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1 Supplementary Materials Supplemental Figure 1. Polymorphism of the distal and proximal promoter. (A) Deduced regulatory region genotypes and LILRB1 expression on NK cells. Three haplotypes and their frequencies within 24 individuals are illustrated for the distal and proximal promoter regions. The numbers underneath indicate the positions relative to the translational start. The lower portion shows distal promoter haplotype for donors listed in ascending order according to LILRB1 expression on NK with each compound genotype coded by color. The LILRB1 expression and proximal promoter genotypes are derived from Davidson et al (1). (B) Transcriptional activity of the two major distal (top) and proximal (bottom) promoter regions in NK92 cells. For each the fragments tested include three of the four SNPs shown in A that are within the active promoter regions. The bottom panel includes three point mutations at the indicated positions. The number of independent experimental tests for each is indicated above the error bar.
2 Supplemental Figure 2. LILRB1 polymorphisms and HCMV in Canadian transplant patients. (A) Incidence of HCMV infection in all post-transplant patients grouped by LILRB1 genotype. (B) Incidence of HCMV disease in post-kidney transplant patients grouped by LILRB1 genotype. (C) Incidence of HCMV infection in post-kidney transplant patients grouped by the genotype of rs and rs
3 Supplemental Figure 3. The association of the HCMV infection or disease (only in the case of D+/R or R+) incidence with different LILRB1 haplotype alleles in kidney transplant patients from STCS.
4 Supplemental Figure 4. Similarities and differences in LILRB1 binding with ligands. (A) The crystal structures of the LILRB1/UL18 (PDB code 3D2U) and LILRB1/HLA-A2 (PDB code 4NO0) complexes reveal significant overall similarities. The positions of the four LILRB1 variable residues Pro45, Ala70, Thr119 and Ile132 are indicated. (B) Superposition of the D1 domain of the four crystal structures of LILRB1 reveals a significant flexibility in the angle formed between the D1 and D2 domains. The free LILRB1-PATI (PDB code 1G0X), free LILRB1-PTTI (PDB code 1UGN), free LILRB1-LAIS (PDB code 1VDG) and the LILRB1-PATI/HLA-A2 complex (PDB code 4NO0) are colored in red, cyan, green and blue, respectively.
5 Supplemental Figure 5. Anti-MHC-I blocking of the killing of cells expressing HLA-G by YTS cells expressing LILRB1 variants. Lysis was determined in the presence of 10μg/ml α-mhc-i (W6/32) or isotype control at an E:T of 10:1. Figure shows the representative result of 3 independent tests.
6 Supplemental Figure 6. FACs data of the LILRB1 mutants binding with HLA-Cw15, HLA-G and UL18. FACs data shown is the representative data of LILRB1 mutants at one same concentration. The binding with HLA-Cw15, HLA-G was using LILRB1 mutants at 50μg/ml, and the binding with UL18 was using LILRB1 mutants at 100μg/ml.
7 Supplemental Figure 7. LILRB1 residues 70 and 132 impact indirectly on MHC binding. (A) The indirect effect of the A70T polymorphism, shown for the HLA-A2/LILRB1 complex, is due to the conformation modification of the side chain of residue E184 and the removal of a salt bridge formed with residue K91 in β2m. (B) The S132I modification abrogates the hydrogen bond formed between the side chain of S132 and the HLA residue N146. As a result of this modification, the side chains of the HLA residues Y177 and W185 are also affected, changing the shape of the section of LILRB1 that connects the D1 and D2 domains and thus the interface that contacts LILRB1. Furthermore, the S132I modification results in a movement of residue E184, which is localized closer to the β2m subunit, resulting in favorable interactions.
