TACI triggers immunoglobulin class switching by activating B cells through the adaptor protein MyD88

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1 Revised NI-A13051-T, He et al. TACI triggers immunoglobulin class switching by activating B cells through the adaptor protein MyD88 Bing He, 1* Raul antamaria, 2* Weifeng Xu, 1 Montserrat Cols, 1 Kang Chen, 1 Irene Puga, 2 Meimei han 1, Huabao Xiong, 1 James B. Bussel, 3 April Chiu, 4 Anne Puel, 5,6 Jeanine Reichenbach, 7 László Marodi, 8 Rainer Döffinger, 9 Julia Vasconcelos, 10 Andrew Issekutz, 11 Jens Krause, 12 Graham Davies, 13 Xiaoxia Li, 14 Bodo Grimbacher, 15 Alessandro Plebani, 16 Eric Meffre, 17 Capucine Picard, 5,6 Charlotte Cunningham-Rundles, 1 Jean-Laurent Casanova, 5,6,18 and Andrea Cerutti 1,2,19 UPPLEMENTARY TABLE 1 2 UPPLEMENTARY FIGURE LEGEND 1 16 UPPLEMENTARY FIGURE 1 16 Page 1

2 UPPLEMENTARY TABLE Table 1. Patients with lesions of genes encoding TACI, MyD88 or IRAK-4 proteins. Patient Gene ex Age ubstitution Protein expression IgM * IgG * IgA * IgG to P23 # B140 TACI F 16 yr A181E/WT Yes 4 [88-184] 371 [ ] 14 [ ] NA A.II.2 TACI M 67 yr 144X/144X No NA 397 [ ] NA NA L54 TACI F 60 yr R72H/WT Yes NA 2 [ ] NA NA M150 TACI M 37 yr V220A/WT Yes 24 [88-184] 202 [ ] <3 [ ] NA M120 TACI M 44 yr C104R/WT Yes <17 [88-184] <7 [ ] <22 [ ] NA M139 TACI F 41 yr C104R/WT Yes 54 [88-184] 475 [ ] 47 [ ] NA P81 TACI F 45 yr C104R/194X Yes 24 [88-184] 200 [ ] <6 [ ] No response W155 TACI F 26 yr A181E/WT Yes 34 [88-184] 164 [ ] <7 [ ] NA P-3 MyD88 F 15 yr R196C/R196C Yes 69 [88-172] 1250 [ ] 136 [ ] NA P-10 MyD88 F 4 mo E52del/E52del No 93 [40-84] 620 [ ] 52 [10-58] NA P-12 MyD88 M 5 mo E52del/E52del No 32 [40-84] 379 [ ] 22 [10-58] NA P-2 IRAK4 M 14 yr Q293X/BAC210N13del No 64 [88-172] 1440 [ ] 88 [ ] No response P-8 IRAK4 M 6 yr G>T/ A>G No 52 [66-118] 1360 [ ] 242 [78-162] No response P-24 IRAK4 M 16 yr 1240insA/ _ del NA 191 [88-184] 760 [ ] 49 [ ] Response ## P-36 IRAK4 F 3 yr Y430X/ G>T No 159 [54-114] 882 [ ] 119 [46-110] Response ## P-37 IRAK4 F 12 yr G298D/G298D NA 128 [88-172] 1590 [ ] 137 [ ] NA * mg/dl (normal range in brackets [ ]: IgM; IgG; IgA). # Pneumovax 23 (normal response = increased x 3 of titer after one month vaccination) ## Response obtained after PCV7 (conjugate) and P23 (non-conjugate) vaccinations Not available Page 2

