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1 ARTICLE NUMBER: DOI: /NMICROBIOL Microbially cleaved immunoglobulins are sensed by the innate immune receptor LILRA2 Kouyuki Hirayasu, Fumiji Saito, Tadahiro Suenaga, Kyoko Shida, Noriko Arase, Keita Oikawa, Toshifumi Yamaoka, Hiroyuki Murota, Hiroji Chibana, Ichiro Nakagawa, Tomoko Kubori, Hiroki Nagai, Yuji Nakamaru, Ichiro Katayama, Marco Colonna, Hisashi Arase* *Corresponding author. Hirayasu et al. Page S1 NATURE MICROBIOLOGY 1

2 DOI: /NMICROBIOL Supplementary Figure 1. Detection of IgM in the immunoprecipitated samples in Figure 1b. Cell lysates prepared from M. hyorhinis-infected Daudi cells were immunoprecipitated with LILRA2-Fc or control (LILRA3)-Fc and the precipitants were analyzed by Western blot with an anti-igm Ab. Data are representative examples from at least three independent experiments. Hirayasu et al. Page S2 2 NATURE MICROBIOLOGY

3 DOI: /NMICROBIOL SUPPLEMENTARY INFORMATION Supplementary Figure 2. IgM expression on Daudi cells. M. hyorhinis-infected and uninfected Daudi cells were stained with an anti-igm Ab and analyzed by flow cytometry. The red histograms indicate staining with an anti-human IgM antibody. The shaded histogram indicates control staining. Data are representative examples from at least three independent experiments. Hirayasu et al. Page S3 NATURE MICROBIOLOGY 3

4 DOI: /NMICROBIOL Supplementary Figure 3. Igκ light chain is not cleaved by M. hyorhinis infection. Igκ light chains in the cell lysates from M. hyorhinis-infected and uninfected Daudi cells were examined by Western blot. Data are representative examples from at least three independent experiments. Hirayasu et al. Page S4 4 NATURE MICROBIOLOGY

5 DOI: /NMICROBIOL SUPPLEMENTARY INFORMATION Supplementary Figure 4. LILRA2 binds to N-terminally truncated immunoglobulins. (a) LILRA2 binding to various N-terminally truncated IgM. IgM with various lengths of N-terminal truncation were transfected into 293T cells and the transfectants were stained with an anti-igm Ab or LILRA2-Fc (red histogram). Shaded histograms indicate control staining. The truncation position is shown as the number of amino acid (aa) residues from the IgM constant region. (b) LILRA2 binding to each class of cleaved immunoglobulins. IgM, IgG1, IgG2, IgG3, IgG4 and IgA1 containing the same Daudi-VDJ region were co-transfected together with Daudi Ig light chain and CD79α/β into M. hyorhinis-infected 293T cells. Red histograms indicate staining with LILRA2-Fc or anti-human Ig antibodies. Shaded histograms indicate control staining. Data are representative examples from at least three independent experiments. Hirayasu et al. Page S5 NATURE MICROBIOLOGY 5

6 DOI: /NMICROBIOL Supplementary Figure 5. Cleavage of each Ig class by M. hyorhinis infection. IgG1, IgG2, IgG3, IgG4 and IgA1 containing the same Daudi-VDJ region were co-transfected together with Daudi Ig light chain and CD79α/β into M. hyorhinis-infected and uninfected 293T cells. Transfected cell lysates were examined by Western blot analysis using anti-human IgG and IgA antibodies. The arrows indicate cleaved Igs. Data are representative examples from at least three independent experiments. Hirayasu et al. Page S6 6 NATURE MICROBIOLOGY

7 DOI: /NMICROBIOL SUPPLEMENTARY INFORMATION Supplementary Figure 6. Cleavage of IgM isolated from single human B cells by M. hyorhinis infection. Each IgM heavy chain and light chain pair from single human B cells was co-transfected together with CD79α/β into M. hyorhinis-infected and uninfected 293T cells. Transfected cell lysates were analyzed by Western blot using an anti-human IgM antibody. Data for seven IgM pairs (clone: D, 13, 19, 43, 1, 2 and 5) are shown. The arrows indicate cleaved IgM. +: IgM that binds to LILRA2; : IgM that does not bind to LILRA2. Data are representative examples from at least three independent experiments. Hirayasu et al. Page S7 NATURE MICROBIOLOGY 7

8 DOI: /NMICROBIOL Supplementary Figure 7. Fine mapping of the epitopes on cleaved Ig that is recognized by LILRA2. (a) An amino acid sequence alignment between #1-1 (LILRA2-non-binding) and #1-9 (LILRA2-binding) light chains was shown. Positioning of amino acids is according to Kabat- Chothia numbering system. There are only four amino acid differences in the amino acid residues 9, 10, 30A and 95 between them. (b) Amino acid residues 9, 10, 30A and 95 of #1-1 light chain were mutated to amino acids corresponding to #1-9, and the mutated #1 light chain was transfected together with membrane-bound IgM heavy chain and CD79α/β into M. hyorhinis-infected 293T cells. Cell surface IgM expressions and LILRA2-Fc bindings on the transfectants were shown. Data are representative examples from at least three independent experiments. Hirayasu et al. Page S8 8 NATURE MICROBIOLOGY