8 Total N=67 CMV Disease (N=17) No CMV Disease p-value Age, y, mean (SD) 48.1 (14.4) 47.7 (14.6) 49.3 (14.2) Sex, M/F, No. 50/17 13/4 37/ Type of transplant Kidney 31 (46.3) 7 (41.2) 34 (48.0) Kidney-pancreas 8 (11.9) 1 (5.9) 7 (14.0) Liver 15 (22.4) 5 (29.4) 10 (20.0) Lung 7 (10.4) 2 (11.8) 5 (10.0) Heart 3 (4.5) 0 3 (6.0) Other 3 (4.5) 2 (11.8) 1 (2.0) Antiviral Prophylaxis Intravenous Ganciclovir 10 (14.9) 3 (17.6) 7 (14.0) Valganciclovir 65 (97.0) 17 (100.0) 48 (96.0) Oral ganciclovir 2 (3.0) 0 2 (4.0) Duration of prophylaxis, d, 98 (96-150) 98 (96-125) 98 (92-153) Induction therapy None 4 (6.0) 1 (5.9) 3 (6.0) Basiliximab 37 (55.2) 9 (52.9) 28 (56.0) Thymoglobulin 26 (38.8) 7 (41.2) 19 (38.0) Alemtuzumab 0 Maintenance Steroids 57 (85.1) 15 (88.2) 42 (84.0) Tacrolimus 58 (86.6) 14 (82.4) 44 (88.0) Cyclosporin 6 (9.0) 3 (17.6) 3 (6.0) MMF/MPA 58 (86.6) 13 (76.5) 45 (90.0) Azathioprine 1 (1.5) 0 1 (2.0) mtor inhibitors 5 (7.5) 3 (17.6) 2 (4.0) Other 1 (1.5) 1 (5.9) rs AA 3 (4.5) 1 (5.9) 2 (4.0) GA 24 (35.8) 7 (41.2) 17 (34.0) GG 40 (59.7) 9 (52.9) 31 (62.0) rs CC 7 (10.4) 3 (17.6) 4 (8.0) GC 26 (38.8) 6 (35.3) 20 (40.0) GG 34 (50.7) 8 (47.1) 26 (52.0) rs AA 34 (50.7) 8 (47.1) 26 (52.0) AG 26 (38.8) 6 (35.3) 20 (40.0) GG 7 (10.4) 3 (17.6) 4 (8.0) rs AA 57 (85.1) 14 (82.4) 43 (86.0) AG 10 (14.9) 3 (17.6) 7 (14.0) GG 0 rs CC 6 (9.0) 3 (17.6) 3 (6.0) TC 26 (38.8) 6 (35.3) 20 (40.0) TT 35 (52.2) 8 (47.1) 27 (54.0) Supplemental Table 1. Canadian Patients Characteristics Abbreviations: CMV, Cytomegalovirus; MMF, mycophenolate mofetil; MPA, mycophenolic acid; mtor, mammalian target of rapamycin
9 All organs Kidney SNP Any CMV constellation Only D+ or/and R+ Any CMV constellation Only D+ or/and R+ N=1018 N=776 N=651 N=479 CMV infection LILRB1 rs A P=0.4 P=0.4 P=0.25 P=0.06 B LILRB1 rs A P=0.2 P=0.2 P=0.02 P=0.005 CMV disease LILRB1 rs A P=0.8 P= LILRB1 rs A P=1.0 P= Supplemental Table 2. Univariate analysis of the association between LILRB1 SNPs and CMV infection in STCS. A P values assessed by log-rank test, recessive mode B P=0.2 when all the patients with rs (N=1118)
10 CMV infection in kidney CMV D+/R- or R+ Univariate analysis Multivariate analysis Variable HR (95%CI) P value A HR (95%CI) P value A LILRB genotypes available Recipient male sex 0.93 ( ) Recipient age (per year) 1.02 ( ) ( ) < LILRB1 genotypes rs AA vs. GG/GA 1.74 ( ) ( ) 0.01 rs CC vs. TT/TC 1.56 ( ) CMV serostatus D+/R- reference D-/R ( ) D+/R ( ) ( ) Corticosteroids 2.25 ( ) ( ) 0.03 Ciclosporine 2.33 ( ) < ( ) Tacrolimus 0.47 ( ) < Rejection episodes 1.46 ( ) ( ) Exposure to antiviral drugs 0.27 ( ) < ( ) < LILRB and KIR genotypes available (N=263) Recipient male sex 0.71 ( ) Recipient age (per year) 1.02 ( ) ( ) 0.02 LILRB1 genotypes rs AA vs. GG/GA 2.13 ( ) ( ) 0.02 rs CC vs. TT/TC 1.64 ( ) KIR haplotype B B 1.40 ( ) KIR gene KIR2DS ( ) ( ) 0.14 CMV serostatus D+/R- reference D-/R ( ) ( ) D+/R ( ) Corticosteroids 2.76 ( ) ( ) 0.03 Ciclosporine 3.18 ( ) < ( ) Tacrolimus 0.37 ( ) Rejection episodes 1.25 ( ) Exposure to antiviral drugs 0.33 ( ) ( ) Supplemental Table 3. Multivariate analysis in kidney HCMV D+/R- or R+ transplant recipients in STCS. A P value assessed by multivariate Cox regression model. B KIR haplotype B was defined by the presence of one or more of the following genes: KIR2DL5, KIR2DS1, KIR2DS2, KIR2DS3, KIR2DS5 and KIR3DS1 (2, 3).
11 Reference 1. Davidson, C.L., Li, N.L., and Burshtyn, D.N LILRB1 polymorphism and surface phenotypes of natural killer cells. Human immunology 71: Gonzalez, A., Schmitter, K., Hirsch, H., Garzoni, C., Van Delden, C., Boggian, K., Mueller, N., Berger, C., Villard, J., and Manuel, O KIR-associated protection from CMV replication requires pre-existing immunity: a prospective study in solid organ transplant recipients. Genes and immunity 15: Robinson, J., Waller, M.J., Stoehr, P., and Marsh, S.G IPD the immuno polymorphism database. Nucleic acids research 33:D523-D526.