3 Table 2. Primers used to generate TACI, MyD88 and TIRAP constructs. Construct Human WT TACI Human D3 TACI Human D4 TACI Human A181E TACI Human 194X TACI Human R202H TACI Human TACI V220A Human P226A TACI Human E228R TACI Human 231R TACI Human C233G TACI Human WT GT-TACI Human GT-D1 TACI Human GT-D2 TACI Human GT-D3 TACI Human GT-D4 TACI Human GT-D5 TACI Human GT-D6 TACI Human GT-D7 TACI Human GT-A181E TACI Human GT-194X TACI Human GT-R202H TACI Human GT-P219A TACI Human GT-V220A TACI Primer sequence GGGAATTCATGAGTGGCCTGGGCCGGAGCAGG * A GGGCGGCCGCTTAATGGTGATGGTGATGATGTGCACCTG GGCCCCCCTC GGGAATTCATGAGTGGCCTGGGCCGGAGCAGG A CCGCGGCCGCTTACGCCCTGCACTCAGGG GGGAATTCATGAGTGGCCTGGGCCGGAGCAGG A CCGCGGCCGCTTATGGCTCGGGGGATGT TGCTGCTTCCTGGTGGAGGTGGCCTGCTTC A GAAGCAGGCCACCTCCACCAGGAAGCAGCA GGGGGATCCCTGCTAATGCCAGCCCCGCTC A GAGCGGGGCTGGCATTAGCAGGGATCCCCC CCCCGCTCAAGGCCCCATCAAAGTCCGGCCAAG A CTTGGCCGGACTTTGATGGGGCCTTGAGCGGGG AGCCGGCAGCCCTGCGAGCACATCCCCCG A TCGGGGGATGTGCTCGCAGGGCTGCCGGCT CACATCCCCCGAGGCAGTGGAGACCTGCAG A CTGCAGGTCTCCACTGCCTCGGGGGATGTG CCCGAGCCAGTGCGAACCTGCAGCTTCTGC A GCAGAAGCTGCAGGTTCGCACTGGCTCGGG CCAGTGGAGACCTGCAGATTCTGCTTCCCT A AGGGAAGCAGAATCTGCAGGTCTCCACTGG GAGACCTGCAGCTTCGGCTTCCCTGAGTGC A GCACTCAGGGAAGCCGAAGCTGCAGGTCTC CCGGAATTCGTGGCCCTG A GGGCGGCCGCTTATGCACCTGGGCCCCCCTC CCGGAATTCGTGGCCCTG A CCGCGGCCGCTTAGATGTGTGGGCAAGG CCGGAATTCGTGGCCCTG A CCGCGGCCGCTTAAGCACAAGTGGGGTCG CCGGAATTCGTGGCCCTG A CCGCGGCCGCTTACGCCCTGCACTCAGGG CCGGAATTCGTGGCCCTG A CCGCGGCCGCTTATGGCTCGGGGGATGT CCGGAATTCGTGGCCCTG A CCGCGGCCGCTTAGGCTTCCATCGCGTGA CCGGAATTCGTGGCCCTG A GGGCGGCCGCTTAGCAGGGATCCCCCCTC CCGGAATTCGTGGCCCTG A GGGCGGCCGCTTAGAGGAAGCAGGCCAC TGCTGCTTCCTGGTGGAGGTGGCCTGCTTC A GAAGCAGGCCACCTCCACCAGGAAGCAGCA GGGGGATCCCTGCTAATGCCAGCCCCGCTC A GAGCGGGGCTGGCATTAGCAGGGATCCCCC CCCCGCTCAAGGCCCCATCAAAGTCCGGCCAAG A CTTGGCCGGACTTTGATGGGGCCTTGAGCGGGG GGAAGCCGGCAGCGCTGTGAGCACATCC A GGATGTGCTCACAGCGCTGCCGGCTTCC AGCCGGCAGCCCTGCGAGCACATCCCCCG A TCGGGGGATGTGCTCGCAGGGCTGCCGGCT Page 3

