The CD46 and Jagged1 interaction is critical for human adaptive T H

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1 The CD46 and Jagged1 interaction is critical for human adaptive T H 1 immunity Gaëlle Le Friec, Devon Sheppard, Pat Whiteman, Christian M. Karsten, Salley Al-Tilib Shamoun, Adam Laing, Laurence Bugeon, Margaret J. Dallman, Teresa Melchionna, Chandramouli Chillakuri, Richard A. Smith, Christian Drouet, Lionel Couzi, Veronique Fremeaux-Bacchi, Jörg Köhl, Simon N. Waddington, James M. McDonnell, Alastair Baker, Penny A. Handford, Susan M. Lea and Claudia Kemper. Supplementary Figures 1 to 6 and Supplementary Tables 1 and 2 Nature Immunology: doi:1.138/ni.2454

2 a Supplementary Figure 1 b Notch1 Notch2 Notch3 Notch4 N Extracellular domain Cytoplasmic membrane Intracellular domain EGF like domain RAM domain ANK domain PEST sequence Cysteine rich LIN domain NLS TAD domain c Jagged1 Jagged2 d J 1 DSL EGF3 J-1DSL-EGF3 Extracellular domain Delta like1 Delta like3 Delta like4 Cytoplasmic membrane Intracellular domain DSL domain EGF like domain Cysteine rich domain Transmembrane domain PDZ domain Supplementary Figure 1. Schematic of CD46, Notch receptors and Notch ligands including their protein modules and domains. (a) CD46 protein structure. The four major isoforms of CD46 all contain CCPs 1 to 4 at the N-terminus followed by an alternatively spliced extracellular BC region and mutually exclusive usage of the CYT1 or CYT2 cytoplasmic domains. Indicated are the Jagged1 (red) and the C3b/C4b binding sites (green). (b) Structures of the Notch receptors. The protein domain expressed and used in this study (N ) has been marked by a red box. (c) Structures of the Notch ligand family members. The J-1 DSL-EGF3 domain used in this study is marked with a red box. (d) CD46 12 with residues involved in Jagged1 interface (red) is on the left, in complex with adenovirus knob protein 11 (3O8E.pdb), with adenovirus knob protein 21 (3L89.pdb), with measles virus haemagglutinin (3INB.pdb), and non-complexed (1CKL.pdb) from top to bottom, highlighting the reorientation of CCP2 with respect to CCP1 in presenting distinctive contiguous binding surfaces for each virus. Nature Immunology: doi:1.138/ni.2454

3 Supplementary Figure 2a and b a Media MFI: 28.9 MFI: 6.6 MFI: 49.8 MFI: 8.8 MFI: 15.1 MFI: 2.9 MFI: 24.9 MFI: 37. -secretase inhibitor MFI: 2.3 MFI: 16.7 MFI: 36.6 MFI: 78.7 Marimastat MFI: 11.1 MFI: 22. MFI: 55. MFI: 95.8 TAPI-2 Events CD46 Notch1 b IFN- (ng/ml) nd nd nd nd nd 8 7 IL-1 (ng/ml) CD28 +CD46 +CD28 +CD46 +CD28 +CD46 +CD28 +CD46 Media + -secret. + Marim. + TAPI-2 inhibitor Nature Immunology: doi:1.138/ni.2454

