Anti-inflammatory activity of IgG1 mediated by Fc galactosylation and association of FcγRIIB and dectin-1

Size: px

Start display at page:

Download "Anti-inflammatory activity of IgG1 mediated by Fc galactosylation and association of FcγRIIB and dectin-1"

Ruby Norman
5 years ago
Views:

1 Anti-inflmmtory ctivity of IgG1 medited y Fc glctosyltion nd ssocition of FcγRIIB nd dectin-1 Christin M. Krsten, Mnoj K. Pndey, Juli Figge, Regin Kilchenstein, Philip R. Tylor, Mrcel Ross, Jcqueline U. McDonld, Selind J. Orr, Mrkus Berger, Dominique Petzold, Veroniqué Blnchrd, André Winkler, Constnze Hess, Delyth M. Reid, Irin V. Mjoul, Richrd T. Strit, Nthniel L. Hrris, Griele Köhl, Ev Wex, Rlf Ludwig, Detlef Zillikens, Flk Nimmerjhn, Fred D. Finkelmn, Gordon D. Brown, Mrc Ehlers & Jörg Köhl Numer of neutrophils (Χ 1 5 ) 4 2 C ut 17. IC (µg kg - 1 ody weight) Supplementry Figure 1. IgG1 ICs suppress C5-medited peritonel inflmmtion in dose-dependent mnner. BALB/c WT mice were treted i.v. with the indicted concentrtions of 17. ICs min prior to the i.p. injection of C5 (2 x 1-7 M). Our dt suggest rod dynmic rnge of inhiition strting t <.5 µg kg -1 ody weight nd reching mximum of inhiition t 5 µg kg -1 ody weight. Dt re mens ± s.e.m. (n=5-8/group).

2 8 CD11 expression (Δ MFI) 4 2, C5 (nm) Supplementry Figure 2. C5 promotes upregultion of CD11 expression in BM neutrophils. BM cells from Fcer1g / mice were incuted with incresing concentrtions of rhc5. BM neutrophils were defined s in Fig. 1g y their FSC/SSC pttern nd the expression of the Gr-1 mrker. The increse in CD11 expression on BM neutrophils ws clculted s the men fluorescence intensity (MFI) in the presence of C5 the MFI mesured in the sence of C5 (=ΔMFI). Dt re mens ± s.e.m. (n=).

3 c Numer of neutrophils per mm C Numer of neutrophils per mm CXCL Numer of neutrophils per mm 2 C5 (1-9 M) CXCL2 (1-9 M) d WT Fcer1g / Fcgr2 / Numer of mcrophges per mm 2 e untreted * ut * * * ut IC (µg ml 1 ) 17. IC 17. (µg IC µg/ml ml 1 ) 17. IC (µg ml 1 ) IC + Src kinse inh. I IC + Src kinse inh. I Numer of mcrophges per mm 2 1 untreted IC ut WT Fcer1g / Fcgr2 / + Src kinse inh. I IC + Src kinse inh. I Numer of mcrophges per mm untreted IC + Src kinse inh. I IC + Src kinse inh. I Numer of neutrophils per mm * * Numer of neutrophils per mm * Numer of neutrophils per mm ut ut ut IC (µg ml 1 ) 17. IC (µg ml 1 ) 17. IC ( µg ml 1 ) Supplementry Figure. Migrtion of BM neutrophils or peritonel (PE) mcrophges towrds C5 nd CXCL2 nd the impct of 17. ICs on cell migrtion. BM cells were incuted for min with the indicted concentrtions of () C5 or () CXCL2. (c) Typicl dose response curve for the inhiitory effect of 17. ICs on C5 (1-8 M)-medited migrtion of BM-neutrophils from WT mice. We clculted hlf-mximl inhiitory concentrtion (IC ) of.48 µg ml -1 y three-prmeter sigmoidl curve fitting. Peritonel mcrophges (d) or BM cells (e) were pre-incuted with the indicted concentrtion of 17. ICs. Then, cells were llowed to migrte towrds (d) C5 (1-8 M) or (e) CXCL2 (1-7 M) for min. 17. ICs inhiit C5 or CXCL2-medited migrtion of cells from WT nd Fcerg1 / ut not from Fcgr2 / mice (n=5/group). Dt re mens + s.e.m. * P<.5, P<.1, * P<.1.

