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

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Presentation on theme: "Heterozygosity for transmembrane activator and calcium modulator ligand interactor A144E causes haploinsufficiency and pneumococcal susceptibility in."— Presentation transcript:

1 Heterozygosity for transmembrane activator and calcium modulator ligand interactor A144E causes haploinsufficiency and pneumococcal susceptibility in mice  Haifa H. Jabara, BSc, John J. Lee, MD, Erin Janssen, MD, PhD, Sumana Ullas, MS, Kyriaki Liadaki, PhD, Lilit Garibyan, MD, PhD, Halli Benson, BS, Tatyana Sannikova, BS, Richard Bram, MD, PhD, Lennart Hammarstrom, MD, Anthony C. Cruz, BS, Richard Siegel, MD, PhD, John Manis, MD, Richard Malley, MD, Raif S. Geha, MD  Journal of Allergy and Clinical Immunology  Volume 139, Issue 4, Pages e4 (April 2017) DOI: /j.jaci Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions

2 Fig 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 sodium thiocyanate (NaSCN; left panel) and NaSCN molarity, resulting in 50% inhibition of the OD (right panel). C and D, E coli–specific IgM (Fig 1, C) and tetanus toxoid (TT)–specific IgG (Fig 1, D) in sera from Swedish asymptomatic subjects carrying a heterozygous TACI A181E mutation (n = 14) and healthy control subjects (n = 20). Data are expressed as OD at 405 nm or international units per milliliter. Sera were used at 1:100 dilution in Fig 1, A (for IgM), B, and D, and 1:500 dilution in Fig 1, A (for IgG). Columns or symbols and bars represent means ± SEMs. *P < .05, **P < .01, and ***P < .001, Student t test. ns, Not significant. Journal of Allergy and Clinical Immunology  , e4DOI: ( /j.jaci ) Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions

3 Fig 2 The A144E mutation impairs TACI surface expression, but not mRNA expression, in mouse B cells. A, Quantitative RT-PCR analysis of Tnfrsf13b mRNA levels in purified B220+ splenic B cells from TACI+/+ (+/+), TACI+/− (+/−), TACI+/A144E (+/mut), TACIA144E/A144E (mut/mut), and TACI−/− (−/−) mice. mRNA expression of Tnfrsf13b compared with that of the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (Gapdh) is shown as a percentage of the ratio in B cells from TACI+/+ WT control mice. B and C, Representative fluorescence-activated cell sorting analysis (Fig 2, B) and pooled data (Fig 2, C) of the mean fluorescence intensity (MFI) of TACI surface expression on gated B220+ splenic B cells. D and E, Representative fluorescence-activated cell sorting analysis (Fig 2, D) and pooled data (Fig 2, 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 Fig 2, A, C, and E, represent means ± SEMs (n = 3-5 mice per group). *P < .05, **P < .01, and ***P < .001, Student t test. n.d., Not detected. Journal of Allergy and Clinical Immunology  , e4DOI: ( /j.jaci ) Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions

4 Fig 3 Proliferation and in vitro immunoglobulin production in response to APRIL are impaired in B cells from A144E TACI mutant mice. Proliferation in response to APRIL (A) and IgG1 secretion in response to APRIL plus IL-4 (B) by splenic B cells from TACI+/+ (+/+) TACI+/− (+/−), TACI+/A144E (+/mut), TACIA144E/A144E (mut/mut), and TACI−/− (−/−) mice. Stimulation with anti-CD40 plus IL-4 was used as a control. Columns and bars represent means ± SEMs (n = 5-7 mice per group). *P < .05 and **P < .01, Student t test. Journal of Allergy and Clinical Immunology  , e4DOI: ( /j.jaci ) Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions

5 Fig 4 Serum immunoglobulin levels and antibody responses to TNP-Ficoll in A144E TACI mutant mice. A, Serum IgM, IgG, and IgA levels from nonimmunized 8- to 12-week-old TACI+/+ (+/+), TACI+/− (+/−), TACI+/A144E (+/mut), TACIA144E/A144E (mut/mut), and TACI−/− (−/−) mice. Each symbol represents an individual mouse, and horizontal lines indicate means (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 Fig 4, B and C, are presented as OD readings at 405 nm. Symbols and bars represent means ± SEMs (n = 5-11 mice per group). *P < .05, **P < .01, and ***P < .001. ns, Not significant. The Student t test was used in Fig 4, A, and 2-way ANOVA was used in Fig 4, B. Journal of Allergy and Clinical Immunology  , e4DOI: ( /j.jaci ) Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions

