The TH1 phenotype of follicular helper T cells indicates an IFN-γ-associated immune dysregulation in patients with CD21low common variable immunodeficiency.

BACKGROUND: A subgroup of patients with common variable immunodeficiency (CVID) experience immune dysregulation manifesting as autoimmunity, lymphoproliferation, and organ inflammation and thereby increasing morbidity and mortality. Therefore treatment of these complications demands a deeper comprehension of their cause and pathophysiology. OBJECTIVES: On the basis of the identification of an interferon signature in patients with CVID with secondary complications and a skewed follicular helper T-cell differentiation in defined monogenic immunodeficiencies, we sought to determine the profile of CD4 memory T cells in blood and secondary lymphatic tissues of these patients. METHODS: We quantified TH1/TH2/TH17 CD4 memory T cells in blood and lymph nodes of patients with CVID using flow cytometry, analyzed their function, and correlated all findings to the burden of immune dysregulation. RESULTS: Patients with CVID with immune dysregulation had a skewed memory CD4 T-cell differentiation toward a CXCR3+CCR6- TH1 phenotype both in blood and lymph nodes. Consistent with our phenotypic findings, we observed a higher IFN-γ production in peripheral CD4 memory T cells and lymph node-derived follicular helper T cells of patients with CVID compared with those of healthy control subjects. Increased IFN-γ production was accompanied by a poor germinal center output, an accumulation of T-box transcription factor (T-bet)+ B cells in lymph nodes, and an accumulation of T-bet+CD21low B cells in peripheral blood of affected patients. CONCLUSION: Identification of excessive IFN-γ production by blood and lymph node-derived T cells of patients with CVID with immune dysregulation will offer new therapeutic avenues for this subgroup. CD21low B cells might serve as a marker of this IFN-γ-associated dysregulation.

Effective Immunological Guidance of Genetic Analyses Including Exome Sequencing in Patients Evaluated for Hemophagocytic Lymphohistiocytosis.

We report our experience in using flow cytometry-based immunological screening prospectively as a decision tool for the use of genetic studies in the diagnostic approach to patients with hemophagocytic lymphohistiocytosis (HLH). We restricted genetic analysis largely to patients with abnormal immunological screening, but included whole exome sequencing (WES) for those with normal findings upon Sanger sequencing. Among 290 children with suspected HLH analyzed between 2010 and 2014 (including 17 affected, but asymptomatic siblings), 87/162 patients with "full" HLH and 79/111 patients with "incomplete/atypical" HLH had normal immunological screening results. In 10 patients, degranulation could not be tested. Among the 166 patients with normal screening, genetic analysis was not performed in 107 (all with uneventful follow-up), while 154 single gene tests by Sanger sequencing in the remaining 59 patients only identified a single atypical CHS patient. Flow cytometry correctly predicted all 29 patients with FHL-2, XLP1 or 2. Among 85 patients with defective NK degranulation (including 13 asymptomatic siblings), 70 were Sanger sequenced resulting in a genetic diagnosis in 55 (79%). Eight patients underwent WES, revealing mutations in two known and one unknown cytotoxicity genes and one metabolic disease. FHL3 was the most frequent genetic diagnosis. Immunological screening provided an excellent decision tool for the need and depth of genetic analysis of HLH patients and provided functionally relevant information for rapid patient classification, contributing to a significant reduction in the time from diagnosis to transplantation in recent years.

Early-onset Evans syndrome, immunodeficiency, and premature immunosenescence associated with tripeptidyl-peptidase II deficiency.