4 Human GT-P226A TACI Human GT-E228R TACI Human GT-E229R TACI Human GT-231R TACI Human GT-C233G TACI Human GT-CD40 Human GT-BAFF-R Human GT-BCMA Human FLAG-MyD88 Human FLAG-(1-260)-MyD88 Human FLAG-(1-160)-MyD88 Human FLAG-(1-109)-MyD88 Human FLAG-( )-MyD88 Human FLAG-( )-MyD88 Human MyD88-HA Human TIRAP-HA Mouse GT-TACI Mouse GT-( )-TACI A A A A A A A A A A A A A A A A A A CACATCCCCCGAGGCAGTGGAGACCTGCAG CTGCAGGTCTCCACTGCCTCGGGGGATGTG CCCGAGCCAGTGCGAACCTGCAGCTTCTGC GCAGAAGCTGCAGGTTCGCACTGGCTCGGG CCCGAGCCAGTGGAGAGGTGCAGCTTCTGC GCAGAAGCTGCACCTCTCCACTGGCTCGGG CCAGTGGAGACCTGCAGATTCTGCTTCCCT AGGGAAGCAGAATCTGCAGGTCTCCACTGG GAGACCTGCAGCTTCGGCTTCCCTGAGTGC GCACTCAGGGAAGCCGAAGCTGCAGGTCTC GGGAATTCAAAAAGGTGGCCAAGAAGCCAACC GGGCGGCCGCTTACTGTCTCTCCTGCACTGAGAT GGGAATTCAGCTGGAGGCGGCGACAGCGGCGG GGGCGGCCGCTTATTGTTGCTCAGGGCCGGCCGTCTT GGGAATTCACCTGTTTGGGACTGAGCTTAATA GGGCGGCCGCTTACCTAGCAGAAATTGATTTCTC GGGAATTCGCTGCAGGAGGTCCCGGCGC CCGGTACCTTAGGGCAGGGACAAGGCCT GGGAATTCGCTGCAGGAGGTCCCGGCGC CCGGTACCTTAGCTGGGGAACTCTTTCTT GGGAATTCGCTGCAGGAGGTCCCGGCGC CCGGTACCTTAGCAGATGAAGGCATCGAA GGGAATTCGCTGCAGGAGGTCCCGGCGC CCGGTACCTTAAATGCTGGGTCCCAGCT GCGAATTCAGAGGAGGATTGCCAAAAG CTGGTACCTTAGGGCAGGGACAAGGCCTT GTCCGAATTCACGTTTCGATGCCTTCATCTG CTGGTACCTTAGGGCAGGGACAAGGCCTT CCAAGCTTAGGAGGGCCATCATGGCTGCAGG GATAGGATCCGGGCAGGGACAAG GGAAGCTTCCACGCTATGGCATCATCGAC GCGGATCCAAGTAGATCAGATACTGTAG CCGGAATTCAGCGACCAGCTGACTCTC ATGCGGCCGCTTAAGTTGCCGGACGAGC CCGGAATTCAGCGACCAGCTGACTCTC ATGCGGCCGCTTAAGCCTCTGTCACGGGGCG * Underlined sequences indicate restriction sites. Page 4

5 UPPLEMENTARY FIGURE Figure 1. TACI cross-linking induces IgG class switching by cooperating with cytokines. ELIA measuring IgG secretion by circulating pre-switched IgD + B cells exposed to a control antibody (ctrl) or anti-taci immobilized on the surface of adherent CD32 (Fcγ receptor II)-expressing L cells. Cultures were carried out for 7 d in the presence or absence (control) of IL-4, IL-10 or IL-21. Data summarize three experiments (error bars, s.e.m.). Page 5

6 Figure 2. TACI expression increases on B cells exposed to TLR ligands. (a) Flow cytometry of TLR5, TLR7 and TLR9 expression by splenic follicular (FO) IgD hi CD27 B cells (red gate in dot plot and red profiles) and splenic IgD lo CD27 + MZ B cells (blue gate in dot plot and blue profiles). Bars delimitate positive cells compared to control with isotype-matched antibody with irrelevant binding activity (black profiles). (b) Flow cytometry of TACI expression by splenic FO B cells and splenic MZ B cells in the presence (blue profiles) or absence (red profiles) of TLR5, TLR7 and TLR9 engagement by flagellin, imiquimod and CpG DNA, respectively. Isotype-matched antibodies with irrelevant binding activity were used as negative control (black profiles). Data represent one of three experiments with similar results. Page 6

