Heterozygosity for transmembrane activator and calcium modulator ligand interactor A144E causes haploinsufficiency and pneumococcal susceptibility in mice

Abstract

Background: The B-cell receptor transmembrane activator and calcium modulator ligand interactor (TACI) is important for T-independent antibody responses. One in 200 blood donors are heterozygous for the TACI A181E mutation.

Objective: We sought to investigate the effect on B-cell function of TACI A181E heterozygosity in reportedly healthy subjects and of the corresponding TACI A144E mutation in mice.

Methods: Nuclear factor κB (NF-κB) activation was measured by using the luciferase assay in 293T cells cotransfected with wild-type and mutant TACI. TACI-driven proliferation, isotype switching, and antibody responses were measured in B cells from heterozygous TACI A144E knock-in mice. Mouse mortality was monitored after intranasal pneumococcal challenge.

Results: Levels of natural antibodies to the pneumococcal polysaccharide component phosphocholine were significantly lower in A181E-heterozygous than TACI-sufficient Swedish blood donors never immunized with pneumococcal antigens. Although overexpressed hTACI A181E and mTACI A144E acted as dominant-negative mutations in transfectants, homozygosity for A144E in mice resulted in absent TACI expression in B cells, indicating that the mutant protein is unstable when naturally expressed. A144E heterozygous mice, such as TACI^+/- mice, expressed half the normal level of TACI on their B cells and exhibited similar defects in a proliferation-inducing ligand-driven B-cell activation, antibody responses to TNP-Ficoll, production of natural antibodies to phosphocholine, and survival after intranasal pneumococcal challenge.

Conclusion: These results suggest that TACI A181E heterozygosity results in TACI haploinsufficiency with increased susceptibility to pneumococcal infection. This has important implications for asymptomatic TACI A181E carriers.

Keywords: B cells; Transmembrane activator and calcium modulator ligand interactor; common variable immunodeficiency; natural antibodies; pneumococcal susceptibility.

Figure 1. Blood donor carriers of the TACI A181E mutation have impaired natural antibody responses

A, Phosphocholine (PC) specific IgM and IgG antibody, B. Inhibition of anti-PC IgG binding to PC by increasing concentrations of NaSCN (left panel) and NaSCN molarity resulting in 50% inhibition of the O.D. (right panel). C, E. coli specific IgM and D, TT specific IgG in sera from Swedish asymptomatic subjects carrying a heterozygous TACI A181E mutation (n=14) and healthy controls (n=20). Data are expressed as OD at 405 nm or IU/ml. Sera were used at 1:100 dilution in A (for IgM), B, and D, and 1:500 dilution in A (IgG). Columns or symbols and bars in A–D represent means ± SEM. * p<0.5, ** p<0.01, *** p<0.001 by Student’s t-test.

Figure 2. The A144E mutation impairs TACI surface expression, but not mRNA expression, in mouse B cells

A, qRT-PCR analysis of Tnfrsf13b mRNA levels in purified B220⁺ splenic B cells from TACI^+/+ (+/+), TACI^+/− (+/−), TACI^+/A144E (+/mut), TACI^A144E/A144E (mut/mut), and TACI^−/− (−/−) mice. The mRNA expression of Tnfrsf13b compared to that of the housekeeping gene Gapdh is shown as a percentage of the ratio in B cells from TACI^+/+ WT controls. B and C, Representative FACS analysis (B) and pooled data (C) of the mean fluorescence intensity (MFI) of TACI surface expression on gated B220⁺ splenic B cells. D and E, Representative FACS analysis (D) and pooled data (E) of intracellular TACI expression in gated B220⁺ peripheral B cells. MFI data presented are the net value of TACI expression (MFI of TACI minus MFI of isotype control). Columns and bars in A, C and E represent mean ± SEM (n=3–5 mice per group). * p<0.05, ** p<0.01, *** p<0.001 by Student’s t-test n.d.= not detected.

Figure 3. Proliferation and in vitro immunoglobulin production in response to APRIL are impaired in B cells from A144E TACI mutant mice

A and B, Proliferation in response to APRIL (A) and IgG1 secretion in response to APRIL+IL-4 (B) by splenic B cells from TACI^+/+ (+/+) TACI^+/− (+/−), TACI^+/A144E (+/mut), TACI^A144E/A144E (mut/mut), and TACI^−/− (−/−) mice. Stimulation with anti-CD40+IL-4 was used as a control. Columns and bars represent means ± SEM (n= 5–7 mice per group). * p<0.05, ** p<0.01 by Student’s t-test.

Figure 4. Serum immunoglobulin levels and antibody responses to TNP-Ficoll in A144E TACI mutant mice

A, Serum IgM, IgG and IgA levels from non-immunized 8–12 week-old TACI^+/+ (+/+), TACI^+/− (+/−), TACI^+/A144E (+/mut), TACI^A144E/A144E (mut/mut) and TACI^−/− (−/−) mice. Each symbol represents an individual mouse and the horizontal lines indicate the mean (n=11–15 mice per group). B, IgM and IgG anti-TNP responses 14 days after immunization with TNP-Ficoll. C. IgG anti-NP response after immunization with NP-OVA. Data in B and C are presented as the optical density (OD) readings at 405 nm. Symbols and bars represent mean ± SEM (n= 5–11 mice per group). * p<0.05, ** p<0.01, *** p<0.001, ns= not significant. Student’s t-test was used in A, and two-way ANOVA was used in B.

Figure 5. Decreased levels of natural antibodies to phosphocholine and increased susceptibility to pneumococcal inhalation challenge in A144E TACI mutant mice

A, IgM (left) and IgG (right) PC specific antibody levels in sera from non-immunized 8–12 week-old TACI^+/+ (+/+) TACI^+/− (+/−), TACI^+/A144E (+/mut), and TACI^−/− (−/−) mice expressed as OD at 405 nm. n=7 each group. B, Kaplan-Meier survival curves following intranasal challenge of 8–12 week-old unimmunized naïve TACI^+/+ (+/+), TACI^+/− (+/−), TACI^+/A144E (+/mut) and TACI^−/− (−/−) mice with a S. pneumoniae serotype 3 strain (n= 12–23 per group). Symbols and bars in A indicate mean ± SEM and symbols in B represent the percent of surviving mice. * p<0.05, ** p<0.01, *** p<0.001. Two-way ANOVA was used in A and Log-rank (Mantel-Cox) test was used in B.