Causative mechanisms and clinical impact of immunoglobulin deficiencies in ataxia telangiectasia.

BACKGROUND: Ataxia telangiectasia (AT) is characterized by cerebellar ataxia, telangiectasia, immunodeficiency, and increased cancer susceptibility and is caused by mutations in the ataxia telangiectasia mutated (ATM) gene. The immunodeficiency comprises predominantly immunoglobulin deficiency, mainly IgA and IgG2, with a variable severity. So far, the exact mechanisms underlying the immunoglobulin deficiency, especially the variable severity, remain unelucidated. OBJECTIVE: We characterized the clinical impact of immunoglobulin deficiencies in AT and elucidated their mechanisms in AT. METHODS: We analyzed long-term immunoglobulin levels, immunophenotyping, and survival time in our cohort (n = 87, median age 16 years; maximum 64 years). Somatic hypermutation and class-switch junctions in B cells were analyzed by next-generation sequencing. Furthermore, an in vitro class-switching induction assay was performed, followed by RNA sequencing, to assess the effect of ATM inhibition. RESULTS: Only the hyper-IgM AT phenotype significantly worsened survival time, while IgA or IgG2 deficiencies did not. The immunoglobulin levels showed predominantly decreased IgG2 and IgA. Moreover, flow cytometric analysis demonstrated reduced naive B and T lymphocytes and a deficiency of class-switched IgG2 and IgA memory B cells. Somatic hypermutation frequencies were lowered in IgA- and IgG2-deficient patients, indicating hampered germinal center reaction. In addition, the microhomology of switch junctions was elongated, suggesting alternative end joining during class-switch DNA repair. The in vitro class switching and proliferation were negatively affected by ATM inhibition. RNA sequencing analysis showed that ATM inhibitor influenced expression of germinal center reaction genes. CONCLUSION: Immunoglobulin deficiency in AT is caused by disturbed development of class-switched memory B cells. ATM deficiency affects both germinal center reaction and choice of DNA-repair pathway in class switching.

Persistent Hypogammaglobulinemia after Receiving Rituximab Post-HSCT Is Not Caused by an Intrinsic B Cell Defect.

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 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 naïve 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.

IL-7 receptor signaling drives human B-cell progenitor differentiation and expansion.

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 α chain (IL-7Rα) 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-7Rα 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-7Rα 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.

Deciphering imprints of impaired memory B-cell maturation in germinal centers of three patients with common variable immunodeficiency.

Common 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.

CD45RB Glycosylation and Ig Isotype Define Maturation of Functionally Distinct B Cell Subsets in Human Peripheral Blood.

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 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.

T and NK Cells in IL2RG-Deficient Patient 50 Years After Hematopoietic Stem Cell Transplantation.

The first successful European hematopoietic stem cell transplantation (HSCT) was performed in 1968 as treatment in a newborn with IL2RG deficiency using an HLA-identical sibling donor. Because of declining naive T and natural killer (NK) cells, and persistent human papilloma virus (HPV)-induced warts, the patient received a peripheral stem cell boost at the age of 37 years. NK and T cells were assessed before and up to 14 years after the boost by flow cytometry. The boost induced renewed reconstitution of functional NK cells that were 14 years later enriched for CD56dimCD27+ NK cells. T-cell phenotype and T-cell receptor (TCR) repertoire were simultaneously analyzed by including TCR Vß antibodies in the cytometry panel. Naive T-cell numbers with a diverse TCR Vß repertoire were increased by the boost. Before and after the boost, clonal expansions with a homogeneous TIGIT and PD-1 phenotype were identified in the CD27- and/or CD28- memory population in the patient, but not in the donor. TRB sequencing was applied on sorted T-cell subsets from blood and on T cells from skin biopsies. Abundant circulating CD8 memory clonotypes with a chronic virus-associated CD57+KLRG1+CX3CR1+ phenotype were also present in warts, but not in healthy skin of the patient, suggesting a link with HPV. In conclusion, we demonstrate in this IL2RG-deficient patient functional NK cells, a diverse and lasting naive T-cell compartment, supported by a stem cell boost, and an oligoclonal memory compartment half a century after HSCT.

