Sigune Goldacker

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. CD21low 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. CD21low 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. CD21low 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 CD21low B cells represent a human innate-like B cell population.

Tackling the heterogeneity of CVID.

Purpose of reviewCommon variable immunodeficiency is clinically the most relevant primary immunodeficiency of the adult. Its heterogeneity has hindered progress in the pathogenetic understanding of the majority of common variable immunodeficiency patients. This abstract summarizes recent aspects of the field and emphasizes the need for a commonly accepted approach to classify common variable immunodeficiency. Recent findingsIn the last 2 years, the first genetic defects underlying common variable immunodeficiency, including ICOS, TACI, BAFF-R and CD19, have been identified. The analysis of dendritic cells demonstrated alterations in a majority of patients in addition to the disturbed T and B-cell function. Several changes of the adaptive immune system might be secondary to an underlying chronic inflammatory setting possibly due to a HHV8 infection in a subgroup of patients with granulomatous disease, autoimmune phenomena and T-cell dysfunction. The occurrence of granulomatous inflammation is associated with a worse prognosis compared with common variable immunodeficiency patients without granuloma. SummaryThe pathogenesis of common variable immunodeficiency includes disturbances of the adaptive as well as innate immune system. Identified monogenic defects account for about 10% of cases, leaving the majority of defects undefined and certainly in part epigenetic. To combine the known aspects of the pathogenesis of common variable immunodeficiency to a conclusive picture, the clinical and immunologic phenotyping of patients needs to be standardized.

T- and B-lymphocyte abnormalities in bone marrow biopsies of common variable immunodeficiency

In common variable immunodeficiency (CVID) defects in early stages of B-cell development, bone marrow (BM) plasma cells and T lymphocytes have not been studied systematically. Here we report the first morphologic and flow cytometric study of B- and T-cell populations in CVID BM biopsies and aspirates. Whereas the hematopoietic compartment showed no major lineage abnormalities, analysis of the lymphoid compartment exhibited major pathologic alterations. In 94% of the patients, BM plasma cells were either absent or significantly reduced and correlated with serum immunoglobulin G levels. Biopsies from CVID patients had significantly more diffuse and nodular CD3(+) T lymphocyte infiltrates than biopsies from controls. These infiltrates correlated with autoimmune cytopenia but not with other clinical symptoms or with disease duration and peripheral B-cell counts. Nodular T-cell infiltrates correlated significantly with circulating CD4(+)CD45R0(+) memory T cells, elevated soluble IL2-receptor and neopterin serum levels indicating an activated T-cell compartment in most patients. Nine of 25 patients had a partial block in B-cell development at the pre-B-I to pre-B-II stage. Because the developmental block correlates with lower transitional and mature B-cell counts in the periphery, we propose that these patients might form a new subgroup of CVID patients.

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 Cγ2 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.

Outcomes of splenectomy in patients with common variable immunodeficiency (CVID): a survey of 45 patients

Splenectomy has been used in patients with common variable immunodeficiency disorders (CVID), mainly in the context of refractory autoimmune cytopenia and suspected lymphoma, but there are understandable concerns about the potential of compounding an existing immunodeficiency. With increasing use of rituximab as an alternative treatment for refractory autoimmune cytopenia, the role of splenectomy in CVID needs to be re‐examined. This retrospective study provides the largest cohesive data set to date describing the outcome of splenectomy in 45 CVID patients in the past 40 years. Splenectomy proved to be an effective long‐term treatment in 75% of CVID patients with autoimmune cytopenia, even in some cases when rituximab had failed. Splenectomy does not worsen mortality in CVID and adequate immunoglobulin replacement therapy appears to play a protective role in overwhelming post‐splenectomy infections. Future trials comparing the effectiveness and safety of rituximab and splenectomy are needed to provide clearer guidance on the second‐line management of autoimmune cytopenia in CVID.

Outcome of allogeneic stem cell transplantation in adults with common variable immunodeficiency

induction, making a role for secreted mediators unlikely (not shown). Another factor influencing FOXP3 induction is the strength of the T-cell receptor (TCR) signal and costimulation. In our settings the TCR signal was standardized by plate-bound anti-CD3, but costimulation is provided by the APCs. High costimulation through CD28 signaling prevents the upregulation of FOXP3. We therefore investigated the role of the CD28 ligands CD80 and CD86 on APCs. We observed no difference in the expression of these molecules. When blocking antibodies toward CD80, CD86, or both were added to the cultures, this did not result in a difference in FOXP3 expression (Fig 2, A). This finding was confirmed by adding increasing concentrations of cytotoxic T lymphocyte–associated antigen 4-immunoglobulin (CTLA4Ig) to the cultures, blocking both CD80 and CD86, which had no obvious effect (data not shown). Inhibition of downstream signaling pathways of TCR and CD28 on T-cell activation is able to promote FOXP3 induction as well. The phosphoinositide 3-kinase/protein kinase B (PKB) pathway is central in T-cell differentiation. Prevention of activation of this pathway by limited costimulation or signaling through inhibitory molecules, such as programmed death 1 (PD-1), promotes FOXP3 upregulation. We hypothesized that differential downstream signaling on activation might be pivotal to the high levels of FOXP3 found on day 6 of activation and onward (Fig 1, D). We therefore investigated the role of PD-1 and PKB in the induction of FOXP3 in CB-naive T cells. On activation, CB CD4 T cells have an increased expression of the PD-1 molecule compared with APB (Fig 2, B). When we blocked the interaction between PD-1 and its ligand PD-L1 by including a blocking mAb to PD-L1 in the culture, a significant reduction in the percentage of FOXP3 T cells was observed (Fig 2, C), isotype mAb had no effect (not shown). A PD-1–blocking mAb showed comparable results. As a consequence, the phosphorylation status of PKB on activation by different concentrations of anti-CD3 was lower for CB than APB cells (Fig 2, D). Additionally, CB T cells required approximately 10-fold more TCR triggering than APB T cells before an increase in PKB phosphorylation was observed. Shortly after birth, the immune system of the newborn encounters all kinds of neoantigens. The low percentages of Treg cells found ex vivo in CB are not likely to play a major role in maintaining tolerance to these neoantigens. However, we here show that PD-1 signaling in CB T cells facilitates their differentiation into induced functional FOXP3 Treg cells through a mechanism involving reduced PKB signaling. This phenomenon might well represent a mechanism that is developed to ensure active tolerance in the neonatal immune system. Sytze de Roock, MSc* Sanne B. E. A. Hoeks, MD* Linda Meurs Anouk Steur Maarten O. Hoekstra, MD, PhD Berent J. Prakken, MD, PhD Marianne Boes, PhD Ism e M. de Kleer, MD, PhD