4 c Supplementary Figure 2c and d Jurkat Jurkat BC1 Jurkat BC2 Events Isotype MFI 3 Jurkat MFI 2 MFI 13 Jurkat BC1 MFI 31 Jurkat BC2 Isotype ctrl Jurkat Jurkat BC1 Jurkat BC2 Events MFI 2 MFI 12 MFI 18 MFI 7 MFI 13 MFI 21 MFI 1 MFI 9 MFI 19 Isotype ctrl CD46-GFP Notch1 d IFN- (pg/ml) Jurkat Jurkat BC1 Jurkat BC2 ** ** 7 IL-1 (pg/ml) ** * ** * CD3/CD46 CD3/CD46 CD3/CD46 + a-cd anti Notch sol Notch + Media + -Notch + sn Supplementary Figure 2. Interference with Notch signalling disturbs CD46-mediated T H 1 induction and regulation. (a) Interference with ADAM or -secretase activity deregulates CD46 and Notch processing on CD3+CD46-activated purified T cells. Cells were activated as indicated and then analysed by FACS for CD46 and Notch expression. Note that the usually observed Notch surface down-regulation upon direct Notch stimulation with activating antibodies is also impaired in the presence of TAPI-2 or Marimastat (data not shown). Data represent one of three similarly performed experiments. (b) Interference with ADAM or -secretase activity deregulates IFN- and IL-1 secretion. Secretion of IFN- and IL-1 by purified CD4 + T cells (measured using the CBA assay) activated for 36h with the depicted immobilized antibody combinations (and 5 U/ml rhil-2) with or without a presenilin/γ-secretase inhibitor or the broad-spectrum matrix metalloproteinases inhibitors TAPI-2 or Marimastat. (c) Non-manipulated Jurkat T cells express negligible surface CD46 while Jurkat T cells stably transfected with BC1 (Jurkat BC1) or BC2 (Jurkat BC2) CD46 isforms express comparable surface levels of CD46 (upper panel). All Jurkat T cells express similar levels of Notch1 (lower three panels). Note that these data have been observed consistently over time, the protein expression levels are therefore stable. (d) CD46 CYT-1 and Notch1 signals are required for IFN- and IL-1 production. IFN- and IL-1 production by Jurkat, Jurkat BC1 or Jurkat BC2 cells activated for 4d with depicted immobilized stimulating antibodies with or without either 2 g/ml anti- Notch (function blocking) or 1 g/ml soluble N Data are representative of five independent experiments. *, p <.5; **, p <.1 when compared to CD3+CD46-activated Jurkat T cells without addition of -Notch or sn Nature Immunology: doi:1.138/ni.2454

5 Supplementary Figure 3 Resting CD4+ T cell Activated CD4+ T cell TCR Notch1 T CD4+ CD46 Delta-like1 Jagged1 DC 1 - C3b production DC + IL-2 + IL C3b binds to CD CD46 engagement induces: CD46 downregulation and IL-2 secretion CD46 mediated signalling events 4 - Modulation of Notch system expression: Upregulation Notch1 and Jagged1 Downregulation Delta like1 + IL-2 + IL-2 + IL-2 + IL T cell / T cell interactions Notch system transactivation Supplementary Figure 3. Model suggestion for the CD46 and Notch system crosstalk in T H 1 regulation. On resting CD4 + T cells, CD46 binds Jagged1 thereby limiting potential Jagged1 and Notch1 interactions (as the CD46 and Jagged1 interaction is of higher affinity compared to the Jagged and Notch1 interaction). This may favour a Notch1 and DLL1 cis interaction, which has been shown to inhibit T cell activation. Thus, similarly to DLL1, in the absence of T cell activation conditions, CD46 expression on the T cell surface may function as stop signal. In antigen presence or infection and TCR engagement, T cells and APCs generate the CD46 ligand C3b. Binding of C3b to CD46 initiates CD46-mediated signalling events including T cell migratory capacity and cluster formation, substantial CD46 and DLL1 down-regulation while maintaining Notch1 and Jagged1 surface availability. This change in surface expression of CD46 and Notch receptors and ligands may release the brake and allow for Notch1 and DLL1 interaction in trans (which generates IFN- ) as well as for Notch1 and Jagged1 binding in cis or trans (necessary for IL-1 induction). The exact role of IL-2 in this model, which is required for CD3+CD46-mediated IFN- to IL-1 switching, awaits clarification. In line with this model is our observation that both IFN- and IL-1 production is blocked substantially when T cell migration and cluster formation is inhibited by function-blocking antibodies to CCL5, RANTES or CCR1 (data not shown). Nature Immunology: doi:1.138/ni.2454 IFN- IL-1

6 Supplementary Figure 4a - d a IP: + GSI - CD3 Isot. b Activation: + + (kda) CD46, BC1 + BC2 85 CD46, C1 + C2 44 Detection: -E-catenin (kda) Detection: -E-catenin c d Isotype control CD3-activated CD3 + CD46-activated Nature Immunology: doi:1.138/ni.2454