Autoimmune cytopenia is a frequent manifestation of primary immunodeficiencies. Two siblings presented with Evans syndrome, viral infections, and progressive leukopenia. DNA available from one patient showed a homozygous frameshift mutation in tripeptidyl peptidase II (TPP2) abolishing protein expression. TPP2 is a serine exopeptidase involved in extralysosomal peptide degradation. Its deficiency in mice activates cell death programs and premature senescence. Similar to cells from naïve, uninfected TPP2-deficient mice, patient cells showed increased major histocompatibility complex I expression and most CD8(+) T-cells had a senescent CCR7-CD127(-)CD28(-)CD57(+) phenotype with poor proliferative responses and enhanced staurosporine-induced apoptosis. T-cells showed increased expression of the effector molecules perforin and interferon-γ with high expression of the transcription factor T-bet. Age-associated B-cells with a CD21(-) CD11c(+) phenotype expressing T-bet were increased in humans and mice, combined with antinuclear antibodies. Moreover, markers of senescence were also present in human and murine TPP2-deficient fibroblasts. Telomere lengths were normal in patient fibroblasts and granulocytes, and low normal in lymphocytes, which were compatible with activation of stress-induced rather than replicative senescence programs. TPP2 deficiency is the first primary immunodeficiency linking premature immunosenescence to severe autoimmunity. Determination of senescent lymphocytes should be part of the diagnostic evaluation of children with refractory multilineage cytopenias.

B-cell signaling in persistent polyclonal B lymphocytosis (PPBL).

Persistent polyclonal B lymphocytosis (PPBL) is a benign hematological disorder characterized by a selective expansion of circulating polyclonal marginal zone (MZ)-like B cells. Previous reports demonstrated that cases of PPBL showed poor activation, proliferation and survival of B cells in vitro, yet the underlying defect remains unknown. Here we report for the first time an attenuated activation of the canonical NF-κB (nuclear factor of kappa light polypeptide gene enhancer in B cells) and mitogen-activated protein kinase/extracellular signal-regulated kinase pathway after CD40 stimulation. This defect was selective, as alternative NF-κB signaling after CD40 stimulation and both B-cell receptor- and Toll-like receptor 9-mediated activation remained unaffected. Reduced canonical NF-κB activation resulted in decreased IκBα and CD40 expression in resting cells. In PPBL patients, expression of Bcl-xL in MZ-like B cells did not increase upon activation, consistent with the high apoptosis rates of PPBL-derived B cells that were observed in vitro. The B-cell phenotype of mice with selective knockouts of early components of the CD40 signaling pathway resembles PPBL, but sequencing corresponding genes in sorted MZ-like B cells of PPBL patients did not reveal relevant genetic alterations. Nevertheless, the frequently observed mutations in early signaling components of the NF-κB pathway in MZ lymphomas underline the relevance of our findings for the pathogenesis of PPBL.

Hypomorphic homozygous mutations in phosphoglucomutase 3 (PGM3) impair immunity and increase serum IgE levels.

BACKGROUND: Recurrent bacterial and fungal infections, eczema, and increased serum IgE levels characterize patients with the hyper-IgE syndrome (HIES). Known genetic causes for HIES are mutations in signal transducer and activator of transcription 3 (STAT3) and dedicator of cytokinesis 8 (DOCK8), which are involved in signal transduction pathways. However, glycosylation defects have not been described in patients with HIES. One crucial enzyme in the glycosylation pathway is phosphoglucomutase 3 (PGM3), which catalyzes a key step in the synthesis of uridine diphosphate N-acetylglucosamine, which is required for the biosynthesis of N-glycans. OBJECTIVE: We sought to elucidate the genetic cause in patients with HIES who do not carry mutations in STAT3 or DOCK8. METHODS: After establishing a linkage interval by means of SNPchip genotyping and homozygosity mapping in 2 families with HIES from Tunisia, mutational analysis was performed with selector-based, high-throughput sequencing. Protein expression was analyzed by means of Western blotting, and glycosylation was profiled by using mass spectrometry. RESULTS: Mutational analysis of candidate genes in an 11.9-Mb linkage region on chromosome 6 shared by 2 multiplex families identified 2 homozygous mutations in PGM3 that segregated with disease status and followed recessive inheritance. The mutations predict amino acid changes in PGM3 (p.Glu340del and p.Leu83Ser). A third homozygous mutation (p.Asp502Tyr) and the p.Leu83Ser variant were identified in 2 other affected families, respectively. These hypomorphic mutations have an effect on the biosynthetic reactions involving uridine diphosphate N-acetylglucosamine. Glycomic analysis revealed an aberrant glycosylation pattern in leukocytes demonstrated by a reduced level of tri-antennary and tetra-antennary N-glycans. T-cell proliferation and differentiation were impaired in patients. Most patients had developmental delay, and many had psychomotor retardation. CONCLUSION: Impairment of PGM3 function leads to a novel primary (inborn) error of development and immunity because biallelic hypomorphic mutations are associated with impaired glycosylation and a hyper-IgE-like phenotype.

