Clinical and Immunological Diversity of Recombination Defects

__abstract__ The immune system is capable to detect a large variability of pathogens and distinguish them from the body’s own healthy tissue. The immune system consists of the innate- and adaptive immune system. The adaptive immune system consists of B-and T-lymphocytes, which can recognize pathogens with an antigen-specific receptor. B-lymphocytes can adapt their responses during an infection to improve recognition of the pathogen and they generate long-term immunological memory. These antigen-specific receptors are called B-cell receptor (BR) and T-cell receptor (TR), respectively. Since the number of possible antigens is innumerable, an enormous diversity of the antigen-specific receptors is required. To achieve this, the BR and TR contain a variable domain that is unique for each individual T or B cell. This variable domain is generated by recombination of the antigen receptor genes in a process which is called V(D

Human IgG2- and IgG4-expressing memory B cells display enhanced molecular and phenotypic signs of maturity and accumulate with age

The mechanisms involved in sequential immunoglobulin G (IgG) class switching are still largely unknown. Sequential IG class switching is linked to higher levels of somatic hypermutation (SHM) in vivo, but it remains unclear if these are generated temporally during an immune response or upon activation in a secondary response. We here aimed to uncouple these processes and to distinguish memory B cells from primary and secondary immune responses. SHM levels and IgG subclasses were studied with 454 pyrosequencing on blood mononuclear cells from young children and adults as models for primary and secondary immunological memory. Additional sequencing and detailed immunophenotyping with IgG subclass-specific antibodies was performed on purified IgG+ memory B-cell subsets. In both children and adults, SHM levels were higher in transcripts involving more downstream-located IGHG genes (esp. IGHG2 and IGHG4). In adults, SHM levels were significantly higher than in children, and downstream IGHG genes were more frequently utilized. This was associated with increased frequencies of CD27+ IgG+ memory B cells, which contained higher levels of SHM, more IGHG2 usage, and higher expression levels of activation markers than CD27-IgG+ memory B cells. We conclude that secondary immunological memory accumulates with age and these memory B cells express CD27, high levels of activation markers, and carry high SHM levels and frequent usage of IGHG2. These new insights contribute to our understanding of sequential IgG subclass switching and show a potential relevance of using serum IgG2 levels or numbers of IgG2-expressing B cells as markers for efficient generation of memory responses

Evaluation of the antigen-experienced B-cell receptor repertoire in healthy children and adults

Upon antigen recognition via their B cell receptor (BR), B cells migrate to the germinal center where they undergo somatic hypermutation (SHM) to increase their affinity for the antigen, and class switch recombination (CSR) to change the effector function of the secreted antibodies. These steps are essential to create an antigen-experienced BR repertoire that efficiently protects the body against pathogens. At the same time, the BR repertoire should be selected to protect against responses to self-antigen or harmless antigens. Insights into the processes of SHM, selection, and CSR can be obtained by studying the antigen-experienced BR repertoire. Currently, a large reference data set of healthy children and adults, which ranges from neonates to the elderly, is not available. In this study, we analyzed the antigen-experienced repertoire of 38 healthy donors (HD), ranging from cord blood to 74 years old, by sequencing IGA and IGG transcripts using next generation sequencing. This resulted in a large, freely available reference data set containing 412,890 IGA and IGG transcripts. We used this data set to study mutation levels, SHM patterns, antigenic selection, and CSR from birth to elderly HD. Only small differences were observed in SHM patterns, while the mutation levels increase in early childhood and stabilize at 6 years of age at around 7%. Furthermore, comparison of the antigen-experienced repertoire with sequences from the naive immune repertoire showed that features associated with autoimmunity such as long CDR3 length and IGHV4-34 usage are reduced in the antigen-experienced repertoire. Moreover, IGA2 and IGG2 usage was increased in HD in higher age categories, while IGG1 usage was decreased. In addition, we studied clonal relationship in the different samples. Clonally related sequences were found with different subclasses. Interestingly, we found transcripts with the same CDR1-CDR3 sequence, but different subclasses. Together, these data suggest that a single antigen can provoke a B-cell response with BR of different subclasses and that, during the course of an immune response, some B cells change their isotype without acquiring additional SHM or can directly switch to different isotypes

ImmunoGlobulin galaxy (IGGalaxy) for simple determination and quantitation of immunoglobulin heavy chain rearrangements from NGS

Background: Sequence analysis of immunoglobulin heavy chain (IGH) gene rearrangements and frequency analysis is a powerful tool for studying the immune repertoire, immune responses and immune dysregulation in health and disease. The challenge is to provide user friendly, secure and reproducible analytical services that are available for both small and large laboratories which are determining VDJ repertoire using NGS technology. Results: In this study we describe ImmunoGlobulin Galaxy (IGGalaxy)- a convenient web based application for analyzing next-generation sequencing results and reporting IGH gene rearrangements for both repertoire and clonality studies. IGGalaxy has two analysis options one using the built in igBLAST algorithm and the second using output from IMGT; in either case repertoire summaries for the B-cell populations tested are available. IGGalaxy supports multi-sample and multi-replicate input analysis for both igBLAST and IMGT/HIGHV-QUEST. We demonstrate the technical validity of this platform using a standard dataset, S22, used for benchmarking the performance of antibody alignment utilities with a 99.9 % concordance with previous results. Re-analysis of NGS data from our samples of RAG-deficient patients demonstrated the validity and user friendliness of this tool. Conclusions: IGGalaxy provides clinical researchers with detailed insight into the repertoire of the B-cell population per individual sequenced and between control and pathogenic genomes. IGGalaxy was developed for 454 NGS results but is capable of analyzing alternative NGS data (e.g. Illumina, Ion Torrent). We demonstrate the use of a Galaxy virtual machine to determine the VDJ repertoire for reference data and from B-cells taken from immune deficient patients. IGGalaxy is available as a VM for download and use on a desktop PC or on a server

