameter flow cytometry (FCM) immunophenotyping is an important tool in the diagnostic screening and classification of primary immunodeficiencies (PIDs). The EuroFlow Consortium... Show moreIntroductionMultiparameter flow cytometry (FCM) immunophenotyping is an important tool in the diagnostic screening and classification of primary immunodeficiencies (PIDs). The EuroFlow Consortium recently developed the PID Orientation Tube (PIDOT) as a universal screening tool to identify lymphoid-PID in suspicious patients. Although PIDOT can identify different lymphoid-PIDs with high sensitivity, clinical validation in a broad spectrum of patients with suspicion of PID is missing. In this study, we investigated the diagnostic performance of PIDOT, as part of the EuroFlow diagnostic screening algorithm for lymphoid-PID, in a daily practice at a tertiary reference center for PID. MethodsPIDOT was tested in 887 consecutive patients suspicious of PID at the Ghent University Hospital, Belgium. Patients were classified into distinct subgroups of lymphoid-PID vs. non-PID disease controls (non-PID DCs), according to the IUIS and ESID criteria. For the clinical validation of PIDOT, comprehensive characterization of the lymphoid defects was performed, together with the identification of the most discriminative cell subsets to distinguish lymphoid-PID from non-PID DCs. Next, a decision-tree algorithm was designed to guide subsequent FCM analyses. ResultsThe mean number of lymphoid defects detected by PIDOT in blood was 2.87 times higher in lymphoid-PID patients vs. non-PID DCs (p < 0.001), resulting in an overall sensitivity and specificity of 87% and 62% to detect severe combined immunodeficiency (SCID), combined immunodeficiency with associated or syndromic features (CID), immune dysregulation disorder (ID), and common variable immunodeficiency (CVID). The most discriminative populations were total memory and switched memory B cells, total T cells, TCD4+cells, and naive TCD4+cells, together with serum immunoglobulin levels. Based on these findings, a decision-tree algorithm was designed to guide further FCM analyses, which resulted in an overall sensitivity and specificity for all lymphoid-PIDs of 86% and 82%, respectively. ConclusionAltogether, our findings confirm that PIDOT is a powerful tool for the diagnostic screening of lymphoid-PID, particularly to discriminate (S)CID, ID, and CVID patients from other patients suspicious of PID. The combination of PIDOT and serum immunoglobulin levels provides an efficient guide for further immunophenotypic FCM analyses, complementary to functional and genetic assays, for accurate PID diagnostics. Show less
Neirinck, J.; Emmaneel, A.; Buysse, M.; Philippé, J.; Gassen, S. van; Saeys, Y.; ... ; Bonroy, C. 2022
IntroductionMultiparameter flow cytometry (FCM) immunophenotyping is an important tool in the diagnostic screening and classification of primary immunodeficiencies (PIDs). The EuroFlow Consortium... Show moreIntroductionMultiparameter flow cytometry (FCM) immunophenotyping is an important tool in the diagnostic screening and classification of primary immunodeficiencies (PIDs). The EuroFlow Consortium recently developed the PID Orientation Tube (PIDOT) as a universal screening tool to identify lymphoid-PID in suspicious patients. Although PIDOT can identify different lymphoid-PIDs with high sensitivity, clinical validation in a broad spectrum of patients with suspicion of PID is missing. In this study, we investigated the diagnostic performance of PIDOT, as part of the EuroFlow diagnostic screening algorithm for lymphoid-PID, in a daily practice at a tertiary reference center for PID. MethodsPIDOT was tested in 887 consecutive patients suspicious of PID at the Ghent University Hospital, Belgium. Patients were classified into distinct subgroups of lymphoid-PID vs. non-PID disease controls (non-PID DCs), according to the IUIS and ESID criteria. For the clinical validation of PIDOT, comprehensive characterization of the lymphoid defects was performed, together with the identification of the most discriminative cell subsets to distinguish lymphoid-PID from non-PID DCs. Next, a decision-tree algorithm was designed to guide subsequent FCM analyses. ResultsThe mean number of lymphoid defects detected by PIDOT in blood was 2.87 times higher in lymphoid-PID patients vs. non-PID DCs (p < 0.001), resulting in an overall sensitivity and specificity of 87% and 62% to detect severe combined immunodeficiency (SCID), combined immunodeficiency with associated or syndromic features (CID), immune dysregulation disorder (ID), and common variable immunodeficiency (CVID). The most discriminative populations were total memory and switched memory B cells, total T cells, TCD4+cells, and naive TCD4+cells, together with serum immunoglobulin levels. Based on these findings, a decision-tree algorithm was designed to guide further FCM analyses, which resulted in an overall sensitivity and specificity for all lymphoid-PIDs of 86% and 82%, respectively. ConclusionAltogether, our findings confirm that PIDOT is a powerful tool for the diagnostic screening of lymphoid-PID, particularly to discriminate (S)CID, ID, and CVID patients from other patients suspicious of PID. The combination of PIDOT and serum immunoglobulin levels provides an efficient guide for further immunophenotypic FCM analyses, complementary to functional and genetic assays, for accurate PID diagnostics. Show less