Next generation sequencing reveals a novel HLA-C allele, HLA-C*16:201.

HLA-C*16:201 differs from HLA-HLA-C*16:01:01 by one nucleotide substitution in codon 73 (ACT ->GCT).

Next-generation sequencing reveals a novel HLA-C allele, HLA-C*15:255.

HLA-C*15:255 differs from HLA-C*15:02:01 by five nucleotide substitutions located in exons 4 and 5.

A novel HLA-DPB1 allele, HLA-DPB1*1447:01, identified by next-generation sequencing.

HLA-DPB1*1447:01 differs from HLA-DPB1*04:01:01 by a single nucleotide in exon 2 from A to C at position 134.

A novel null-allele, HLA-B*35:574N, containing a mutation in the start-codon.

HLA-B*35:574N contains a single nucleotide substitution at nucleotide position 2 (ATG to ACG).

B cell receptor-mediated calcium signaling is impaired in B lymphocytes of type Ia patients with common variable immunodeficiency.

Several lines of evidence have demonstrated B cell intrinsic activation defects in patients with common variable immunodeficiency (CVID). The rapid increase of intracellular free calcium concentrations after engagement of the BCR represents one crucial element in this activation process. The analysis of 53 patients with CVID for BCR-induced calcium flux identified a subgroup of patients with significantly reduced Ca2+ signals in primary B cells. This subgroup strongly corresponded to the class Ia of the Freiburg classification. Comparison at the level of defined B cell subpopulations revealed reduced Ca2+ signals in all mature B cell populations of patients with CVID class Ia when compared with healthy individuals and other groups of patients with CVID but not in circulating transitional B cells. BCR-induced Ca2+ responses were the lowest in CD21low B cells in patients as well as healthy donors, indicating an additional cell-specific mechanism inhibiting the Ca2+ flux. Although proximal BCR signaling events are unperturbed in patients' B cells, including normal phospholipase Cgamma2 phosphorylation and Ca2+ release from intracellular stores, Ca2+ influx from the extracellular space is significantly impaired. CD22, a negative regulator of calcium signals in B cells, is highly expressed on CD21low B cells from patients with CVID Ia and might be involved in the attenuated Ca2+ response of this B cell subpopulation. These data from patients with CVID suggest that a defect leading to impaired BCR-induced calcium signaling is associated with the expansion of CD21low B cells, hypogammaglobulinemia, autoimmune dysregulation, and lymphadenopathy.

B-cell activating factor receptor deficiency is associated with an adult-onset antibody deficiency syndrome in humans.

B-cell survival depends on signals induced by B-cell activating factor (BAFF) binding to its receptor (BAFF-R). In mice, mutations in BAFF or BAFF-R cause B-cell lymphopenia and antibody deficiency. Analyzing BAFF-R expression and BAFF-binding to B cells in common variable immunodeficiency (CVID) patients, we identified two siblings carrying a homozygous deletion in the BAFF-R gene. Removing most of the BAFF-R transmembrane part, the deletion precludes BAFF-R expression. Without BAFF-R, B-cell development is arrested at the stage of transitional B cells and the numbers of all subsequent B-cell stages are severely reduced. Both siblings have lower IgG and IgM serum levels but, unlike most CVID patients, normal IgA concentrations. They also did not mount a T-independent immune response against pneumococcal cell wall polysaccharides but only one BAFF-R-deficient sibling developed recurrent infections. Therefore, deletion of the BAFF-R gene in humans causes a characteristic immunological phenotype but it does not necessarily lead to a clinically manifest immunodeficiency.