7 Supplementary Figure 4e and f e f IFN- (ng/ml) Ctrl. sir -E-catenin sir * Events DLL1 Jur.BC1 mock Jur.BC1 mock Jur.BC1 Ctrl shr Jur.BC1 DDL1 shr 5 IL-1 (ng/ml) CD28 * Change in IL-1 secretion (%) Jurkat mock Jur.BC1 mock Jur.BC1 ctrl.r * Jur.BC1 DLL1R + Supplementary Figure 4. -E-catenin interacts with CD46. (a) -E-catenin interacts with the intracellular domain(s) of CD46 in resting human CD4 + T cells. -E-catenin was identified in a yeast-two-hybrid screen as CD46 CYT-2 interacting protein. To assess for a CD46 and -E-catenin interaction in human CD4 + T cells, cell lysates of nonactivated () or CD3 or CD3+CD46-activated CD4 + T cells were either left untreated or used for immunoprecipitation (IP) with a CD46-specific mab, an CD3-specific mab or isotype control (Isot.) mab. Immunoblotting of the IP eluates with an anti- -E-catenin antibody revealed an association between CD46 and -E-catenin. Note that in CD3+CD46- activated T cells, the -E-catenin and CD46 IP is less efficient, likely because CD46 CYT1 and -2 are processed by - secretase complex upon T cell activation. In line with this, activation of T cells in presence of a -secretase inhibitor (GSI) increased -E-catenin pull-down. Data are representative of two similarly performed experiments. (b) -E-catenin protein knock down in CD4 + T cells via sir. CD4 + T cells were transfected with Cy 3 -labeled sir targeting -E-catenin and transfection efficiency assessed via FACS 12h post transfection (left panel). Protein knockdown was measured via FACS 36h post transfection (right panel). Cell viability was routinely above 8% (not shown). Shown is one representative result of six independently performed experiments. Transfection efficiency and protein knock down reached statistical significance when the mean fluorescence intensities (MFI) of the histograms in each experiment were compared. (c) -Ecatenin protein knockdown reduces CD46 down-regulation (top left FACS panel) but (d) does not affect CD3, CD25, CD28 or CD69 expression and (e) impedes IFN- secretion and IL-1 induction in CD3+CD46+IL-2-activated CD4 + T cells. Mean ± SD (n = 4). (f) DLL1 protein knockdown in CD46-BC1-transfected Jurkat T cells increases CD46- mediated IL-1 production. Upper panel, decreased expression of DLL1 on the cell surface of Jurkat-BC1 cells (Jur.BC1) after DLL1 shr transfection and CD3+CD46 activation ( cells have barely detectable DLL1 expression, not shown). Lower panel, IL-1 secretion at d5 by Jurkat cells and Jurkat-BC1 cells treated as depicted. Data represent mean ± SD (n = 3). *, p <.1 when compared to control sir-treated cells. Nature Immunology: doi:1.138/ni.2454

8 Supplementary Figure 5a a + -CD CD28 + Nature Immunology: doi:1.138/ni.2454

9 Supplementary Figure 5b - d b a 1 8" 6" 4" 2" " Human CD45 + cells (%) 1" Mouse A = HD1 T cells Mouse B = HD2 T cells Mouse C = HD3 T cells Mouse D = CD46-1 T cells cb IFN- (ng/ml) " d2 d7 d14 d2 d7 d14 1" 8" 8 6" 6 4" 4 2" 2 Mouse A = HD1 T cells Mouse B = HD2 T cells Mouse C = HD3 T cells Mouse D = CD46-1 T cells d c 1 Weight change (%) d d3 d5 d7 d12 d15 d21 Mouse A = HD1 T cells Mouse B = HD1 T cells Mouse C = HD1 T cells Mouse D = CD46-1 T cells Supplementary Figure 5. CD4 + T cells from CD46-deficient patient present with defective expression regulation of Jagged1, CD46, CD127 or CD132 and fail to induce GvHD. (a) T cells isolated from freshly-drawn blood of two healthy donors (HD3 and 4) and three patients with CD46 mutations (CD46-1 to CD46-3) were either left non-activated () or activated with immobilized antibodies to CD3 and CD46 in the presence of 25 U/ml rhil-2. Depicted surface maker expression was measured by FACS analysis 36h post activation. Each experimental condition was performed and analysed in duplicate with differences in measured mean fluorescence intensities (MFIs) < 4% in all cases. The MFI value for the isotype control (Isot.) in each experiment is shown as range observed over all experiments performed. Note, that the marker profile of the two HDs shown are representative of twelve age- and gender-matched individuals assessed through the course of the study. (b-d) PBMCs from three different healthy donors (HD 1 to HD3) or from Patient CD46-1 wereactivated72hwithimmobilizedantibodiestocd3andcd28and1x1 7 of expanded T cells injected into NOD- SCID- 2M / mice. (b) Human T cell engraftment was assessed via presence of hcd45-positive cells at time points indicated as well as by measurement of (c) hifn- in serum. Disease onset and progression was monitored by assessing (d) the weight change until sacrifice of the animals and confirmed using immunohistological analysis of intestinal tissue (data not shown)., Mouse B was sacrificed at day 15 at humane end point. Nature Immunology: doi:1.138/ni.2454