7 Figure 3. TACI triggers IgG class switching by cooperating with CD40L. (a) Flow cytometry of IgG, IgA and CD27 on primary circulating IgD + B cells stimulated for 7 days with a control antibody (ctrl), anti-taci, IL-10 and/or CD40L. Numbers indicate percentages. (b) IgG secretion by circulating IgD + B cells exposed to CD40L and ctrl or anti-taci in the presence or absence of IL-10. (c) Immunofluorescence of IgD (green), AID, Pax5 or Ki-67 (red), and nuclei (blue) in the spleen from a healthy donor (HD) carrying WT TACI (upper panels) or a CVID patient carrying a heterozygous C104R/194X TACI substitution (lower panels). (d) Flow cytometric analysis of the IgD /IgD + B cell ratio in the peripheral blood of healthy subjects, CVID patients with TACI defects as well as control HIGM1 patients with CD40L defects or HIGM2 patients with AID defects. The CVID patients reported here correspond to those Page 7

8 listed in upplementary Table 1, except patient A.II.2, whose ratio is not available. *P <0.05 and **P <0.005, compared to HD (two-tailed paired tudent s t-test). Data represent one of three experiments with similar results (a-c) or summarize three or more experiments (d). Page 8

9 Figure 4. TACI interacts with MyD88. Left gels: Immunoblotting (IB) of HA or TRAF6 (loading control) from lysates of 293 cells expressing or not HA-MyD88. Right gels: GT-TACI immunoprecipitation (IP) of lysates from 293 cells expressing or not FLAG-MyD88, followed by IB of HA or GT (loading control). Data represent one of three experiments with similar results. Page 9

10 Figure 5. TACI co-localizes with MyD88, TRAF6, CAML and flotillin-1 upon exposure of B cells to APRIL. Confocal microscopy of TACI (red), TRAF6 (green) and MyD88 (blue) (a), TACI (red), MyD88 (green) and CAML (blue) (b), or TACI (red), MyD88 (green) and flotillin-1 (blue) (c) in primary circulating IgD + B cells exposed to medium (ctrl) or APRIL for 15 min. Arrowheads point to colocalization. Data represent one of five experiments with similar results. Page 10

11 Figure 6. TACI ligands do not induce binding of p50, p65 and c-rel to the I γ 1 promoter of B cells from MyD88 KO mice. EMA showing I γ 1 promoter-bound C3 protein complex from primary MyD88 KO mouse B cells cultured with BAFF for 3 h. C3, complex 3; N, non-specific band (loading control). Data represent one of three experiments with similar results. Page 11

12 Figure 7. TACI requires MyD88 to initiate C γ 1 gene transcription. QRT-PCR of germline I γ 1-C γ 1 transcripts from primary mouse WT (red) or MyD88 KO naive B cells of (blue) incubated with medium alone (ctrl), APRIL or IL-4 for 3, 6, 12, 24 or 48 h. Results are normalized to ACTB (encoding β-actin) mrna; RE, relative expression compared to B cells incubated with a control antibody (ctrl). The data shown are from one of three experiments giving similar results. Page 12

13 Figure 8. TACI encompasses five highly conserved cytoplasmic domains. Aligned cytoplasmic domains of TACI proteins from multiple species; HCD, highly conserved cytoplasmic domain. Upper graphics show degree of similarity and mean hydrophobicity. Page 13

14 Figure 9. CAML has two transmembrane domains and interacts with TRAF5 and TRAF6. (a) Amino acid sequence of CAML. Green, red and pink arrows indicate cytoplasmic, transmembrane and extracellular domains; blue box corresponds to TRAF-binding site (B). (b) Immunoblotting (IB) of CAML, TRAF5 and TRAF6 after immunoprecipitation (IP) of total lysates from 2E2 B cells incubated with an irrelevant IgG antibody (ctrl) or anti-caml. kda, kilodaltons. Arrowheads points to specific CAML and TRAF5 bands. Page 14

15 Figure 10. TACI requires its MyD88-binding and TRAF2-binding sites to initiate signaling. (a) chematic of TACI structure. ED, extracellular domain; TD, transmembrane domain; CD, cytoplasmic domain; B, binding site. Numbers indicate amino acid positions. (b) Immunoprecipitation (IP) of lysates from 2E2 B cells with WT GT-TACI or various GT-TACI mutants, followed by immunoblotting (IB) of MyD88, TRAF2, TRAF5, TRAF6, CAML or GT (loading control). (c) NF-κB and AP1 reporter assays in 293 cells expressing no TACI, WT TACI, or various TACI mutants. Bottom gel is an IB of TACI from 293 cell lysates. *P < 0.05 and **P < 0.05, compared to WT TACI (one-tailed unpaired tudent s t-test). Data represent one of three experiments with similar results (b) or summarize three experiments (c; error bars, s.e.m.). Page 15