Zuiderwijk, M.O.; Burg, M. van der; Bekker, V.; Schoenaker, M.H.D. 2022
Necrotizing enterocolitis (NEC) is a leading cause of mortality in premature infants. However, the pathophysiology and influence of regulatory T cells (Tregs) have not been sufficiently elucidated.... Show moreNecrotizing enterocolitis (NEC) is a leading cause of mortality in premature infants. However, the pathophysiology and influence of regulatory T cells (Tregs) have not been sufficiently elucidated. We performed a scoping review to investigate current knowledge on the influence of Tregs in NEC, and to investigate the predictive value of Treg number in NEC development. Pubmed, Embase, Prospero and Cochrane Library were searched during December 2020. Primary research articles discussing Tregs and NEC development written in English were selected. Two reviewers screened title and abstract for relevance, after which full-text screening was performed. A total of 20 articles were selected-13 of the articles discussed studies performed in animal models, while 8 used human neonate data. One study discussed both animal and human data. It was shown that after NEC diagnosis or induction, Treg levels were decreased while Th17 levels were increased. No studies were found which investigated the predictive value of Treg number in NEC development. A reduced Treg level is found in animals and neonates with NEC. The question remains whether this effect is a factor on the causal pathway of NEC development or a bystander effect. Future research focusing on the pathophysiological timeline of NEC and the involvement of Tregs is required for better understanding of this disease. Show less
Kaiser, F.M.P.; Gruenbacher, S.; Oyaga, M.R.; Nio, E.; Jaritz, M.; Sun, Q.; ... ; Busslinger, M. 2022
The genetic causes of primary antibody deficiencies and autism spectrum disorder (ASD) are largely unknown. Here, we report a patient with hypogammaglobulinemia and ASD who carries biallelic... Show moreThe genetic causes of primary antibody deficiencies and autism spectrum disorder (ASD) are largely unknown. Here, we report a patient with hypogammaglobulinemia and ASD who carries biallelic mutations in the transcription factor PAX5. A patient-specific Pax5 mutant mouse revealed an early B cell developmental block and impaired immune responses as the cause of hypogammaglobulinemia. Pax5 mutant mice displayed behavioral deficits in all ASD domains. The patient and the mouse model showed aberrant cerebellar foliation and severely impaired sensorimotor learning. PAX5 deficiency also caused profound hypoplasia of the substantia nigra and ventral tegmental area due to loss of GABAergic neurons, thus affecting two midbrain hubs, controlling motor function and reward processing, respectively. Heterozygous Pax5 mutant mice exhibited similar anatomic and behavioral abnormalities. Lineage tracing identified Pax5 as a crucial regulator of cerebellar morphogenesis and midbrain GABAergic neurogenesis. These findings reveal new roles of Pax5 in brain development and unravel the underlying mechanism of a novel immunological and neurodevelopmental syndrome. Show less
Sikonja, J.; Groselj, U.; Scarpa, M.; Marca, G. la; Cheillan, D.; Kolker, S.; ... ; Bonham, J.R. 2022
Although individual rare disorders are uncommon, it is estimated that, together, 6000+ known rare diseases affect more than 30 million people in Europe, and present a substantial public health... Show moreAlthough individual rare disorders are uncommon, it is estimated that, together, 6000+ known rare diseases affect more than 30 million people in Europe, and present a substantial public health burden. Together with the psychosocial burden on affected families, rare disorders frequently, if untreated, result in a low quality of life, disability and even premature death. Newborn screening (NBS) has the potential to detect a number of rare conditions in asymptomatic children, providing the possibility of early treatment and a significantly improved long-term outcome. Despite these clear benefits, the availability and conduct of NBS programmes varies considerably across Europe and, with the increasing potential of genomic testing, it is likely that these differences may become even more pronounced. To help improve the equity of provision of NBS and ensure that all children can be offered high-quality screening regardless of race, nationality and socio-economic status, a technical meeting, endorsed by the Slovenian Presidency of the Council of the European Union, was held in October 2021. In this article, we present experiences from individual EU countries, stakeholder initiatives and the meeting's final conclusions, which can help countries attempting to establish new NBS programmes or expand existing provision. Show less