4 Numer of neutrophils per mm t untreted C 17. IC C nti-fcγriib IC B nti-fcγriib C Isotype-Control IC Supplementry Figure 4. Phrmcologicl trgeting of FcγRIIB locks the inhiitory effect of IgG1 ICs on C5-medited neutrophil migrtion. BM cells from C57BL/ Fcer1g / mice were preincuted with.5 µg ml -1 FcγRIIB-specific A (Ly17.2) or PBS for min, followed y min incution with 17. ICs (.1 µg ml -1 ) or with PBS. Pretretment of BM cells with the FcγRIIB-specific A olished the inhiitory effect of the IgG1 ICs (n=/group). Dt re mens + s.e.m. P<.1.

5 Time (min) (nm) Counts P-ERK1/2 PE 17. IC.1 µg ml IC 1 µg ml Counts P-ERK 1/2 PE Supplementry Figure 5. Kinetics of C5-medited ERK1/2 phosphoryltion in BMderived Gr-1 + neutrophils from WT mice nd the impct of 17. IC tretment on ERK phosphoryltion of neutrophils from Fcgr2 / mice. () BM cells from WT mice were incuted with rhc5 ( nm) for the indicted times. To determine C5-medited ERK1/2 phosphoryltion, cells were first gted on Gr-1 + neutrophils nd nlyzed using PE-leled p-erk1/2-specific A. The mximum ERK phosphoryltion occurred lredy one min fter C5 tretment nd stedily declined during the next 9min. () BM cells from Fcgr2-deficient mice were incuted for min with the indicted concentrtions of 17. ICs. Then, BM cells were stimulted for one min with C5 nd ERK phosphoryltion of Gr1 hi neutrophils ws ssessed. White histogrm: ERK phosphoryltion in the sence of C5 (ckground); grey histogrm: ERK phosphoryltion in response to C5; mgent histogrm: ERK phosphoryltion in response to C5 nd the indicted concentrtions of 17. ICs. Results in nd re representtive of three independent experiments.

6 Ly-G FcγRIIB Dectin-1 Merge Lminrin Curdln Untreted Supplementry Figure. Curdln drives rpid internliztion of Dectin-1 in BM neutrophils. BM-derived cells were stined for Ly-G (green, FITC), FcγRIIB (red, PE) nd Dectin-1 (lue, Alex47). Then, such cells were incuted with 1 µg ml -1 curdln or lminrin for min t 7 C, wshed nd pictures were recorded using n Olympus FV 1 lser scnning microscope. Curdln tretment leds to internliztion of Dectin-1 wheres lminrin displces the nti-dectin-1 A. Results re representtive of t lest three independent experiments.

7 MyPH8.B.Clec7 / + pmxs-iz MyPH8.B.Clec7 / + Dectin-1B.98% 8.% Ly-B.2-PerCP c Dectin-1-Alex % 92.25% CD88-PE 11.4% 7.2% FcγRIIB-Biotin: SAv-Alex45 Supplementry Figure 7. Phenotypic chrcteristics of the Clec7-deficient neutrophil cell line MyPH8-B.Clec7 /. () Neutrophils derived in vitro from MyPH8-B.Clec7 / cells, retrovirlly trnsduced with vector pmxs-iz express no Dectin-1. () Following retrovirl trnsduction with the Dectin-1B isoform, the in vitro-derived neutrophils from MyPH8- B.Clec7 / cells express Dectin-1. (c) MyPH8-B.Clec7 / cells express C5R nd FcγRIIB independent of whether they re trnsduced with vector pmxs-iz (left pnel) or with Dectin-1B (right pnel). MyPH8-B.Clec7 / cells were gted on Ly-B.2 + in vitro-generted neutrophils. Smples were cquired on CyAn ADP nlyser ( lser) (Beckmn-Coulter) nd nlyzed using Summit softwre (Beckmn-Coulter).