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

Delineating Human B Cell Precursor Development With Genetically Identified PID Cases as a Model

B-cell precursors (BCP) arise from hematopoietic stem cells in bone marrow (BM). Identification and characterization of the different BCP subsets has contributed to the understanding of normal B-cell development. BCP first rearrange their immunoglobulin (Ig) heavy chain (IGH) genes to form the pre-B-cell receptor (pre-BCR) complex together with surrogate light chains. Appropriate signaling via this pre-BCR complex is followed by rearrangement of the Ig light chain genes, resulting in the formation, and selection of functional BCR molecules. Consecutive production, expression, and functional selection of the pre-BCR and BCR complexes guide the BCP differentiation process that coincides with corresponding immunophenotypic changes. We studied BCP differentiation in human BM samples from healthy controls and patients with a known genetic defect in V(D)J recombination or pre-BCR signaling to unravel normal immunophenotypic changes and to determine the effect of differentiation blocks caused by the specific genetic defects. Accordingly, we designed a 10-color antibody panel to study human BCP development in BM by flow cytometry, which allows identification of classical preB-I, preB-II, and mature B-cells as defined via BCR-related markers with further characterization by additional markers. We observed heterogeneous phenotypes associated with more than one B-cell maturation pathway, particularly for the preB-I and preB-II stages in which V(D)J recombination takes place, with asynchronous marker expression patterns. Next Generation Sequencing of complete IGH gene rearrangements in sorted BCP subsets unraveled their rearrangement status, indicating that BCP differentiation does not follow a single linear pathway. In conclusion, B-cell development in human BM is not a linear process, but a rather complex network of parallel pathways dictated by V(D)J-recombination-driven checkpoints and pre-BCR/BCR mediated-signaling occurring during B-cell production and selection. It can also be described as asynchronous, because precursor B-cells do not differentiate as full population between the different stages, but rather transit as a continuum, which seems influenced (in part) by V-D-J recombination-driven checkpoints

Activated PI3Kδ syndrome, an immunodeficiency disorder, leads to sensorimotor deficits recapitulated in a murine model.

The phosphoinositide-3-kinase (PI3K) family plays a major role in cell signaling and is predominant in leukocytes. Gain-of-function (GOF) mutations in the PIK3CD gene lead to the development of activated PI3Kδ syndrome (APDS), a rare primary immunodeficiency disorder. A subset of APDS patients also displays neurodevelopmental delay symptoms, suggesting a potential role of PIK3CD in cognitive and behavioural function. However, the extent and nature of the neurodevelopmental deficits has not been previously quantified. Here, we assessed the cognitive functions of two APDS patients, and investigated the causal role of the PIK3CD GOF mutation in neurological deficits using a murine model of this disease. We used p110δE1020K knock-in mice, harbouring the most common APDS mutation in patients. We found that APDS patients present with visuomotor deficits, exacerbated by autism spectrum disorder comorbidity, whereas p110δE1020K mice exhibited impairments in motor behaviour, learning and repetitive behaviour patterning. Our data indicate that PIK3CD GOF mutations increase the risk for neurodevelopmental deficits, supporting previous findings on the interplay between the nervous and the immune system. Further, our results validate the knock-in mouse model, and offer an objective assessment tool for patients that could be incorporated in diagnosis and in the evaluation of treatments

What Works When Treating Granulomatous Disease in Genetically Undefined CVID? A Systematic Review

Background: Granulomatous disease is reported in at least 8–20% of patients with common variable immunodeficiency (CVID). Granulomatous disease mainly affects the lungs, and is associated with significantly higher morbidity and mortality. In half of patients with granulomatous disease, extrapulmonary manifestations are found, affecting e.g. skin, liver, and lymph nodes. In literature various therapies have been reported, with varying effects on remission of granulomas and related clinical symptoms. However, consensus recommendations for optimal management of extrapulmonary granulomatous disease are lacking. Objective: To present a literature overview of the efficacy of currently described therapies for extrapulmonary granulomatous disease in CVID (CVID+EGD), compared to known treatment regimens for pulmonary granulomatous disease in CVID (CVID+PGD). Methods: The following databases were searched: Embase, Medline (Ovid), Web-of-Science Core Collection, Cochrane Central, and Google Scholar. Inclusion criteria were 1) CVID patients with granulomatous disease, 2) treatment for granulomatous disease reported, and 3) outcome of treatment reported. Patient characteristics, localization of granuloma, treatment, and association with remission of granulomatous disease were extracted from articles. Results: We identified 64 articles presenting 95 CVID patients with granulomatous disease, wherein 117 different treatment courses were described. Steroid monotherapy was most frequently described in CVID+EGD (21 out of 53 treatment courses) and resulted in remission in 85.7% of cases. In CVID+PGD steroid monotherapy was described in 15 out of 64 treatment courses, and was associated with remission in 66.7% of cases. Infliximab was reported in CVID+EGD in six out of 53 treatment courses and was mostly used in granulomatous disease affecting the skin (four out of six cases). All patients (n = 9) treated with anti-TNF-α therapies (infliximab and etanercept) showed remission of extrapulmonary granulomatous disease. Rituximab with or without azathioprine was rarely used for CVID+EGD, but frequently used in CVID+PGD where it was associated with remission of granulomatous disease in 94.4% (17 of 18 treatment courses). Conclusion: Although the number of CVID+EGD patients was limited, data indicate that steroid monotherapy often results in remission, and that anti-TNF-α treatment is effective for granulomatous disease affecting the skin. Also, rituximab with or without azathioprine was mainly described in CVID+PGD, and only in few cases of CVID+EGD