9 DOI: /NMICROBIOL SUPPLEMENTARY INFORMATION Supplementary Figure 8. Amino acid sequence alignments of various LILRA2-binding and LILRA2-non-binding light chains. Amino acid sequences around N-terminal and C-terminal V regions of light chains are shown. Positioning of amino acids is according to Kabat-Chothia numbering system. Conserved amino acids are highlighted by yellow. Hirayasu et al. Page S9 NATURE MICROBIOLOGY 9

10 DOI: /NMICROBIOL Supplementary Figure 9. LILRA2 recognition sites on immunoglobulin light chain. Structure of the Fab fragment of human IgM (PDB ID: 1DN0) that has light chain sequence similar to #1-9 light chain is illustrated using MacPyMOL software (DeLano Scientific LLC). Light chain and heavy chain are shown in green and yellow, respectively. Amino acids (Q95P and S10T) involved in binding to LILRA2 (Supplementary Figure 7) are shown in red. Hirayasu et al. Page S10 10 NATURE MICROBIOLOGY

11 DOI: /NMICROBIOL SUPPLEMENTARY INFORMATION Supplementary Figure 10. Mutagenesis analyses of N-terminal and C-terminal V regions of light chains. Amino acid residues (position 9-10 and 91-95) of V regions of LILRA2-nonbinding light chains (1-1, 1-2, K4) were mutated to amino acids corresponding to LILRA2- binding light chains (1-14, 1-9, and 1-3). The mutated light chains as well as wild type light chains were transfected into M. hyorhinis-infected 293T cells together with membrane-bound IgM heavy chain and CD79α/β. Cell surface IgM expressions and LILRA2-Fc bindings on the transfectants were shown. Data are representative examples from at least three independent experiments. Hirayasu et al. Page S11 NATURE MICROBIOLOGY 11

12 DOI: /NMICROBIOL Supplementary Figure 11. Microbially cleaved Ig without its Fc portion did not stimulate LILRA2 reporter cells. (a) LILRA2 reporter cells that do not express Fc receptor were stimulated with immobilized full-length IgG (K13-IgG3) or its Fab fragment with or without M. hyorhinis-infection. (b) A F(ab ) 2 fragment of truncated IgG (K13-IgG3) generated by pepsin treatment bound to LILRA2-expressing reporter cells. (c) LILRA2 reporter cells or parental reporter cells were stimulated with truncated IgG (K13-IgG3) or its F(ab ) 2 fragment that was immobilized on the plate. Data are representative examples from at least three independent experiments. Hirayasu et al. Page S12 12 NATURE MICROBIOLOGY

13 DOI: /NMICROBIOL SUPPLEMENTARY INFORMATION Supplementary Figure 12. Commensal bacteria from skin, nasal cavity and oral cavity of a healthy individual did not cleave IgM and IgG. Staphylococcus epidermidis, Staphylococcus capitis, Staphylococcus lugdunensis, Neisseria flavescens, and Veillonella dispar were isolated from skin, nasal cavity, and oral cavity of a healthy individual. IgM and IgG were incubated with the culture supernatants of these bacteria and then analyzed by Western blot. Data are representative examples from at least three independent experiments. Hirayasu et al. Page S13 NATURE MICROBIOLOGY 13

14 DOI: /NMICROBIOL Supplementary Figure 13. Identification of the L. pneumophila IgM-cleaving protease. (a) Flow chart outlining the identification of the IgM-cleaving protease in L. pneumophila culture supernatants. L. pneumophila culture supernatants were separated by ultrafiltration using molecular weight membranes of 10, 30 and 50 kda. The fraction containing proteins smaller than 50 kda and larger than 30 kda showed IgM-cleaving activity by Western blot. IgMcleaving fraction 1 was further fractionated by anion exchange chromatography on a Mono Q column. The IgM-cleaving fraction purified by anion exchange chromatography was subjected to mass spectrometry to identify the IgM-cleaving protease. (b) IgM-cleaving fraction obtained by ultrafiltration was separated by anion exchange chromatography. The IgM cleaving activity of each fraction was determined by Western blot. The fraction that showed the strongest IgMcleaving activity (red arrow) was analyzed by mass spectrometry. Hirayasu et al. Page S14 14 NATURE MICROBIOLOGY

15 DOI: /NMICROBIOL SUPPLEMENTARY INFORMATION Supplementary Figure 14. The cleavage patterns of immunoglobulins induced by MspA are different between immunoglobulin classes. IgG and IgA were treated with recombinant MspA and were analyzed by Western blot. Data are representative examples from at least three independent experiments. Hirayasu et al. Page S15 NATURE MICROBIOLOGY 15