Numer of neutrophils per mm 2 1 1 * untreted 1 Time of preincution (min) c d Numer of neutrophils per mm 2 1 1 untreted * * + 17. IC + picetnnol + 17.

8 Numer of neutrophils per mm * untreted 1 Time of preincution (min) c d Numer of neutrophils per mm untreted * * IC + picetnnol IC + picetnnol Numer of neutrophils per mm untreted IC + By IC + By 1- Numer of neutrophils per mm untreted * * * IC + Syk Inhiitor IC + Syk Inhiitor e Supplementry Figure 8. Impct of preincution time, tyrosine nd Syk kinse ctivity on the inhiitory effect of IgG1 ICs nd pull-down of p-syk with Syk-inding phospho-petide. () BM cells from WT mice were preincuted with 17. ICs (.1 µg ml -1 ) for the indicted times. 17. ICs significntly inhiit C5-medited chemotxis of BM cells fter only one min of preincution. Specific lockde of Syk tyrosine kinse using () picetnnol, (c) By 1-, or (d) Syk inhiitor (ll t 1 µm) reversed the inhiitory effect of 17. ICs (.1 µg ml -1 ). (n=-5/group). Dt re mens + s.e.m. * P<.5, P<.1, * P<.1. (e) Representtive peptide pull-down (pclec2 peptide 21 ) experiment showing 17. IC-driven phosphoryltion of Syk in Clec7 / neutrophil cell line trnsfected with Dectin-1B isoform. Dt re representtive of two independent experiments.

Ros2-promoter. () BM cells from such mice were cultured in RPMI for 48h. Then they were treted with 1 µg ml -1 of the Dectin-1 gonist curdln.

9 Supplementry Figure 9. Tmoxifen tretment of BM cells from conditionl Syk knockout mice results in functionl Syk depletion. We used inducile Syk knockout mice (Syk flox/flox ) tht comprise floxed Syk gene nd tmoxifen-inducile Cre recominse under the control of the uiquitously ctive Ros2-promoter. () BM cells from such mice were cultured in RPMI for 48h. Then they were treted with 1 µg ml -1 of the Dectin-1 gonist curdln. This trement resulted in strong phosphoryltion of Syk fter min. () Addition of tmoxifen during the 48h culture rogted the curdln-induced phosphoryltion of Syk. Results re representtive of three independent experiments.

Untreted 1 min min 5 min WT Fcgr2 -/- Untreted 1 min min 5 min WT Fcgr2 -/- Supplementry Figure 1. 17. ICs promote trnsient phosphoryltion of Syk nd SHIP.

10 Untreted 1 min min 5 min WT Fcgr2 -/- Untreted 1 min min 5 min WT Fcgr2 -/- Supplementry Figure ICs promote trnsient phosphoryltion of Syk nd SHIP. BM cells from BALB/c WT or Fcgr2 / mice were incuted with 17. ICs for the indicted times. Untreted BM neutrophils show no () Syk or () SHIP phosphoryltion. In, contrst, 17. ICs tretment for one or three min induced () Syk or () SHIP phosphoryltion which declined fter 5min. Results in nd re representtive of t lest three independent experiments.