AKT Hyperphosphorylation and T Cell Exhaustion in Down Syndrome.

Down syndrome (DS) is associated with increased susceptibility to infections, auto-immunity, immunodeficiency and haematological malignancies. The exact underlying immunological pathophysiology is still unclear. The immunophenotype and clinical characteristics of DS resemble those of Activated PI3K Delta Syndrome (APDS), in which the PI3K/AKT/mTOR pathway is overactivated. We hypothesized that T cell exhaustion and the hyperactivation of the AKT signalling pathway is also present in immune cells of children with DS. In this observational non-interventional cohort study we collected blood samples of children with DS (n=22) and healthy age-matched controls (n=21) for flowcytometric immunophenotyping, phospho-flow AKT analysis and exhaustion analysis of T cells. The median age was 5 years (range 1-12y). Total T and NK cells were similar for both groups, but absolute values and transitional B cells, naive memory B cells and naive CD4+ and CD8+ T cells were lower in DS. pAKT and AKT were increased for CD3+ and CD4+ T cells and CD20+ B cells in children with DS. Total AKT was also increased in CD8+ T cells. Children with DS showed increased expression of inhibitory markers Programmed cell dealth-1 (PD-1), CD244 and CD160 on CD8+ T cells and increased PD-1 and CD244+ expression on CD4+ T cells, suggesting T cell exhaustion. Children with DS show increased pAKT and AKT and increased T cell exhaustion, which might contribute to their increased susceptibility to infections, auto immunity and haematological malignancies.

Lessons learned from the diagnostic work-up of a patient with the bare lymphocyte syndrome type II.

A patient presented severe combined immunodeficiency (SCID)-like symptoms. The presence of a substantial number of CD4+ T-cells in the peripheral blood was not explained by maternal engraftment. Genetic analysis revealed a novel RFXANK mutation, c.232C > T, resulting in a stop codon, with consequently defective transcription of MHC class II resulting in bare lymphocyte syndrome (BLS) type II. The initial unawareness of complete absence of MHC class II expression and normal T-cell receptor excision circles (TREC)-levels delayed the final diagnosis. After identification of the genetic defect the patient was scheduled for hematopoietic stem cell transplantation (HSCT). Here, we present and discuss the diagnostic and therapeutic approach of a novel case of BLS type II in relation to T-cell development.

Primary Ovarian Failure in Addition to Classical Clinical Features of Coats Plus Syndrome in a Female Carrying 2 Truncating Variants of CTC1.

Coats plus syndrome is an autosomal recessive multisystemic and pleiotropic disorder affecting the eyes, brain, bone, and gastrointestinal tract, usually caused by compound heterozygous variants of the conserved telomere maintenance component 1 gene (CTC1), involved in telomere homeostasis and replication. So far, most reported patients are compound heterozygous for a truncating mutation and a missense variant. The phenotype is believed to result from telomere dysfunction, with accumulation of DNA damage, cellular senescence, and stem cell depletion. Here, we report a 23-year-old female with prenatal and postnatal growth retardation, microcephaly, osteopenia, recurrent fractures, intracranial calcification, leukodystrophy, parenchymal brain cysts, bicuspid aortic valve, and primary ovarian failure. She carries a previously reported maternally inherited pathogenic variant in exon 5 (c.724_727del, p.(Lys242Leufs*41)) and a novel, paternally inherited splice site variant (c.1617+5G>T; p.(Lys480Asnfs*17)) in intron 9. CTC1 transcript analysis showed that the latter resulted in skipping of exon 9. A trace of transcripts was normally spliced resulting in the presence of a low level of wild-type CTC1 transcripts. We speculate that ovarian failure is caused by telomere shortening or chromosome cohesion failure in oocytes and granulosa cells, with early decrease in follicular reserve. This is the first patient carrying 2 truncating CTC1 variants and the first presenting primary ovarian failure.

Second Tier Testing to Reduce the Number of Non-actionable Secondary Findings and False-Positive Referrals in Newborn Screening for Severe Combined Immunodeficiency.