12 Supplemental Acknowledgements The members of the Swiss Transplant Cohort Study are: Patrizia Amico, Universitätsspital Basel (USB) John-David Aubert, Centre Hospitalier Universitaire Vaudois (CHUV) Vanessa Banz, Universitätsspital Insel Bern Guido Beldi, Universitätsspital Zürich (USZ Christian Benden, Universitätsspital Zürich (USZ) Christoph Berger, Universitätsspital Basel (USB) Isabelle Binet, Kantonsspital St. Gallen Pierre-Yves Bochud, Centre Hospitalier Universitaire Vaudois (CHUV) Elsa Boëly, Hôpitaux Universitaire de Genève (HUG) Heiner Bucher, Universitätsspital Basel (USB) Thierry Carell, Universitätsspital Insel Bern Emmanuelle Catana, Centre Hospitalier Universitaire Vaudois (CHUV) Yves Chalandon, Universitaire de Genève (HUG) Sabina de Geest, Universitätsspital Basel (USB) Olivier de Rougemont, Universitätsspital Zürich (USZ) Michael Dickenmann, Universitätsspital Basel (USB) Michel Duchosal, Centre Hospitalier Universitaire Vaudois (CHUV) Laure Elkrief, Hôpitaux Universitaire de Genève (HUG) Thomas Fehr, Universitätsspital Zürich (USZ) Sylvie Ferrari-Lacraz, Hôpitaux Universitaire de Genève (HUG) Christian Garzoni, External non- transplant center Paola Gasche Soccal, Centre Hospitalier Universitaire Vaudois (CHUV) Christophe Gaudet, Hôpitaux Universitaire de Genève (HUG) Emiliano Giostra, Hôpitaux Universitaire de Genève (HUG) Déla Golshayan, Centre Hospitalier Universitaire Vaudois (CHUV) Karine Hadaya, Hôpitaux Universitaire de Genève (HUG) Jörg Halter, Universitätsspital Basel (USB) Dominik Heim, Universitätsspital Basel (USB)
13 Christoph Hess, Universitätsspital Basel (USB) Sven Hillinger, Universitätsspital Zürich (USZ) Hans H. Hirsch, Universitätsspital Basel (USB) Günther Hofbauer, Universitätsspital Zürich (USZ) Uyen Huynh-Do, Universitätsspital Insel Bern Franz Immer, Swiss Transplant Richard Klaghofer, Zurich University Hospital Michael Koller (Head of the data center), Universitätsspital Basel (USB) Bettina Laesser, Universitätsspital Insel Bern Roger Lehmann, Universitätsspital Zürich (USZ) Christian Lovis, Hôpitaux Universitaire de Genève (HUG) Pietro Majno; Universitaire de Genève (HUG) Oriol Manuel, Centre Hospitalier Universitaire Vaudois (CHUV) Hans-Peter Marti, University Hospital of Bern Pierre Yves Martin, Hôpitaux Universitaire de Genève (HUG) Pascal Meylan, (Head, Biological samples management group), Centre Hospitalier Universitaire Vaudois (CHUV) Paul Mohacsi, Universitätsspital Insel Bern Philippe Morel, Hôpitaux Universitaire de Genève (HUG) Ulrike Mueller, Universitätsspital Basel (USB) Nicolas J Mueller (Chairman Scientific Committee), Universitätsspital Zürich (USZ) Helen Mueller-McKenna (Head of local data management), Universitätsspital Zürich (USZ) Antonia Müller, University Hospital Zurich Thomas Müller, Universitätsspital Zürich (USZ) Beat Müllhaupt, Universitätsspital Zürich (USZ) Manuel Pascual (Executive office), Centre Hospitalier Universitaire Vaudois (CHUV) Jakob Passweg, University Hospital, Geneva (HUG) Klara Posfay-Barbe, University Hospital, Geneva (HUG) Juliane Rick, Universitätsspital Basel (USB) Eddy Roosnek, Hôpitaux Universitaire de Genève (HUG) Anne Rosselet, Centre Hospitalier Universitaire Vaudois (CHUV)
14 Silvia Rothlin, Kantonsspital St. Gallen Frank Ruschitzka, Universitätsspital Zürich (USZ) Urs Schanz, Universitätsspital Zürich (USZ) Stefan Schaub, Universitätsspital Basel (USB) Aurelia Schnyder, Universitätsspital Basel (USB) Christian Seiler, Universitätsspital Basel (USB) Jan Sprachta; Universitätsspital Basel (USB) Susanne Stampf, Universitätsspital Basel (USB) Jürg Steiger (Head, Executive Office), Universitätsspital Basel (USB) Guido Stirnimann, Universitätsspital Insel Bern Christian Toso, Hôpitaux Universitaire de Genève (HUG Christian Van Delden (Executive office), Hôpitaux Universitaire de Genève (HUG) Jean-Pierre Venetz, Centre Hospitalier Universitaire Vaudois (CHUV) Jean Villard, Hôpitaux Universitaire de Genève (HUG) Madeleine Wick (STCS coordinator), Universitätsspital Basel (USB) Markus Wilhelm, University Hospital Zurich Patrick Yerly, Centre Hospitalier Universitaire Vaudois (CHUV)
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