11 Fc glycn 2D structure Mss (m/z) Glß1-4GlcNAcß1-2Mnα1 Mnα1 GlcNAcß1-2Mnα1 GlcNAcß1-2Mnα1 GlcNAcß1-2Mnα1 GlcNAcß1-2Mnα1 Glß1-4GlcNAcß1-2Mnα1 GlcNAcß1-2Mnα1 Glß1-4GlcNAcß1-2Mnα1 GlcNAcß1-2Mnα1 Glß1-4GlcNAcß1-2Mnα1 Glß1-4GlcNAcß1-2Mnα1 Glß1-4GlcNAcß1-2Mnα1 Glß1-4GlcNAcß1-2Mnα1 NeuAcα2-Glß1-4GlcNAcß1-2Mnα1 GlcNAcß1-2Mnα1 NeuGcα2-Glß1-4GlcNAcß1-2Mnα1 GlcNAcß1-2Mnα1 NeuAcα2-Glß1-4GlcNAcß1-2Mnα1 Glß1-4GlcNAcß1-2Mnα1 NeuGcα2-Glß1-4GlcNAcß1-2Mnα1 Glß1-4GlcNAcß1-2Mnα1 NeuAcα2-Glß1-4GlcNAcß1-2Mnα1 NeuAcα2-Glß1-4GlcNAcß1-2Mnα1 NeuGcα2-Glß1-4GlcNAcß1-2Mnα1 NeuGcα2-Glß1-4GlcNAcß1-2Mnα1 Mnß1-4GlcNAcß1-4GlcNAc-R Mnß1-4GlcNAcß1-4GlcNAc-R Fucα1 Mnß1-4GlcNAcß1-4GlcNAc-R Mnß1-4GlcNAcß1-4GlcNAc-R Fucα1 Mnß1-4GlcNAcß1-4GlcNAc-R Mnß1-4GlcNAcß1-4GlcNAc-R Fucα1 Mnß1-4GlcNAcß1-4GlcNAc-R Fucα1 Mnß1-4GlcNAcß1-4GlcNAc-R Fucα1 Mnß1-4GlcNAcß1-4GlcNAc-R Fucα1 Mnß1-4GlcNAcß1-4GlcNAc-R Fucα1 Mnß1-4GlcNAcß1-4GlcNAc-R Fucα1 Mnß1-4GlcNAcß1-4GlcNAc-R Fucα1 Mnß1-4GlcNAcß1-4GlcNAc-R Supplementry Figure 11. Relevnt N-glycns found in this study. N-glycns re formed of pentscchride core structure (GlcNAc2Mn). The heterogeneity of these N-glycns derives from dditions of GlcNAc, Gl nd ll silic cids (N-cetylneurminic cid (NeuAc) medited y humn silyltrnsferses or N-glycolylneurminic cid y mouse silytrnsferses (NeuGc). We show monoglctosyltion only on the lph1, rnch or mono-silyltion on the lph2, rnch s exmples. In ntiodies, core fucosyltion occurs only with n lph1, linkge. Species differences re indicted y colors (humn=lue nd mouse=red). The numers show the moleculr mss of the distinct N-glycn structures relesed upon PNGse F tretment s determined y MALDI-TOF. The moleculr mss of 2.5 for the G2S2 structure is the clculted mss. Fuc: (fucose).

12 c 17. 7B4 (IgG1) 7B4 (IgG2) d e f H5 LoGlH5 HiGlH5 g SilH5 Intensity Mss (m/z) Supplementry Figure 12. MALDI-TOF spectr of N-glycns relesed from Asn297 of different TNP-specific ntiodies fter PNGse F digestion. N-glycn composition of () A 17. (IgG1; G1 nd G2 = 81.8%), () A 7B4 (IgG1; G1 nd G2 = 24.%), (c) A 7B4 (IgG2; G1 nd G2 = 84.5%), (d) A H5 (IgG1; G1 nd G2 = 29.7%), (e) in vitro deglctosylted A H5 (LoGlH5; IgG1; G1 nd G2 = 8.8%), (f) in vitro glctosylted A H5 (HiGlH5, IgG1; G1 nd G2 = 9.9 %); nd (g) in vitro silylted A H5 (SilH5; IgG1; G1 nd G2 = 9.%). Numers indicte the moleculr mss of relesed glycns, the 2D structures of which re depicted in Supplementry Fig. 11. *: contmintion