PURPOSE: Newborn screening (NBS) for severe combined immunodeficiency (SCID) is based on the detection of T-cell receptor excision circles (TRECs). TRECs are a sensitive biomarker for T-cell lymphopenia, but not specific for SCID. This creates a palette of secondary findings associated with low T-cells that require follow-up and treatment or are non-actionable. The high rate of (non-actionable) secondary findings and false-positive referrals raises questions about the harm-benefit-ratio of SCID screening, as referrals are associated with high emotional impact and anxiety for parents. METHODS: An alternative quantitative TREC PCR with different primers was performed on NBS cards of referred newborns (N = 56) and epigenetic immune cell counting was used as for relative quantification of CD3 + T-cells (N = 59). Retrospective data was used to determine the reduction in referrals with a lower TREC cutoff value or an adjusted screening algorithm. RESULTS: When analyzed with a second PCR with different primers, 45% of the referrals (25/56) had TREC levels above cutoff, including four false-positive cases in which two SNPs were identified. With epigenetic qPCR, 41% (24/59) of the referrals were within the range of the relative CD3 + T-cell counts of the healthy controls. Lowering the TREC cutoff value or adjusting the screening algorithm led to lower referral rates but did not prevent all false-positive referrals. CONCLUSIONS: Second tier tests and adjustments of cutoff values or screening algorithms all have the potential to reduce the number of non-actionable secondary findings in NBS for SCID, although second tier tests are more effective in preventing false-positive referrals.

EuroFlow Standardized Approach to Diagnostic Immunopheneotyping of Severe PID in Newborns and Young Children.

The EuroFlow PID consortium developed a set of flow cytometry tests for evaluation of patients with suspicion of primary immunodeficiency (PID). In this technical report we evaluate the performance of the SCID-RTE tube that explores the presence of recent thymic emigrants (RTE) together with T-cell activation status and maturation stages and discuss its applicability in the context of the broader EuroFlow PID flow cytometry testing algorithm for diagnostic orientation of PID of the lymphoid system. We have analyzed peripheral blood cells of 26 patients diagnosed between birth and 2 years of age with a genetically defined primary immunodeficiency disorder: 15 severe combined immunodeficiency (SCID) patients had disease-causing mutations in RAG1 or RAG2 (n = 4, two of them presented with Omenn syndrome), IL2RG (n = 4, one of them with confirmed maternal engraftment), NHEJ1 (n = 1), CD3E (n = 1), ADA (n = 1), JAK3 (n = 3, two of them with maternal engraftment) and DCLRE1C (n = 1) and 11 other PID patients had diverse molecular defects [ZAP70 (n = 1), WAS (n = 2), PNP (n = 1), FOXP3 (n = 1), del22q11.2 (DiGeorge n = 4), CDC42 (n = 1) and FAS (n = 1)]. In addition, 44 healthy controls in the same age group were analyzed using the SCID-RTE tube in four EuroFlow laboratories using a standardized 8-color approach. RTE were defined as CD62L+CD45RO-HLA-DR-CD31+ and the activation status was assessed by the expression of HLA-DR+. Naïve CD8+ T-lymphocytes and naïve CD4+ T-lymphocytes were defined as CD62L+CD45RO-HLA-DR-. With the SCID-RTE tube, we identified patients with PID by low levels or absence of RTE in comparison to controls as well as low levels of naïve CD4+ and naïve CD8+ lymphocytes. These parameters yielded 100% sensitivity for SCID. All SCID patients had absence of RTE, including the patients with confirmed maternal engraftment or oligoclonally expanded T-cells characteristic for Omenn syndrome. Another dominant finding was the increased numbers of activated CD4+HLA-DR+ and CD8+HLA-DR+ lymphocytes. Therefore, the EuroFlow SCID-RTE tube together with the previously published PIDOT tube form a sensitive and complete cytometric diagnostic test suitable for patients suspected of severe PID (SCID or CID) as well as for children identified via newborn screening programs for SCID with low or absent T-cell receptor excision circles (TRECs).

Repertoire Sequencing of B Cells Elucidates the Role of UNG and Mismatch Repair Proteins in Somatic Hypermutation in Humans.