6 Fig 5 Decreased levels of natural antibodies to phosphocholine (PC) and increased susceptibility to pneumococcal inhalation challenge in TACI A144E mutant mice. A, IgM (left) and IgG (right) PC-specific antibody levels in sera from nonimmunized 8- to 12-week-old TACI+/+ (+/+), TACI+/− (+/−), TACI+/A144E (+/mut), and TACI−/− (−/−) mice expressed as OD at 405 nm (n = 7 for each group). B, Kaplan-Meier survival curves after intranasal challenge of 8- to 12-week-old unimmunized naive TACI+/+ (+/+), TACI+/− (+/−), TACI+/A144E (+/mut), and TACI−/− (−/−) mice with an S pneumoniae serotype 3 strain (n = 12-23 per group). Symbols and bars in Fig 5, A, indicate means ± SEMs, and symbols in Fig 5, B, represent the percentage of surviving mice. *P < .05, **P < .01, and ***P < .001. Two-way ANOVA was used in Fig 5, A, and the log-rank (Mantel-Cox) test was used in Fig 5, B. Journal of Allergy and Clinical Immunology  , e4DOI: ( /j.jaci ) Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions

7 Fig E1 Strategy for the generation of TACI A144E knock-in mice. A, Genomic structure of murine Tnfrsf13b and the targeting construct, predicted structure of the targeted allele after homologous recombination, and predicted structure of the targeted allele after Cre-mediated removal of the neo gene by breeding with EIIa-Cre mice. Exons are represented by blue boxes, the neomycin resistance gene (neo) is represented by the red box, and the thymidine kinase gene (tk) is represented by the green box. Black triangles represent loxP sites. Primers a and b were used for genotyping. B, PCR analysis of tail genomic DNA from TACI+/+ (+/+), TACIA144E/A144E (mut/mut), and TACI+/A144E (+/mut) mice. Journal of Allergy and Clinical Immunology  , e4DOI: ( /j.jaci ) Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions

8 Fig E2 TACI A181E and A144E mutants assemble with their WT counterparts and act as dominant-negative mutations in 293T cell transfectants. A and B, Representative immunoblots showing the association of WT and A181E hTACI (Fig E2, A) and WT and A144E mTACI in 293T cell transfectants (Fig E2, B). C and D, NF-κB induction by APRIL in 293T cells transfected with WT hTACI, A181E hTACI or both (Fig E2, A) or with WT mTACI, A144E mTACI, or both (Fig E2, B). Fold induction was calculated by dividing the normalized NF-κB luciferase result of APRIL-treated samples by the NF-κB luciferase result of untreated samples. Blots in Fig E2, A and B, are representative of 3 independent experiments. Results in Fig E2, C and D, represent means and SDs of 3 independent experiments. *P < .05 and ***P < .001, Student t test. Journal of Allergy and Clinical Immunology  , e4DOI: ( /j.jaci ) Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions

9 Fig E3 Normal B-cell development in TACI A144E mice. B-cell subpopulations were examined in the bone marrow, spleen, and peritoneal cavity of 6- to 8-week-old TACI+/+ (+/+), TACI+/− (+/−), TACI+/A144E (+/mut), TACIA144E/A144E (mut/mut), and TACI−/− (−/−) mice. A, Percentages of B-cell subpopulations in the bone marrow. Left panel, CD43+B220hiIgM− pro-B, CD43−B220hiIgM− pre-B, and CD43−B220hiIgMmed immature B cells. Right panel, Mature B220hiIgM+ B cells. B, Percentages of CD3+ T cells and B220+ B cells in the spleen. C, Subpopulations of CD23−CD21hi marginal zone (MZ), CD23hiCD21lo follicular (FO), CD24+CD21− transitional 1 (T1), and CD23+CD21+ transitional 2 (T2) cells among the B220+IgM+ splenic B cells. D, Percentages of subpopulations of CD23− B-1, CD23+ B-2, and CD23-CD5+CD11b+ B-1a cells within the B220+ cell population in the peritoneal lavage cavity. Columns and bars represent means ± SEMs (n = 5 mice per group). *P < .05 and **P < .01. Journal of Allergy and Clinical Immunology  , e4DOI: ( /j.jaci ) Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions

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