13 WT Fcer1g / Fcgr2 / Clec7 / numer Numer of neutrophils per (mm mm 2 ) -1 2 c 1 1 ut untreted * * H5 IC H5 IC HiGlH5 IC HiGl H5 IC * * LoGlH5 IC 7B4 IC LoGl H5 IC 7B4 IC numer of neutrophils (mm 2 ) -1 Numer of neutrophils per mm untreted * * H5 IC * * ut H5 IC HiGl H5 IC LoGl H5 IC 7B4 IC HiGlH5 IC LoGlH5 IC 7B4 IC numer of neutrophils (mm 2 ) -1 Numer of neutrophils per mm ut untreted H5 IC H5 IC HiGlH5 IC HiGl H5 IC LoGlH5 IC LoGl H5 IC 7B4 IC 7B4 IC numer of neutrophils (mm 2 ) -1 Numer of neutrophils per mm ut untreted H5 IC H5 IC HiGl H5 IC HiGlH5 IC LoGl H5 IC LoGlH5 IC 7B4 IC 7B4 IC WT Fcgr2 / Clec7 / dmfi CD dmfi CD dmfi CD c5 c5 C5 + HiGl IC C5 + HiGlH5 IC C5 + LoGl IC C5 + LoGlH5 IC 5 c5 IC C5 + HiGlH5 IC IC C5 + LoGlH5 IC c5 C C5 + HiGlH5 IC C C5 + LoGlH5 IC c WT Fcgr2 / Clec7 / * 2 * 2 2 C 2+ pek height 1 1 C 2+ pek height 1 1 C 2+ pek height untreted C5 C5 + HiGlH5 IC C5 + LoGlH5 IC untreted C5 C5 + HiGlH5 IC C5 + LoGlH5 IC untreted C5 C5 + HiGlH5 IC C5 + LoGlH5 IC Supplementry Figure 1. Impct of HiGlH5 ICs on C5-medited chemotxis, upregultion of CD11 nd increse in [C 2+ ] i. (), BM cells from the indicted mouse strins were treted with either H5 ICs, HiGlH5 ICs, LoGlH5 ICs or low glctosylted 7B4 IgG1 ICs. HiGlH5 ICs ut not LoGlH5 ICs or 7B4 ICs inhiit C5-medited chemotxis of neutrophils from WT nd Fcerg1 / mice. HiGlH5 ICs exert no inhiitory effect on neutrophils from Fcgr2 / or Clec7 / mice. () HiGlH5 ICs suppress C5-medited upregultion of CD11 expression on WT ut not on neutrophils from Fcgr2 / or Clec7 / mice. LoGlH5 ICs exert no inhiitory effect. dmfi CD11: increse in MFI medited y the indicted tretment s compred with untreted control. (c) C5-medited increse in [C 2+ ] i is locked y HiGlH5 ICs in BM cells from WT mice ut not from Fcgr2 / or Clec7 / mice. (n=/group). Dt re mens + s.e.m. *P<.5, P<.1, * P<.1.

14 H5 HiGlH5 LoGlH5 H5 HiGlH5 LoGlH5 µl ml 1 µl ml 1 Supplementry Figure 14. In vitro glctosyltion or deglctosyltion of TNP-specific IgG1 H5 does not influence ntigen inding. () Mouse IgG1 ELISA of nïve H5 A, in vitro glctosylted (HiGlH5) nd in vitro deglctosylted (LoGlH5) TNP-specific IgG1 As. ELISA pltes were coted with polyclonl mouse IgG1-specific A nd developed with polyclonl mouse-specific IgG1 coupled to horserdish peroxidse. (), TNP rectivity of H5, HiGlH5 nd LoGlH5 TNP-specific IgG1 As (n=/group).

15 Numer of neutrophils per mm untreted * SilH5 IC H5 IgG1 SilH5 IgG1 Numer of neutrophils per mm 2 4 2,5,1,5,1,5 7B4 IgG2 IC µg ml 1 c Numer of neutrophils per mm ,5,1,5,1,5 7B4 IgG2 IC µg ml -1 Supplementry Figure 15. Impct of silylted IgG1 H5 (SilH5) ICs, monomeric H5 IgG1 nd 7B4 IgG2 ICs on C5-medited chemotxis. () BM cells from WT mice were incuted with SilH5 ICs, H5 IgG1 or SilH5 IgG1 As for min. The result shows tht (i) silyltion does not ffect the inhiitory effect of HiGlH5 ICs; nd (ii) IC formtion of IgG1 As is essentil for the inhiitory effect. Of note, the silyltrnsferse lmost completely silylted the i-glctosylted glycns (2% out 29%) ut only frction of the monoglctosylted glycns (2% of 4%). At this point, we cnnot exclude tht higher silyltion of mono-glctosylted glycns would lter the inhiitory effect of highly glctosylted H5 ICs. Highly glctosylted TNP-specific IgG2 7B4 ICs do not lock C5-medited migrtion of BM neutrophils from () WT or (c) Fcer1g / mice.