The generation of high-affinity antibodies depends on somatic hypermutation (SHM). SHM is initiated by the activation-induced cytidine deaminase (AID), which generates uracil (U) lesions in the B-cell receptor (BCR) encoding genes. Error-prone processing of U lesions creates a typical spectrum of point mutations during SHM. The aim of this study was to determine the molecular mechanism of SHM in humans; currently available knowledge is limited by the number of mutations analyzed per patient. We collected a unique cohort of 10 well-defined patients with bi-allelic mutations in genes involved in base excision repair (BER) (UNG) or mismatch repair (MMR) (MSH2, MSH6, or PMS2) and are the first to present next-generation sequencing (NGS) data of the BCR, allowing us to study SHM extensively in humans. Analysis using ARGalaxy revealed selective skewing of SHM mutation patterns specific for each genetic defect, which are in line with the five-pathway model of SHM that was recently proposed based on mice data. However, trans-species comparison revealed differences in the role of PMS2 and MSH2 in strand targeting between mice and man. In conclusion, our results indicate a role for UNG, MSH2, MSH6, and PMS2 in the generation of SHM in humans comparable to their function in mice. However, we observed differences in strand targeting between humans and mice, emphasizing the importance of studying molecular mechanisms in a human setting. The here developed method combining NGS and ARGalaxy analysis of BCR mutation data forms the basis for efficient SHM analyses of other immune deficiencies.

Identification of CVID Patients With Defects in Immune Repertoire Formation or Specification.

Common variable immune deficiency disorder (CVID) is the most clinically relevant cause of antibody failure. It is a highly heterogeneous disease with different underlying etiologies. CVID has been associated with a quantitative B cell defect, however, little is known about the quality of B cells present. Here, we studied the naïve and antigen selected B-cell receptor (BCR) repertoire in 33 CVID patients using next generation sequencing, to investigate B cells quality. Analysis for each individual patient revealed whether they have a defect in immune repertoire formation [V(D)J recombination] or specification (somatic hypermutation, subclass distribution, or selection). The naïve BCR repertoire was normal in most of the patients, although alterations in repertoire diversity and the junctions were found in a limited number of patients indicating possible defects in early B-cell development or V(D)J recombination in these patients. In contrast, major differences were found in the antigen selected BCR repertoire. Here, most patients (15/17) showed a reduced frequency of somatic hypermutation (SHM), changes in subclass distribution and/or minor alterations in antigen selection. Together these data show that in our CVID cohort only a small number of patients have a defect in formation of the naïve BCR repertoire, whereas the clear majority of patients have disturbances in their antigen selected repertoire, suggesting a defect in repertoire specification in the germinal centers of these patients. This highlights that CVID patients not only have a quantitative B cell defect, but that also the quality of, especially post germinal center B cells, is impaired.

Immunodeficiency in Bloom's Syndrome.

Bloom's syndrome (BS) is an autosomal recessive disease, caused by mutations in the BLM gene. This gene codes for BLM protein, which is a helicase involved in DNA repair. DNA repair is especially important for the development and maturation of the T and B cells. Since BLM is involved in DNA repair, we aimed to study if BLM deficiency affects T and B cell development and especially somatic hypermutation (SHM) and class switch recombination (CSR) processes. Clinical data of six BS patients was collected, and immunoglobulin serum levels were measured at different time points. In addition, we performed immune phenotyping of the B and T cells and analyzed the SHM and CSR in detail by analyzing IGHA and IGHG transcripts using next-generation sequencing. The serum immunoglobulin levels were relatively low, and patients had an increased number of infections. The absolute number of T, B, and NK cells were low but still in the normal range. Remarkably, all BS patients studied had a high percentage (20-80%) of CD4+ and CD8+ effector memory T cells. The process of SHM seems normal; however, the Ig subclass distribution was not normal, since the BS patients had more IGHG1 and IGHG3 transcripts. In conclusion, BS patients have low number of lymphocytes, but the immunodeficiency seems relatively mild since they have no severe or opportunistic infections. Most changes in the B cell development were seen in the CSR process; however, further studies are necessary to elucidate the exact role of BLM in CSR.

