PMC6299974_01
Unique CARD11 mutation in a four-generation family. (A) Pedigree of patients in a four-generation BENTA family. Squares and circles represent males and females, respectively, and black represents affected patients. (B) Sanger DNA sequencing showing a novel in-frame 12 bp deletion in genomic DNA derived from PBMC isolated from patients II.1, III.1 and IV.1 compared to healthy control (HC). (C) Schematic diagram showing the nucleotide and corresponding amino acid changes comprising the H234LDelta235-8 CARD11 variant found in patients vs. healthy control.
fimmu-09-02944-g0001.jpg
PMC6299974_01_fimmu-09-02944-g0001.jpg
F1
IgH gene rearrangement analysis demonstrated a polyclonal B-cell lymphocytosis pattern (Supplementary Figure 1). The family reported that parents were not related and were of Caucasian origin (Figure 1A). This analysis revealed a heterozygous four amino acid deletion and missense mutation within exon 5 of CARD11 (NM_032415.3 c.701_713delinsT, p.His234_Lys238delinsLeu, hereafter referred to as H234LDelta235-8) (Figures 1B,C). The grandmother and great-grandmother were later confirmed to carry the same pathological mutation (Figure 1B).
Abnormal NF-kappaB activation induced by H234LDelta235-8 CARD11 in vitro. (A) JPM50.6 T cells were transfected with 5 mug of either empty vector (EV), WT, E134G (GOF mutant), or H234LDelta235-8 mutant CARD11-FLAG plasmids alone (top panel) or in the presence of 5 mug WT CARD11 plasmid (bottom panel) as previously described. After 24 h incubation in complete RPMI, transfected cells were stimulated with 1 mug/ml of anti-CD3 and anti-CD28 Abs or left unstimulated for an additional 24 h. GFP expression (reflecting relative NF-kappaB activity) was subsequently measured by flow cytometry; %GFP cells are labeled in each histogram. (B) Quantification of NF-kappaB driven GFP reporter expression in transfected JPM50.6 cells as described in (A). Data are mean +- SEM for mean fluorescence intensity of GFP+ cells for 3 separate transfection experiments. Asterisks denote statistically significant differences (2-way ANOVA) between the stimulated groups indicated. (C) Immunoblots confirming the comparable expression of WT and mutant CARD1-FLAG proteins in cells from (A) at 24 h post-transfection. beta-actin served as a loading control. Data are representative of 3 separate experiments. (D) WT Jurkat T cells were transfected with WT or mutant CARD11-FLAG plasmids as in (A). After 24 h, cells were stimulated with phorbol 12-myristate 13-acetate (PMA) for 20 min and lysed. Immunoprecipitations (IPs) using anti-BCL10 Ab were performed as previously described. BCL10 IPs and input lysates were separated by SDS-PAGE and immunoblotted Abs against CARD11, BCL10, and MALT1. (E) JPM50.6 cells were transfected with WT CARD11-GFP -/+ WT or mutant CARD11-FLAG plasmids as in (A). After 24 h, cells were stimulated with PMA for 20 min and lysed. FLAG IPs and input lysates were immunoblotted for total CARD11 protein; arrows indicate GFP- vs. FLAG-tagged CARD11. Actin served as a loading control for input lysates. IPs in (D,E) are representative of 2 separate experiments each. (F) PBMC from 2 healthy controls (HC) and patients III.1 and IV.1 were stimulated with PMA (20 ng/ml) plus monensin (2 muM) for 20 min. Cells were stained with FITC-conjugated mouse anti-human CD4 mAb, fixed in 1.5% paraformaldehyde and permeabilized in ice cold methanol before staining with AlexaFluor647-conjugated mouse anti-human phospho-p65 (Ser529) or AlexaFluor647-conjugated mouse anti-human IkappaBalpha. NF-kappaB activation was assessed in gated CD4+ T cells by flow cytometry; numbers in each histogram denote % of phospho-p65+ (left) or IkappaBalphahi cells (right). Data are representative of 2 separate experiments.