Circulating CD21low B cells in common variable immunodeficiency resemble tissue homing, innate-like B cells.

The homeostasis of circulating B cell subsets in the peripheral blood of healthy adults is well regulated, but in disease it can be severely disturbed. Thus, a subgroup of patients with common variable immunodeficiency (CVID) presents with an extraordinary expansion of an unusual B cell population characterized by the low expression of CD21. CD21(low) B cells are polyclonal, unmutated IgM(+)IgD(+) B cells but carry a highly distinct gene expression profile which differs from conventional naïve B cells. Interestingly, while clearly not representing a memory population, they do share several features with the recently defined memory-like tissue, Fc receptor-like 4 positive B cell population in the tonsils of healthy donors. CD21(low) B cells show signs of previous activation and proliferation in vivo, while exhibiting defective calcium signaling and poor proliferation in response to B cell receptor stimulation. CD21(low) B cells express decreased amounts of homeostatic but increased levels of inflammatory chemokine receptors. This might explain their preferential homing to peripheral tissues like the bronchoalveolar space of CVID or the synovium of rheumatoid arthritis patients. Therefore, as a result of the close resemblance to the gene expression profile, phenotype, function and preferential tissue homing of murine B1 B cells, we suggest that CD21(low) B cells represent a human innate-like B cell population.

The novel HLA-DQB1*06 null allele, HLA-DQB1*06:423N, identified in a volunteer blood donor.

A single nucleotide exchange in exon 2 at position 370 (C ->T) generates a preterminal STOP encoding a C-terminally truncated protein.

A novel HLA allele, HLA-A*03:420, identified by next-generation sequencing.

HLA-A*03:420 differs from HLA-A*03:01:01:01 by a single nucleotide in exon 1, codon -22.

Bronchoalveolar Lavage Fluid Reflects a TH1-CD21low B-Cell Interaction in CVID-Related Interstitial Lung Disease.

Background: About 20% of patients with common variable immunodeficiency (CVID) suffer from interstitial lung disease (ILD) as part of a systemic immune dysregulation. Current understanding suggests a role of B cells in the pathogenesis based on histology and increased levels of BAFF and IgM associated with active disease corroborated by several reports which demonstrate the successful use of rituximab in CVID-ILD. It is debated whether histological confirmation by biopsy or even video-assisted thoracoscopy is required and currently not investigated whether less invasive methods like a bronchoalveolar lavage (BAL) might provide an informative diagnostic tool. Objective: To gain insight into potential immune mechanisms underlying granulomatous and lymphocytic interstitial lung disease (GLILD) and to define biomarkers for progressive ILD by characterizing the phenotype of B- and T-cell populations and cytokine profiles in BAL fluid (BALF) of CVID-ILD compared to sarcoidosis patients and healthy donors (HD). Methods: Sixty-four CVID, six sarcoidosis, and 25 HD BALF samples were analyzed by flow cytometric profiling of B- and T-cells and for cytokines by ELISA and Multiplexing LASER Bead technology. Results: Both sarcoidosis and CVID-ILD are characterized by a predominantly T-cell mediated lymphocytosis in the BALF. There is an increase in T follicular helper (TFH)-like memory and decrease of regulatory T cells in CVID-ILD BALF. This TFH-like cell subset is clearly skewed toward TH1 cells in CVID-ILD. In contrast to sarcoidosis, CVID-ILD BALF contains a higher percentage of B cells comprising mostly CD21low B cells, but less class-switched memory B cells. BALF analysis showed increased levels of APRIL, CXCL10, and IL-17. Conclusion: Unlike in sarcoidosis, B cells are expanded in BALF of CVID-ILD patients. This is associated with an expansion of TFH- and TPH-like cells and an increase in APRIL potentially supporting B-cell survival and differentiation and proinflammatory cytokines reflecting not only the previously described TH1 profile seen in CVID patients with secondary immune dysregulation. Thus, the analysis of BALF might be of diagnostic value not only in the diagnosis of CVID-ILD, but also in the evaluation of the activity of the disease and in determining potential treatment targets confirming the prominent role of B-cell targeted strategies.