An evaluation of the TREC assay with regard to the integration of SCID screening into the Dutch newborn screening program.

Newborn screening of severe combined immunodeficiency through the detection of T-cell receptor excision circles will provide the opportunity of treating before the occurrence of life-threatening infections. With the EnLite Neonatal TREC assay (PerkinElmer) and end-point PCR, 39 samples (3.0%) of 1295 heel prick cards of the Dutch newborn screening program required a retest after initial analysis. After retest, 21 samples (1.62%) gave TREC levels below cut-off. A significant reduction in TREC levels was observed in heel prick cards stored for three months (n=33) and one year (n=33). Preterm newborns (n=155) showed significantly lower TREC levels and a higher retest-rate than full-term newborns. Peripheral blood spots of 22 confirmed SCID patients and 17 primary immunodeficiency patients showed undetectable or low TREC-levels. These findings suggest that the EnLite Neonatal TREC assay is a suitable method for SCID-screening in the Netherlands, thereby providing guidance in the decisions concerning implementation into the Dutch program.

Antigen Receptor Galaxy: A User-Friendly, Web-Based Tool for Analysis and Visualization of T and B Cell Receptor Repertoire Data.

Antigen Receptor Galaxy (ARGalaxy) is a Web-based tool for analyses and visualization of TCR and BCR sequencing data of 13 species. ARGalaxy consists of four parts: the demultiplex tool, the international ImMunoGeneTics information system (IMGT) concatenate tool, the immune repertoire pipeline, and the somatic hypermutation (SHM) and class switch recombination (CSR) pipeline. Together they allow the analysis of all different aspects of the immune repertoire. All pipelines can be run independently or combined, depending on the available data and the question of interest. The demultiplex tool allows data trimming and demultiplexing, whereas with the concatenate tool multiple IMGT/HighV-QUEST output files can be merged into a single file. The immune repertoire pipeline is an extended version of our previously published ImmunoGlobulin Galaxy (IGGalaxy) virtual machine that was developed to visualize V(D)J gene usage. It allows analysis of both BCR and TCR rearrangements, visualizes CDR3 characteristics (length and amino acid usage) and junction characteristics, and calculates the diversity of the immune repertoire. Finally, ARGalaxy includes the newly developed SHM and CSR pipeline to analyze SHM and/or CSR in BCR rearrangements. It analyzes the frequency and patterns of SHM, Ag selection (including BASELINe), clonality (Change-O), and CSR. The functionality of the ARGalaxy tool is illustrated in several clinical examples of patients with primary immunodeficiencies. In conclusion, ARGalaxy is a novel tool for the analysis of the complete immune repertoire, which is applicable to many patient groups with disturbances in the immune repertoire such as autoimmune diseases, allergy, and leukemia, but it can also be used to address basic research questions in repertoire formation and selection.

Genetic defects in PI3Kδ affect B-cell differentiation and maturation leading to hypogammaglobulineamia and recurrent infections.

BACKGROUND: Mutations in PIK3CD and PIK3R1 cause activated PI3K-δ syndrome (APDS) by dysregulation of the PI3K-AKT pathway. METHODS: We studied precursor and peripheral B-cell differentiation and apoptosis via flowcytometry. Furthermore, we performed AKT-phosphorylation assays and somatic hypermutations (SHM) and class switch recombination (CSR) analysis. RESULTS: We identified 13 patients of whom 3 had new mutations in PIK3CD or PIK3R1. Patients had low total B-cell numbers with increased frequencies of transitional B cells and plasmablasts, while the precursor B-cell compartment in bone marrow was relatively normal. Basal AKT phosphorylation was increased in lymphocytes from APDS patients and natural effector B cells where most affected. PI3K mutations resulted in altered SHM and CSR and increased apoptosis. CONCLUSIONS: The B-cell compartment in APDS patients is affected by the mutations in PI3K. There is reduced differentiation beyond the transitional stage, increased AKT phosphorylation and increased apoptosis. This B-cell phenotype contributes to the clinical phenotype.