Untreted 1 Ly-G 2K Dectin-1 % of mx. 8 4.1 SSC-A SSC 2K 1K 1K 2 K C5 1 1 2 1 1 4 1 5 Pcific Blue-A: - 2K 1 2 1 1 4 1 5 APC-A: Dectin1 8 2K % of mx. 4 89.1 SSC-A SSC 1K 1K 2 K 74.

16 Untreted 1 Ly-G 2K Dectin-1 % of mx SSC-A SSC 2K 1K 1K 2 K C Pcific Blue-A: - 2K APC-A: Dectin1 8 2K % of mx SSC-A SSC 1K 1K 2 K 74. Curdln + C5 c Pcific Blue-A: - 2K 2K APC-A: Dectin1 % of mx. % of Mx SSC-A SSC 1K 1K K Pcific Blue-A: APC-A: Dectin1 Supplementry Figure 1. Curdln pretretment olishes Dectin-1 expression on neutrophils tht migrted into the peritonel cvity in response to C5. () Percentge of Ly-G hi cells (left pnel) in nive WT BALB/c mice nd their expression of Dectin-1 (right pnel). () s in, except tht mice were treted with C5 (2 nm, i.p.) for h. Here, 74.% of Ly-G hi neutrophils tht migrted into the peritoneum express Dectin-1. (c) s in, except tht mice were pretreted with curdln (1 µg, i.v.) min prior to C5 injection. Plese note tht only 1.7% of Ly-G hi neutrophils express Dectin-1. Results re representtive of t lest three independent experiments.

AUC (% ffected surfce re) WT C5R / Wildtype C5R / Supplementry Figure 17. C5R deficiency protects from disese development in experimentl epidermolysis ullos cquisit (EBA).

17 AUC (% ffected surfce re) WT C5R / Wildtype C5R / Supplementry Figure 17. C5R deficiency protects from disese development in experimentl epidermolysis ullos cquisit (EBA). () Determintion of the clinicl severity during the course of experimentl EBA from d4-12 y ssessment of the re under the curve (AUC). () Clinicl phenotypes of two representtive mice from the WT nd the C5R / group re depicted on d12 fter nti-collgen type VII IgG tretment. C57BL/ mice developed widespred listering skin disese (cutneous lesions indicted y white rrows). In contrst, C5R / mice were lmost completely protected from EBA development. Dt re mens + s.e.m. (n = 5-9/group).

Dy PBS HiGl IC LoGl IC Supplementry Figure 18. HiGlH5 ICs reduce the clinicl severity of experimentl EBA induced y trnsfer of rit IgG directed ginst murine collgen type VII.

18 Dy PBS HiGl IC LoGl IC Supplementry Figure 18. HiGlH5 ICs reduce the clinicl severity of experimentl EBA induced y trnsfer of rit IgG directed ginst murine collgen type VII. () Protocol underlying the model of experimentl EBA nd IgG1 ICs tretment scheme. () Clinicl phenotype of three representtive mice from ech tretment group (PBS, HiGlH5- ICs nd LoGlH5 ICs) on d12 fter nti-collgen type VII IgG injection is depicted. Mice treted with PBS or LoGlH5 ICs developed widespred cutneous lesions typicl for experimentl EBA. They re chrcterized y lopeci, listers, erosions, epiderml detchment nd crust formtions. We oserved lesions preferentilly in the fcil re, t the ers, the neck nd the ventrl site of the mice (s indicted y white rrows). In HiGlH5 ICs-treted mice, such lesions were reduced in quntity nd size.

AUC (% ffected surfce re) 4 2 1 untreted C HiGl IC PBS HiGl IC Supplementry Figure 19. HiGl ICs do not reduce the clinicl severity of experimentl EBA in Clec7 / mice.