High levels of SOX5 decrease proliferative capacity of human B cells, but permit plasmablast differentiation.

Currently very little is known about the differential expression and function of the transcription factor SOX5 during B cell maturation. We identified two new splice variants of SOX5 in human B cells, encoding the known L-SOX5B isoform and a new shorter isoform L-SOX5F. The SOX5 transcripts are highly expressed during late stages of B-cell differentiation, including atypical memory B cells, activated CD21low B cells and germinal center B cells of tonsils. In tonsillar sections SOX5 expression was predominantly polarized to centrocytes within the light zone. After in vitro stimulation, SOX5 expression was down-regulated during proliferation while high expression levels were permissible for plasmablast differentiation. Overexpression of L-SOX5F in human primary B lymphocytes resulted in reduced proliferation, less survival of CD138neg B cells, but comparable numbers of CD138+CD38hi plasmablasts compared to control cells. Thus, our findings describe for the first time a functional role of SOX5 during late B cell development reducing the proliferative capacity and thus potentially affecting the differentiation of B cells during the germinal center response.

Autosomal dominant immune dysregulation syndrome in humans with CTLA4 mutations.

The protein cytotoxic T lymphocyte antigen-4 (CTLA-4) is an essential negative regulator of immune responses, and its loss causes fatal autoimmunity in mice. We studied a large family in which five individuals presented with a complex, autosomal dominant immune dysregulation syndrome characterized by hypogammaglobulinemia, recurrent infections and multiple autoimmune clinical features. We identified a heterozygous nonsense mutation in exon 1 of CTLA4. Screening of 71 unrelated patients with comparable clinical phenotypes identified five additional families (nine individuals) with previously undescribed splice site and missense mutations in CTLA4. Clinical penetrance was incomplete (eight adults of a total of 19 genetically proven CTLA4 mutation carriers were considered unaffected). However, CTLA-4 protein expression was decreased in regulatory T cells (Treg cells) in both patients and carriers with CTLA4 mutations. Whereas Treg cells were generally present at elevated numbers in these individuals, their suppressive function, CTLA-4 ligand binding and transendocytosis of CD80 were impaired. Mutations in CTLA4 were also associated with decreased circulating B cell numbers. Taken together, mutations in CTLA4 resulting in CTLA-4 haploinsufficiency or impaired ligand binding result in disrupted T and B cell homeostasis and a complex immune dysregulation syndrome.

Dependence of immunoglobulin class switch recombination in B cells on vesicular release of ATP and CD73 ectonucleotidase activity.

Immunoglobulin (Ig) isotype diversification by class switch recombination (CSR) is an essential process for mounting a protective humoral immune response. Ig CSR deficiencies in humans can result from an intrinsic B cell defect; however, most of these deficiencies are still molecularly undefined and diagnosed as common variable immunodeficiency (CVID). Here, we show that extracellular adenosine critically contributes to CSR in human naive and IgM memory B cells. In these cells, coordinate stimulation of B cell receptor and toll-like receptors results in the release of ATP stored in Ca(2+)-sensitive secretory vesicles. Plasma membrane ectonucleoside triphosphate diphosphohydrolase 1 CD39 and ecto-5'-nucleotidase CD73 hydrolyze ATP to adenosine, which induces CSR in B cells in an autonomous fashion. Notably, CVID patients with impaired class-switched antibody responses are selectively deficient in CD73 expression in B cells, suggesting that CD73-dependent adenosine generation contributes to the pathogenesis of this disease.