fimmu-09-02944-g0002.jpg
PMC6299974_01_fimmu-09-02944-g0002.jpg
F2
H234LDelta235-8 induced constitutive NF-kappaB activation in unstimulated cells, increasing slightly upon TCR/CD28 crosslinking (Figures 2A,B). However, unlike E134G, co-expression of H234LDelta235-8 with WT CARD11 sharply reduced constitutive NF-kappaB activity. Strikingly, H234LDelta235-8 also significantly reduced WT CARD11-dependent NF-kappaB activation following TCR/CD28 stimulation (Figure 2B). CARD11 were similar in transfected cells (Figure 2C). Co-immunoprecipitations in WT Jurkat transfectants revealed a substantial reduction of total CARD11 association with BCL10 and MALT1 following stimulation in the presence of H234LDelta235-8 but not WT or E134G CARD11 (Figure 2D). Moreover, FLAG-tagged CARD11 variants co-precipitated with WT GFP-tagged CARD11 when co-expressed in JPM50.6 cells, indicating WT CARD11 can directly associate with mutant CARD11 protein regardless of stimulation (Figure 2E). Similar to other patients harboring CARD11 DN variants, we also observed marked defects in NF-kappaB p65 phosphorylation and IkappaBalpha degradation in stimulated primary CD4+ T cells from patients III.1 and IV.1 relative to healthy control subjects (Figure 2F). T cells secreted less IL-2 in response to all stimuli (Figure 3F, Supplementary Figure 2). These results are consistent with previous work demonstrating that BENTA patient B-cells exhibit intrinsic differentiation defects despite enhanced survival in vitro, whereas T cells from both BENTA (CARD11 GOF) and CARD11 DN patients are hyporesponsive in vitro.
Impaired patient B cell differentiation and T cell hyporesponsiveness to polyclonal stimuli in vitro. (A) PBMCs from mother (healthy control), III.1 and IV.1 were cultured in cRPMI media with anti-CD40 (1 mug/mL) + IL-4 (200 U/mL) + IL-21 (50 ng/mL) at 37 C and 5% CO2 for 7 days as previously described. B-cell subpopulations were analyzed by flow cytometry (Becton Dickinson LSRII, FlowJo Software) using Zombie aqua fixable viability dye and the following antibodies: PE-Cy7-conjugated mouse anti-human CD19, APC-conjugated mouse anti-human CD27, FITC-conjugated mouse anti-human CD38, PE-conjugated mouse anti-human IgM. Representative density plots are shown, with gates demarcating short-lived plasma cells (PC) (CD19+CD38+IgM-), class-switched (CS) memory B cells (CD19+CD27+IgM-) and marginal zone B-cells (CD19+CD27+IgM+). (B) Bar graph indicating the percentages of baseline naive B cells (CD19+CD27-IgM+) and marginal, CS memory B and short-lived PC after in vitro differentiation in healthy mother and patients. (C) Immunoglobulin production in cell supernatants after 7 days in vitro stimulation of healthy mother and patients' PBMC with anti-CD40+IL-4+IL-21, quantified by ELISA. Data in (A-C) are representative of three independent experiments. (D) Healthy control, II.1, III.1, and IV.1 patient CD4+ T cells were labeled with 1 muM CFSE and stimulated with 2 mug/ml of soluble anti-CD3 and CD28 Abs +- Protein A (2.5 mug/ml), or MACS iBead particles (1:1 bead:cell ratio) loaded with biotinylated anti-CD2, anti-CD3, and anti-CD28 Abs (Miltenyi Biotec) in cRPMI for 5-6 days. Histogram overlays display T cell proliferation based on CFSE dilution on day 5. (E) Bar graph denoting the percentage of dividing T cells in each group in response to various stimuli. (F) IL-2 levels in day 6 T cell culture supernatants were measured by ELISA as previously described. Data in (D-F) are representative of two independent experiments.
fimmu-09-02944-g0003.jpg
PMC6299974_01_fimmu-09-02944-g0003.jpg
F3
CD40 signaling, and IL-4 enhances these effects. Cells from patient (IV.1) and father (III.1) not only displayed poor short-lived plasma cell differentiation (Figure 3A, top panel) compared to the healthy mother (IV.1 0.6%, III.1 2.4%, HC 12.1%), but also decreased CD27+ memory B-cell differentiation including IgM- CD27+ CS memory B-cells (IV.1 2.4%, III.1 4.4%, HC 20%) (Figure 3A, bottom panel). B cell differentiation phenotypes were quantified in Figure 3B. These results were associated with marked decreases in production of IgM (IV.1 0.2 mg/L, III.1 0.4 mg/L, HC 3.7 mg/L), IgA (IV.1 0.0 mg/L, III.1 0.1 mg/L, HC 1.7 mg/L), and IgG (IV.1 0.4 mg/L, III.1 2.1 mg/L, HC 6.5 mg/L) (Figure 3C). Purified T cells from affected family members showed impaired proliferation in response to anti-CD3/CD28 Ab stimulation in vitro, which was largely rescued by more robust stimuli (Ab crosslinking or Ab-conjugated beads) (Figures 3D,E). T cells secreted less IL-2 in response to all stimuli (Figure 3F, Supplementary Figure 2). These results are consistent with previous work demonstrating that BENTA patient B-cells exhibit intrinsic differentiation defects despite enhanced survival in vitro, whereas T cells from both BENTA (CARD11 GOF) and CARD11 DN patients are hyporesponsive in vitro.