19 AUC (% ffected surfce re) untreted C HiGl IC PBS HiGl IC Supplementry Figure 19. HiGl ICs do not reduce the clinicl severity of experimentl EBA in Clec7 / mice. () Determintion of the clinicl severity during the course of experimentl EBA from d4-12 y ssessment of the re under the curve (AUC) in untreted nd HiGl IC-treted Clec7 / mice. () Clinicl phenotypes of two representtive Clec7 / mice from the untreted nd the HiGl IC-treted groups re depicted on d12 fter nti-collgen type VII IgG tretment. Mice from oth tretment groups developed comprle cutneous lesions (in quntity nd size) typicl for experimentl EBA s indicted y white rrows. Dt re mens + s.e.m. (n = -7/group).

20 TNP-2-BSA + nti-migg-pe TNP-2-BSA + IgG1 HiGl H5 IgG1 IC H5 IgG1 IC HiGl H5 IgG1 IC H5 IgG1 IC PE CHO CHO-mFcgr2 Supplementry Figure 2. H5 ICs ind to FcγRIIB independent of the degree of glycn glctosyltion. HiGlH5 ICs nd H5 ICs were incuted with CHO cells (lck) or CHO cells trnsfected with mouse FcγRIIB (CHO mfcgr2, grey) nd stined for FcγRIIB inding using PE-leled murine IgG-specific A. As positive control, CHO or CHO mfcgr2 cells were stined with ICs composed of TNP-2 BSA nd TNP-specific IgG1 A. As negtive control, CHO or CHO mfcgr2 cells were incuted with HiGlH5 ICs or H5 ICs only (lower four histogrms). Results re representtive of two independent experiments.

21 K D = 7.74e-7 Time [s] Concentrtion [M] c d K D = 4.78e-7 Time [s] Concentrtion [M] Supplementry Figure 21. Surfce plsmon resonnce interction nlysis of HiGlH5 nd LoGlH5 ICs with FcγRIIB. The inding of HiGlH5-ICs ( nd ) nd LoGlH5-ICs (c nd d) to recominnt mouse FcγRIIB-Fc ws nlyzed using surfce plsmon resonnce. Ten seril dilutions of IC (1:2) were prepred strting t 1x1-8 M (pink curves in nd c) nd inding curves were determined. The equilirium response (RU increse) ws plotted ginst the concentrtions of HiGlH5 ICs () or LoGlH5 ICs (d) to crete sturtion curve of nlyte inding nd to clculte the dissocition constnt (K D ).

22 Supplementry Figure 22. Assessment of the requirements for FRET using Cy leled HiGlH5 ICs nd Alex47-leled Dectin-1-specific A. () Spectrl overlp of sorption spectrum for Alex47-leled Dectin-1 A cceptor nd emission spectrum for Cy-leled HiGlH5 IC donor. () Single cell imge of neutrophil efore (left) nd fter (right) cceptor lech with four highlighted regions of interest.

, Highly glctosylted IgG1 ICs such s HiGlH5 ICs link FcγRIIB to Dectin-1 resulting in tyrosine phosphoryltion of the ITAM-like motif downstrem of Dectin-1 nd trnsient

23 Supplementry Figure 2. Two step model y which highly glctosylted IgG1-ICs ssocite FcγRIIB with Dectin-1 nd inhiit C5R signling., IgG1-ICs ind to FcγRIIB independent of their N-glycn glctosyltion. Low or glctosylted IgG1 ICs such s LoGlH5 ICs do not drive ssocition of FcγRIIB with Dectin-1., Highly glctosylted IgG1 ICs such s HiGlH5 ICs link FcγRIIB to Dectin-1 resulting in tyrosine phosphoryltion of the ITAM-like motif downstrem of Dectin-1 nd trnsient phosphoryltion of Syk. Further, HiGlH5 ICs drive the ssocition of SHIP with the ITIM within FcγRIIB nd its phosphoryltion. This pthwy inhiits C5-medited ERK1/2 phosphoryltion nd severl cellulr effector functions of C5R.