Glycosylation of CD45RB (RB+) has recently been identified to mark antigen-experienced B cells, independent of their CD27 expression. By using a novel combination of markers including CD45RB... Show moreGlycosylation of CD45RB (RB+) has recently been identified to mark antigen-experienced B cells, independent of their CD27 expression. By using a novel combination of markers including CD45RB glycosylation, CD27 and IgM/IgD isotype expression we segregated human peripheral blood B cell subsets and investigated their IGHV repertoire and in vitro functionality. We observed distinct maturation stages for CD27-RB+ cells, defined by differential expression of non-switched Ig isotypes. CD27-RB+ cells, which only express IgM, were more matured in terms of Ig gene mutation levels and function as compared to CD27-RB+ cells that express both IgM and IgD or cells that were CD27-RB-. Moreover, CD27-RB+IgM+ cells already showed remarkable rigidity in IgM isotype commitment, different from CD27-RB+IgMD+ and CD27-RB- cells that still demonstrated great plasticity in B cell fate decision. Thus, glycosylation of CD45RB is indicative for antigen-primed B cells, which are, dependent on the Ig isotype, functionally distinct. Show less
Background: Severe multilineage cytopenia in childhood caused by bone marrow failure (BMF) often represents a serious condition requiring specific management. Patients are at risk for invasive... Show moreBackground: Severe multilineage cytopenia in childhood caused by bone marrow failure (BMF) often represents a serious condition requiring specific management. Patients are at risk for invasive infections and bleeding complications. Previous studies report low rates of identifiable causes of pediatric BMF, rendering most patients with a descriptive diagnosis such as aplastic anemia (AA). Methods: We conducted a multi-center prospective cohort study in which an extensive diagnostic approach for pediatric patients with suspected BMF was implemented. After exclusion of malignant and transient causes of BMF, patients entered thorough diagnostic evaluation including bone marrow analysis, whole exome sequencing (WES) including copy number variation (CNV) analysis and/or single nucleotide polymorphisms (SNP) array analysis. In addition, functional and immunological evaluation were performed. Here we report the outcomes of the first 50 patients (2017-2021) evaluated by this approach. Results: In 20 patients (40%) a causative diagnosis was made. In this group, 18 diagnoses were established by genetic analysis, including 14 mutations and 4 chromosomal deletions. The 2 remaining patients had short telomeres while no causative genetic defect was found. Of the remaining 30 patients (60%), 21 were diagnosed with severe aplastic anemia (SAA) based on peripheral multi-lineage cytopenia and hypoplastic bone marrow, and 9 were classified as unexplained cytopenia without bone marrow hypoplasia. In total 28 patients had undergone hematopoietic stem cell transplantation (HSCT) of which 22 patients with an unknown cause and 6 patients with an identified cause for BMF. Conclusion: We conclude that a standardized in-depth diagnostic protocol as presented here, can increase the frequency of identifiable causes within the heterogeneous group of pediatric BMF. We underline the importance of full genetic analysis complemented by functional tests of all patients as genetic causes are not limited to patients with typical (syndromal) clinical characteristics beyond cytopenia. In addition, it is of importance to apply genome wide genetic analysis, since defects in novel genes are frequently discovered in this group. Identification of a causal abnormality consequently has implications for the choice of treatment and in some cases prevention of invasive therapies. Show less
Blom, M.; Zetterstrom, R.H.; Stray-Pedersen, A.; Gilmour, K.; Gennery, A.R.; Puck, J.M.; Burg, M. van der 2022
Background: Public health newborn screening (NBS) programs continuously evolve, taking advantage of international shared learning. NBS for severe combined immunodeficiency (SCID) has recently been... Show moreBackground: Public health newborn screening (NBS) programs continuously evolve, taking advantage of international shared learning. NBS for severe combined immunodeficiency (SCID) has recently been introduced in many countries. However, comparison of screening outcomes has been hampered by use of disparate terminology and imprecise or variable case definitions for non-SCID conditions with T-cell lymphopenia.Objectives: This study sought to determine whether standardized screening terminology could overcome a Babylonian confusion and whether improved case definitions would promote international exchange of knowledge.Methods: A systematic literature review highlighted the diverse terminology in SCID NBS programs internationally. While, as expected, individual screening strategies and tests were tailored to each program, we found uniform terminology to be lacking in definitions of disease targets, sensitivity, and specificity required for comparisons across programs.Results: The study's recommendations reflect current evidence from literature and existing guidelines coupled with opinion of experts in public health screening and immunology. Terminologies were aligned. The distinction between actionable and nonactionable T-cell lymphopenia among non-SCID cases was clarified, the former being infants with T-cell lymphopenia who could benefit from interventions such as protection from infections, antibiotic prophylaxis, and live-attenuated vaccine avoidance.Conclusions: By bringing together the previously unconnected public health screening community and clinical immunology community, these SCID NBS deliberations bridged the gaps in language and perspective between these disciplines. This study proposes that international specialists in each disorder for which NBS is performed join forces to hone their definitions and recommend uniform registration of outcomes of NBS. Standardization of terminology will promote international exchange of knowledge and optimize each phase of NBS and follow-up care, advancing health outcomes for children worldwide. Show less