16 Figure 11. DN-MyD88 proteins do not activate NF-κB. NF-κB luciferase reporter assay in 293 cells expressing neither TACI nor MyD88 (ctrl), WT TACI, WT (1-260)-MyD88, or various MyD88 deletion mutants. The data shown are from one of three experiments giving similar results (error bars, s.e.m.). Page 16

17 Figure 12. TACI activates NF-κB via a MyD88-dependent pathway involving IRAK1, IRAK-4, TRAF6, TAK1 and IKK, but not TRAF3. NF-κB reporter assays in 2E2 B cells expressing WT TACI without (no DN) or with DN-MyD88, DN-IRAK-1, DN-IRAK-4, DN-TRAF6, DN-TAK1, DN-IKKβ, or DN-TRAF3, and then exposed to medium alone (ctrl) or BAFF for 48 h. The data shown are from one of three experiments giving similar results. Page 17

18 Figure 13. TACI induction of AID is conserved or only marginally blunted in some patients with MyD88 or IRAK-4 deficiency. QRT-PCR of AICDA transcripts in B cells from three patients with MyD88 or IRAK-4 deficiency expressing normal IgD /IgD + B-cell ratios in their peripheral blood (circled cases in Figure 6a). B cells were cultured for 4 d in the presence or absence of BAFF plus IL-10, APRIL plus IL-10 or CpG DNA plus IL-10. Pink area indicates normal interval of AICDA induction in healthy donors. Results are normalized to ACTB (encoding β-actin) mrna; RE, relative expression compared to B cells incubated with a control antibody (ctrl). Page 18

19 Figure 14. TACI delivers class switch-inducing signals in B cells exposed to BAFF- and APRILexpressing monocytes. (a) Flow cytometry of IgM and IgD on B cells from a healthy donor (HD), an AID-deficient (HIGM2) patient, or a CVID patient carrying compound heterozygous C104R/194X TACI substitutions after exposure to autologous monocytes for 5 d. Bottom-left numbers indicate percentage of class-switched B cells lacking both IgM and IgD. (b) BAFF and APRIL release by monocytes from the same subjects as in a. Data represent one of three experiments with similar results (a) or summarize three experiments (b; error bars, s.d.). Page 19

20 Figure 15. TACI survival signals are slightly attenuated by the lack of MyD88. Flow cytometric analysis of live 7-AAD-negative splenic B cells from wt or MyD88 KO mice cultured for 4 d with medium (control, ctrl), BAFF or APRIL. Page 20

21 Figure 16. TACI signals CR through a TLR-like TIR-independent pathway involving MyD88. Engagement of TACI by BAFF and/or APRIL from dendritic cells triggers oligomerization of TACI and recruitment of MyD88 to the THC domain. ubsequent recruitment of IRAK-1, IRAK-4 and TAK-1 (which forms a complex with two additional proteins known as TAB1 and TAB2), elicits activation of the IKK complex, which in turn triggers phosphorylation and proteasome-dependent degradation of IκBα. This process is further enhanced by binding of TRAF2 to a TRAF2-binding site (TB) in the cytoplasmic Page 21

22 tail of TACI located immediately downstream of THC. Phosphorylation and degradation of IκBα are followed by nuclear translocation of NF-κB, which interacts with cis-regulatory κb sites to initiate transcriptional activation of germline C H gene promoters downstream of C and AICDA gene promoter. These transcriptional events increase and eventually elicit CR in the presence of co-signals provided by cytokines and microbial TLR ligands (antigen). Cytokine receptors induce phosphorylation and nuclear translocation of TAT dimers, which bind to GA motifs proximal to κb sites in C H and AICDA gene promoters. Finally, TLR ligands signal through a canonical TIR-dependent pathway. TLR ligands cooperate with TACI ligands and cytokines to induce antibody secretion. Double arrows indicate receptor dimerization or oligomerization.. Page 22