Association of CLEC16A with human common variable immunodeficiency disorder and role in murine B cells.

Common variable immunodeficiency disorder (CVID) is the most common symptomatic primary immunodeficiency in adults, characterized by B cell abnormalities and inadequate antibody response. CVID patients have considerable autoimmune comorbidity and we therefore hypothesized that genetic susceptibility to CVID may overlap with autoimmune disorders. Here, in the largest genetic study performed in CVID to date, we compare 778 CVID cases with 10,999 controls across 123,127 single nucleotide polymorphisms (SNPs) on the Immunochip. We identify the first non-HLA genome-wide significant risk locus at CLEC16A (rs17806056, P=2.0×10−9) and confirm the previously reported human leukocyte antigen (HLA) associations on chromosome 6p21 (rs1049225, P =4.8×10−16). Clec16a knock down (KD) mice showed reduced number of B cells and elevated IgM levels compared to controls, suggesting that CLEC16A may be involved in immune regulatory pathways of relevance to CVID. In conclusion, the CLEC16A associations in CVID represent the first robust evidence of non-HLA associations in this immunodeficiency condition.

Screening of 181 patients with antibody deficiency for Deficiency of Adenosine Deaminase 2 sheds new light on the disease in adulthood

We aimed to test the relevance of deficiency of adenosine deaminase 2 (DADA2) in patients with antibody deficiency and describe the clinical picture of the disease in adulthood.

Sequential decisions on FAS sequencing guided by biomarkers in patients with lymphoproliferation and autoimmune cytopenia

Clinical and genetic heterogeneity renders confirmation or exclusion of autoimmune lymphoproliferative syndrome difficult. To re-evaluate and improve the currently suggested diagnostic approach to patients with suspected FAS mutation, the most frequent cause of autoimmune lymphoproliferative syndrome, we prospectively determined 11 biomarkers in 163 patients with splenomegaly or lymphadenopathy and presumed or proven autoimmune cytopenia(s). Among 98 patients sequenced for FAS mutations in CD3+TCRα/β+CD4−CD8− “double negative” T cells, 32 had germline and six had somatic FAS mutations. The best a priori predictor of FAS mutations was the combination of vitamin B12 and soluble FAS ligand (cut-offs 1255 pg/mL and 559 pg/mL, respectively), which had a positive predictive value of 92% and a negative predictive value of 97%. We used these data to develop a web-based probability calculator for FAS mutations using the three most discriminatory biomarkers (vitamin B12, soluble FAS ligand, interleukin-10) of the 11 tested. Since more than 60% of patients with lymphoproliferation and autoimmune cytopenia(s) in our cohort did not harbor FAS mutations, 15% had somatic FAS mutations, and the predictive value of double-negative T-cell values was rather low (positive and negative predictive values of 61% and 77%, respectively), we argue that the previously suggested diagnostic algorithm based on determination of double-negative T cells and germline FAS sequencing, followed by biomarker analysis, is not efficient. We propose vitamin B12 and soluble FAS ligand assessment as the initial diagnostic step with subsequent decision on FAS sequencing supported by a probability-calculating tool.

Ill-Defined Germinal Centers and Severely Reduced Plasma Cells are Histological Hallmarks of Lymphadenopathy in Patients with Common Variable Immunodeficiency

Given the severely reduced numbers of circulating class-switched memory B cells and plasmablasts in patients with common variable immunodeficiency (CVID) the germinal center (GC) reaction as the source of both populations is expected to be disturbed in many CVID patients. Therefore immunohistochemical studies were performed on lymph node (LN) biopsies from ten CVID patients with benign lymphoproliferation. According to the Sander classification the majority of patients presented with reactive lymphoid hyperplasia (7/10), 6/10 showed granulomatous inflammation. All cases showed some normal GCs but in 9/10 these concurred to a varying degree with hyperplastic, ill-defined GCs in the same LN. The percentage of ill-defined GCs correlated significantly with the percentage of circulating CD21low B cells suggesting a common origin of both immune reactions. In 9/10 CVID LNs significantly higher numbers of infiltrating CD8+ T cells were found in GCs of CVID patients compared to controls, but no HHV-8 and only in 2/10 LNs EBV infection was detected. Class switched plasma cells (PCs) were severely reduced in 8/10 LNs and if present, rarely found in the medulla of the LN. Based on the presence of large GCs in all examined patients, the reduction of circulating memory B cells and PCs points towards a failure of GC output rather than GC formation in CVID patients with lymphadenopathy.