24 Antiody clone Mss 17. 7B4 7B4 H5 LoGl HiGl SilH5 Glctose/ (IgG1) (IgG2) H5 H5 Silic cid A1G G G G G G G G1S G1S G2S G2S G2S G2S2 Totl Supplementry Tle. Composition of the glycns relesed from N297 of the indicted TNP-specific ntiody clones fter PNGse F digestion. The numers on the left indicte the moleculr mss of the different N-glycns s determined y MALDI-TOF. On the right, the numer of terminl Gl residues (=G; 1=G1; nd 2=G2) nd silic cid residues (Neu5Ac of humn in lue nd Neu5Gc mouse in red; 1=S1 nd 2=S2) is depicted. For N- glycn 2D-structures, plese refer to Supplementry Fig. 11. The numers elow ech A clone depict the percentges of ech individul glycn structure. The moleculr mss of 2.5 for the mouse G2S2 structure is the clculted mss.

25 Supplementry movie 1. Expression of FcγRIIB ut sence of Syk phosphoryltion in untreted BM neutrophils. This movie shows the expression pttern of FcγRIIB (green, nti- FcγRIIB-FITC A) nd of phosphorylted Syk (p-syk, mgent, nti-p-syk-alex58 A) in BM-derived neutrophil min fter incution with PBS s determined y confocl microscopy in n nimted three-dimensionl projection. Cell stins positive for FcγRIIB. No phosphoryltion of Syk is visile in the sence of 17. IC tretment. Similr results were otined for p-ship. Supplementry movie 2. Expression of Dectin-1 ut sence of Syk phosphoryltion in untreted BM neutrophils. This movie shows the expression pttern of Dectin-1 (mgent, nti-dectin-1-apc A) nd phosphorylted Syk (p-syk, green, nti-p-syk-alex58 A) in BM-derived neutrophil min fter incution with PBS s determined y confocl microscopy in n nimted three-dimensionl projection. No phosphoryltion of Syk occurs in the sence of 17. IC tretment. Similr results were otined for p-ship. Supplementry movie. Phosphoryltion of Syk nd co-locliztion of p-syk with FcγRIIB in response to 17. IC tretment. This movie shows the co-locliztion pttern of FcγRIIB (green, nti-fcγriib-fitc A) nd of phosphorylted Syk (p-syk, mgent, nti-p- Syk-Alex58 A) in BM-derived neutrophil min following 17. IC tretment s determined y confocl microscopy in n nimted three-dimensionl projection. 17. IC tretment drives phosphoryltion of Syk; p-syk co-loclizes with FcγRIIB s indicted y the white spots. Similr results were otined for p-ship. Supplementry movie 4. Phosphoryltion of Syk nd co-locliztion of p-syk with Dectin-1 in response to 17. IC tretment. This movie shows the co-locliztion pttern of Dectin-1 (mgent, nti-dectin-1-apc A) nd phosphorylted Syk (p-syk, green, nti-p- Syk-Alex58 A) in BM-derived neutrophil min following 17. IC tretment s determined y confocl microscopy in n nimted three-dimensionl projection. 17. IC tretment drives phosphoryltion of Syk; p-syk co-loclizes with Dectin-1 s indicted y the white spots. Similr results were otined for p-ship. Supplementry movie IC tretment promotes ssocition of FcγRIIB nd Dectin-1. This movie shows the co-locliztion pttern of FcγRIIB (green, nti-fcγriib-fitc A) nd of Dectin-1 (mgent, nti-dectin-1apc A) in BM-derived neutrophil min fter incution with 17. ICs s determined y confocl microscopy in n nimted threedimensionl projection. Co-locliztion of FcγRIIB nd Dectin-1 is indicted y the white spots.

TNF-α (pg/ml) IL-6 (ng/ml)

Xio, et l., Supplementry Figure 1 IL-6 (ng/ml) TNF-α (pg/ml) 16 12 8 4 1,4 1,2 1, 8 6 4 2 med Cl / Pm3CSK4 zymosn curdln Poly (I:C) LPS flgelin MALP-2 imiquimod R848 CpG TNF-α (pg/ml) IL-6 (ng/ml) 2 1.6