16 kda 75 IgM 50 IB: anti-igm S. aureus #1 S. aureus #2 S. aureus #3 S. aureus #4 S. aureus #5 Medium kda DOI: /NMICROBIOL S. aureus #1 S. aureus #2 S. aureus #3 S. aureus #4 S. aureus #5 Medium IB: anti-ig kappa IgG IB: anti-igg IB: anti-ig kappa IgA IB: anti-iga IB: anti-ig kappa Supplementary Figure 15. Staphylococcus aureus cleaves heavy chains of IgM, IgG and IgA but not light chains. IgM, IgG and IgA were incubated with the culture supernatants of five clinical isolates of Staphylococcus aureus and analyzed by Western blot. Heavy chains and their corresponding light chains are displayed in left and right panels, respectively. The arrow indicates cleaved immunoglobulin heavy chains. S. aureus #1 and #2 cleaved IgM, IgG and IgA but not light chains. S. aureus #3, #4 and #5 did not cleave IgM but cleaved IgG and IgA. Data are representative examples from at least three independent experiments. Hirayasu et al. Page S16 16 NATURE MICROBIOLOGY

17 DOI: /NMICROBIOL SUPPLEMENTARY INFORMATION Supplementary Figure 16. No binding of various PIR-As and LILRs to cleaved IgM. Daudi- IgM heavy chain, Daudi Ig light chain and CD79α/β were co-transfected into M. hyorhinisinfected 293T cells and the transfectants were stained with PIR-Fc (a) and LILR-Fc (b) fusion proteins. PIRs are putative homologs of human LILRs in terms of expression pattern and chromosomal location. LILRA2-Fc fusion protein serves as a positive control. Red and shaded histograms indicate staining of the IgM and mock transfectants with each Fc-fusion protein, respectively. Data are representative examples from at least three independent experiments. Hirayasu et al. Page S17 NATURE MICROBIOLOGY 17

18 DOI: /NMICROBIOL Supplementary Figure 17. Antigen-binding to anti-hen egg lysozyme (HEL) B cell receptor was reduced by cleavage of heavy chain. Membrane-form of IgM heavy chain and light chain of anti-hel antibody were transfected together with CD79α/β into M. hyorhinis-infected (cleaved IgM) and uninfected (full-length IgM) 293T cells. Binding of DyLight 649-labeled HEL and LILRA2-Fc to the transfectants were analyzed. Staining control indicates secondary antibody staining. Data are representative examples from at least three independent experiments. Hirayasu et al. Page S18 18 NATURE MICROBIOLOGY

19 DOI: /NMICROBIOL SUPPLEMENTARY INFORMATION Antibody Immune evasion Cleavage Protease Detection of immune disorder Cleaved Ab Microbial Pathogen Defense LILRA2 Innate Activation immune cell Hirayasu et al. Fig. S11 Supplementary Figure 18. Schematic diagram of host and pathogen interaction from the perspective of LILRA2. Antibodies play an important role in host defense against microbial pathogens. However, microbial pathogens seem to have acquired a protease that destroys antibodies in order to evade host immune system. On the other hand, the host immune system appears to have acquired an immune activating receptor, LILRA2, that specifically recognizes microbially cleaved antibodies. Hirayasu et al. Page S19 NATURE MICROBIOLOGY 19

20 DOI: /NMICROBIOL Supplementary Figure 19. Papain and pepsin digestion of IgG. Full-length and truncated IgG3 that was mutated to bind to protein A was digested with papain and pepsin, respectively. Thereafter, undigested IgG3 and Fc fragment were removed by protein A-immobilized sepharose. Non-treated and treated IgG antibodies were analyzed by SDS-PAGE under non-reducing condition and were detected by Oriole staining. Hirayasu et al. Page S20 20 NATURE MICROBIOLOGY

21 DOI: /NMICROBIOL SUPPLEMENTARY INFORMATION Supplementary Figure 20. Production of recombinant MspA in E. coli. C-terminal Histagged full-length MspA (61 kda) was expressed in E. coli using IPTG induction. A full-length MspA is cleaved to form a 38 kda active mature MspA in the periplasm. Each fraction of the E. coli was analyzed by SDS-PAGE, followed by visualization using Oriole staining. A mature 38 kda MspA was cleaved from a full-length MspA and was detected in the soluble fraction, whereas a full-length MspA was present only in the insoluble fraction. An active mature form of MspA was purified from the soluble fraction using TALON metal affinity chromatography. Hirayasu et al. Page S21 NATURE MICROBIOLOGY 21

22 DOI: /NMICROBIOL Supplementary Figure 21. Full images of all the Western/gels results presented in this study. Hirayasu et al. Page S22 22 NATURE MICROBIOLOGY

23 DOI: /NMICROBIOL SUPPLEMENTARY INFORMATION Hirayasu et al. Page S23 NATURE MICROBIOLOGY 23

24 DOI: /NMICROBIOL Supplementary Table 1. Primer information. µ! Hirayasu et al. Page S24 24 NATURE MICROBIOLOGY