10 Supplementary Figure 6a a Nature Immunology: doi:1.138/ni.2454

11 Supplementary Figure 6b - d ba Human CD45 + cells (%) Mouse A = HD1 T cells Mouse B = HD2 T cells Mouse C = AP1-1 T cells Mouse D = AP3-1 T cells Mouse E = AP3-2 T cells IFN- (ng/ml) nd nd nd nd nd nd nd 1 nd nd nd nd nd nd nd nd nd nd d7 d14 d25 D46 b c 7 Mouse A = HD1 T cells 6 Mouse B = HD2 T cells 5 Mouse C = AP1-1 T cells 4 Mouse D = AP3-1 T cells Mouse E = AP3-2 T cells 3 2 d7 d14 d25 D46 dc Weight change (%) d d3 d7 d14 d25 d34 d46 d5 Mouse A = HD1 T cells Mouse B = HD2 T cells Mouse C = AP1-1 T cells Mouse D = AP3-1 T cells Mouse E = AP3-2 T cells Supplementary Figure 6. Alagille Syndrome patients present with defective expression regulation of Jagged1, CD46, CD127 or CD132 and fail to induce GvHD. (a) T cells isolated from freshly-drawn blood of two healthy age-matched donors (HD1 and 2) and four Alagille Syndrome patients (AP1 to AP4) were either left non-activated () or activated with immobilized antibodies to CD3 and CD46 in the presence of 25 U/ml rhil2. Depicted surface marker expression was measured by FACS at 36h post activation. Each experimental condition was performed and analysed in duplicate with differences in measured mean fluorescence intensities (MFIs) < 4% in all cases. The MFI value for the isotype control (Isot.) in each experiment is shown as range observed over all experiments performed. (b-d) PBMCs from two healthy donors (HD1 and HD2, one animal each) or Patients AP1 (two animals) and AP3 (one animal) were activated 72h with immobilizedantibodiestocd3andcd28and1x1 7 of expanded T cells injected into NOD-SCID-IL2RG / mice (one animal per healthy donor and two animals per patient). (b) Human T cells engraftment was assessed via presence of hcd45-positive cells at time point indicated as well as measurement of (c) hifn- in serum. Disease onset and progression was monitored by assessing (d) the weight change until sacrifice of the animals and confirmed using immunohistological analysis of intestinal tissue (data not shown). Note, that for logistic reasons, we were unable to utilize the NOD-SCID- 2M / mouse strain used previously for the injection of T cells from the CD46-deficient and GvHD onset and progression follows distinct kinetics between the two mouse strains. nd, not detectable;, Mouse A was sacrificed at day 49 at humane end point. Nature Immunology: doi:1.138/ni.2454

12 Supplementary Table 1. Features of CD46-deficient patients that participated in this study HD3 HD4 dcd46-1 a dcd46-2 b dcd46-3 Gender Female Male Female Male Male Age (years) Ethnicity Cauc Cauc Cauc Cauc Cauc Mutation Exon 2; 1. Splice site alteration IVS2+2T>G leading to deletion of c144/p48 in frame with residual WT sequence. Homozygous Heterozygous < 1% protein expression on T cells Exon 2; 1. Splice site alteration IVS2+1G>C leading to three aberrant mr transcripts. < 1% T cells positive for protein Exon 2; 1. Missense Cys1Tyr 2. Nonsense Arg25X. No detectable protein on T cells Homozygous Homozygous Compound heterozygous HUS Infections No hospital record of recurrent infections Recurrent infections (chest) until IVIG Recurrent infections (chest) until IVIG a, see reference [1]; b, see reference [11]; Cauc, Caucasian; HD, healthy donor; HUS, Hemolytic uremic syndrome; IVIG, Intravenous Immunoglobulin therapy; X, Stop. Note that the lymphocyte compartment (B cells, CD4 + and CD8 + T cells) is within normal range in patients CD46-1 a, CD46-2 b and CD46-3 (data not shown) Nature Immunology: doi:1.138/ni.2454

13 Nature Immunology: doi:1.138/ni.2454