Hypomorphic Mutations in the BCR Signalosome Lead to Selective Immunoglobulin M Deficiency and Impaired B-cell Homeostasis

Abstract

B cell activation via the B cell receptor (BCR) signalosome involves participation of signaling molecules such as BTK and BLNK. Genetic defects in these molecules are known to impair B cell differentiation and subsequently lead to agammaglobulinemia. Here we identified novel mutations in BTK and BLNK in two unrelated patients that perturb the intrinsic B-cell receptor signaling pathway and lead to selective IgM deficiency, whereas production of other immunoglobulin isotypes and IgG antibody response remain intact. Currently it is unknown how BCR signaling strength affects mature B cell development in humans. Both patients show reduced levels of BCR signalosome phosphorylation as well as impaired BCR-dependent Ca²⁺ influx, which was accompanied by a marked decrease in IgD⁺IgM⁺CD27⁺ MZ-like B-cells. We further describe reduced expression of essential B cell differentiation factors such as BAFF-R and T-Bet in the patients' B-cells, which might contribute to the observed deficiency of MZ-like B cells. MZ-like B cells are known to produce natural IgM antibodies that play an essential role in immune homeostasis. By using surface plasmon resonance (SPR) technology and a synthetic blood group A trisaccharide as antigen we were able to show that both patients lack the presence of anti-blood group A IgM considered to be prototypical natural antibodies whereas IgG levels were normal. Antibody binding dynamics and binding affinity of anti-blood group A IgG were comparable between patients and healthy controls. These results indicate that human IgM deficiency can be associated with signaling defects in the BCR signalosome, defective production of natural IgM antibodies in the blood group A/B/0 system and abnormalities in B cell development.

Keywords: B-cell defects; BLNK; BTK; marginal-zone B cells; natural antibodies; primary immunodeficiency; selective IgM deficiency.

Figure 1

Molecular characterization of novel hypomorphic BTK/BLNK mutations. (A) Gene expression of precursor, secreted and membrane IgM of patients and control EBV-LCL quantified by semi-quantitative cDNA-PCR. (B) Protein expression of monomeric and pentameric IgM of patients and control EBV-LCLs by SDS-PAGE or native-PAGE and detected by western blot. (C) BTK/BLNK Mutation analysis of genomic DNA from peripheral blood. Patient A is hemizygote for a c615G>T in BTK. Patient B is compound heterozygous in BLNK for c328C>G, c472G>T. Healthy controls served as wild type control. (D) Schematic depiction of mutation sites and phosphorylation sites in BTK and BLNK.

Figure 2

Effects of hypomorphic BTK/BLNK mutations on BCR signaling. (A) Representative FACS histograms depicting BTK (left) and BLNK (right) expression of αIgM/αIgD -stimulated EBV-LCLs. Blue histograms represent patients, black histograms represent healthy control and dotted gray histograms represent isotype control. (B) Bar graphs and representative flow cytometry plots showing the expression of pBtk, pBLNK, and pPLCγ2 in αIgM/αIgD -stimulated EBV-LCLs. Blue histograms represent patient A, green histograms represent patient B, black histograms represent healthy control, and dotted gray histograms represent isotype control. Results in bar graphs are expressed as mean fluorescence intensity (MFI, mean ± SD) of stimulated CD20⁺ EBV-LCLs after subtraction of expression of unstimulated CD20⁺ EBV-LCLs (no significant difference was found in basal expression between controls and patients, data not shown). CD20⁺ EBV-LCLs were stimulated with αIgM and αIgD antibodies. Bars represent the mean and standard deviation of five healthy controls and three repeat experiments using cells from the two patients. (ns = statistically not significant, ^*p ≤ 0.05, ^**p ≤ 0.01, Mann Whitney U-test) (C) Kinetics plot showing calcium influx of Fluo-4 loaded peripheral CD19⁺ B cells from patient A and healthy control following activation with αIgM and αIgD antibodies.

Figure 3

Altered B-cell compartment in sIgMD with impaired BCR signaling. (A) Bar graphs and representative flow cytometry plots showing percent of total CD19⁺B cells of Naïve (CD19⁺IgD⁺CD27⁻) Transitional T1/2 (CD19⁺CD27⁻CD24^highCD38^high) Follicular FO (CD19⁺CD27⁻CD24^dimCD38^dim) MZ (CD19⁺CD27⁺IgD⁺IgM⁺), Class Switched Memory (CD19⁺CD27⁺IgD⁻IgM⁻) IgM only (CD19⁺IgD⁻IgM⁺), CD21low (CD19⁺IgM⁺CD21^lowCD38^low), and. Patients are depicted as filled circles (•) and healthy controls (n = 14) as filled squares (■), horizontal bars represent the mean. (B,C) Bar graphs and representative flow cytometry plots showing the expression of BAFF-R, T-Bet, NOTCH2, and TACI. Blue histograms represent patient A, green histograms represent patient B, black histograms represent healthy control and dotted gray histograms represent isotype control. Results are expressed as mean fluorescence intensity (MFI, mean ± SD) on stimulated peripheral CD19⁺ B-cells or stimulated CD20⁺ EBV-LCLs after subtracting expression of unstimulated CD19⁺ B-cells or stimulated CD20⁺ EBV-LCLs (no significant difference was found in basal expression between controls and patients, data not shown). Peripheral CD19⁺ B-cells (B) and CD20⁺ EBV-LCLs (C) were stimulated with αIgM and αIgD antibodies. Bars represent the mean and standard deviation of three experiments. ns, statistically not significant; ^*p ≤ 0.05, Mann Whitney U-test.

Figure 4

Analysis of antibody response in patients with impaired BCR signaling.(A) In healthy individuals (n = 80, circles) and patients A and B (n = 2, squares) antibodies against pneumococcal polysaccharides (anti-PnPs) were determined by ELISA and presented as IgM–Pn23-antibody response (left panel) or IgG–Pn23-antibody response (right panel). Healthy controls and patient A were immunized with the PnPs vaccine Pneumo 23 Vaccine “Pasteur Merieux” (Pn23), and blood samples were drawn 4–6 weeks after vaccination. (B) Serum samples were diluted in HES-EP buffer (1:2) and samples were injected over the blood group A trisaccharide-coupled CM5 sensor chip. Amounts of anti-A antibodies were recorded as sensorgrams in resonance units (RU) against time in FC-I and FC-II. (C) Estimated affinity constant of blood group specific anti-A IgG antibodies is calculated as KD and t_1/2. Horizontal bars represent the mean.