Zoutman, W.H.; Nell, R.J.; Versluis, M.; Pico, I.; Vu, T.H.K.; Verdijk, R.M.; ... ; Velden, P.A. van der 2022
B cells fulfill an important role in the adaptive immunity. Upon activation and immunoglobulin (IG) class switching, these cells function in the humoral immunity compartment as plasma cells. For... Show moreB cells fulfill an important role in the adaptive immunity. Upon activation and immunoglobulin (IG) class switching, these cells function in the humoral immunity compartment as plasma cells. For clinical applications, it can be important to quantify (switched) B cells accurately in a variety of body fluids and tissues of benign, inflammatory and malignant origin. For decades, flow cytometry and immunohistochemistry (IHC) have been the preferred methods for quantification. Although these methods are widely used, both depend on the accessibility of B cell epitopes and therefore require intact (fixed) cells. Whenever samples are low in quantity and/or quality, accurate quantification can be difficult. By shifting the focus from epitopes to DNA markers, quantification of B cells remains achievable. During differentiation and maturation, B cells are subjected to programmed genetic recombination processes like VDJ rearrangements and class switch recombination (CSR), which result in deletion of specific sequences of the IGH locus. These cell type-specific DNA "scars" (loss of sequences) in IG genes can be exploited as B cell markers in digital PCR (dPCR) based quantification methods. Here, we describe a novel, specific and sensitive digital PCR-based method to quantify mature and switched B cells in DNA specimens of benign and (copy number unstable) malignant origin. We compared this novel way of B cell quantitation with flow cytometric and immunohistochemical methods. Through cross-validation with flow cytometric sorted B cell subpopulations, we gained quantitative insights into allelic involvement in different recombination processes in the IGH locus. Our newly developed method is accurate and independent of the cellular context, offering new possibilities for quantification, even for (limited) small samples like liquid biopsies. Show less
Atmar, K.; Tulling, A.J.; Lankester, A.C.; Bartels, M.; Smiers, F.J.; Burg, M. van der; Mohseny, A.B. 2022
BackgroundIn most patients with aplastic anemia (AA), the diagnosis is limited to a description of the symptoms. Lack of understanding of the underlying pathophysiological mechanisms causing bone... Show moreBackgroundIn most patients with aplastic anemia (AA), the diagnosis is limited to a description of the symptoms. Lack of understanding of the underlying pathophysiological mechanisms causing bone marrow failure (BMF), hampers tailored treatment. In these patients, auto-immune cell-mediated destruction of the bone marrow is often presumed to be the causative mechanism. The status of the bone marrow microenvironment, particularly the mesenchymal stromal cell (MSC) component, was recently suggested as a potential player in the pathophysiology of AA. Therefore, functional, and immune modulatory characteristics of bone marrow MSCs might represent important parameters for AA.ObjectiveTo conduct a systematic review to evaluate in vitro functional properties of MSCs derived from patients with AA compared to healthy controls.MethodsAccording to PRISMA guidelines, a comprehensive search strategy was performed by using online databases (Pubmed, ISI Web of Science, Embase, and the Cochrane Library). Studies reporting on phenotypical characterization, proliferation potential, differentiation capacity, immunomodulatory potential, and ability to support hematopoiesis were identified and screened using the Rayyan software tool.Results23 articles were included in this systematic review, describing a total of 324 patients with AA and 285 controls. None of the studies identified a significant difference in expression of any MSC surface marker between both groups. However, AA-MSCs showed a decreased proliferation potential, an increased tendency to differentiate into the adipogenic lineage and decreased propensity towards osteogenic differentiation. Importantly, AA-MSCs show reduced capacity of immunosuppression and hematopoietic support in comparison to healthy controls.ConclusionWe conclude that there are indications for a contribution of MSCs in the pathophysiology of AA. However, the current evidence is of poor quality and requires better defined study populations in addition to a more robust methodology to study MSC biology at a cellular and molecular level. Future studies on bone marrow microenvironment should aim at elucidating the interaction between MSCs, hematopoietic stem cells (HSCs) and immune cells to identify impairments associated with/causing BMF in patients with AA. Show less
