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1 Supplementary appendix This appendix formed part of the original submission and has been peer reviewed. We post it as supplied by the authors. Supplement to: Kennedy GA, Varelias A, Vuckovic S, et al. Addition of interleukin-6 inhibition with tocilizumab to standard graft-versus-host disease prophylaxis after allogeneic stem-cell transplantation: a phase 1/2 trial. Lancet Oncol 2014; published online Nov 14.
2 SUPPLEMENTARY APPENDIX Table of Contents: 1. Methods Figure S Figure S Figure S Figure S Figure S Figure S Table S Figure S p 1
3 METHODS Anti IL-6R mab infusion. Tocilizumab (TCZ), a humanized anti IL-6R mab (Chugai Pharmaceutical Co., Tokyo, Japan, distributed by Roche Australia) was administered as a single dose, 8mg/kg (capped at 800mg), on day -1 of conditioning as an IV infusion over 60 mins using a standard infusion set. TCZ antibody concentrations in patient sera were determined using high sensitivity ELISA by QPS Netherlands (Groningen, The Netherlands). Briefly, biotin-labeled soluble IL-6R (IL-6sR) was bound to streptavidin coated microtiterplates. TCZ in the sample binds to the immobilized biotin-labeled IL-6sR. The bound antibodies were detected by digoxiginated anti-tcz antibodies followed by an anti- DIG-POD (poly) conjugate and ABTS. The quantity of bound peroxidase was determined with the ABTS substrate. The absorbance was measured and was proportional with the TCZ concentration in the sample (wavelength at 405 nm and 490 nm as reference). The absorbance of the reagent blank (Std 0) was subtracted from all other samples and the corrected absorbance used for further calculation. A calibration curve was plotted and the TCZ concentration in the sample determined by interpolation from the standard curve. Cytokine analysis. Human IL-1, IL-4, IL-6, IL-8, IL-10, IL-13, IL-17A, TNF and IFN levels were measured in patient plasma samples using human Flex Array sets (BD Biosciences Pharminogen, San Diego, CA). Soluble IL-6R and soluble gp130 were measured using Quantikine Immunoassays (R&D Systems) according to the manufacturer s instructions. The soluble IL-6R Quantikine Immunoassay is able to detect 3 forms of sil-6r: sil-6r free from IL-6 or TCZ, sil-6r in a complex of IL-6/sIL-6R, and sil-6r in an immune-complex of TCZ/sIL-6R (Nishimoto N et al., Blood. 2008, 112(10): ). PBMC isolation and FACS analysis. Plasma was harvested from whole blood after centrifugation at 600g and stored at -70 C for subsequent cytokine analysis. Peripheral blood mononuclear cells (PBMC) were purified from whole blood using Ficoll-paque centrifugation. Effector and regulatory cell enumeration was performed on freshly isolated PBMC. Quantification of STAT3 phophorylation on specific PBMC subsets was performed on 100ul whole blood. Briefly, cells were surface stained prior to stimulation with either recombinant human IL-6 (100 ng/ml, 5 minutes), G-CSF (100 ng/ml, 15 minutes) or nil. RBC were lyzed using BD FACS lysing solution. Cells were permeabilized with pre-chilled Perm p 2
4 Buffer III (BD Biosciences) for 30 minutes on ice. Cells were stained with Phosflow pstat3-pe mab (Clone 4/P-STAT3) or IgG2a-PE mab (MOPC-173) for 1 hr. For cytokine analysis in PBMC by intracellular staining, stored PBMC were thawed, rested overnight in culture media supplemented with 10 Units/ml recombinant human IL-2 prior to stimulation for 4 hours with phorbol myristate acetate (50ng/mL) and ionomycin (500ng/mL) in the presence of brefeldin A (Biolegend). Subsequently, PBMC were surface-labeled and stained for IFN-γ, IL-17, TNF and IL-4 using the BD Cytofix/Cytoperm kit (BD Biosciences) as per the manufacturer s instructions. Dead cells were excluded from analysis using the Live/Dead Fixable Dead Cell staining kit (Molecular Probes). All samples were aquired on an 18-colour, 4-laser, BD LSR Fortessa (BD Biosciences) using BD FACSDiva (v7.0) software and analysed with FlowJo (v9.7.5) software. The following mabs were purchased from BioLegend (San Diego, CA): CD16 FITC (3G8), CD4 FITC (RPA-T4), CD141 PE (M80), CD11c PE-CY5 (3.9), CD8 PE-Cy7 (RPA-T8), CD20 PE-CY7 (2H7), CD25 PE-CY7 (BC96), CD1c APC (L161), HLA-DR APC-CY7 (L243), CD3 APC- CY7 (HIT3a), CD123 PerCP/CY5.5 (6H6), CD14 Pacific Blue (HCD14), CD3 AF700 (HIT3a), IFN Pacific Blue (4S.B3), IL-17A BV605 (BL168), TNF APC (MAb11), IL-4 PE (8D4-8). The following mabs were purchased from BD Biosciences (San Jose, CA): CD8 PE-CF594 (RPA-T8), CD56 PE-CY7 (B159), CD4 V500 (RPA-T4), CD45 V500 (HI30), CD127 V450 (HIL-7R-M21). CMV DNA quantification. CMV DNA quantification was performed on plasma samples at least once a week from day 0 to CMV DNA quantification was performed with COBAS Amplicator CMV Monitor Test (Roche Diagnostics, Basel, Switzerland) or the Qiagen Artus CMV kit (Qiagen, Doncaster, VIC). Results were linear from 600 to 100,000 copies/ml. Patients did not receive CMV prophylaxis and CMV reactivation was defined as the detection of CMV DNA at 600 copies/ml. Patients were thereafter pre-emptively treated with ganciclovir as defined in the study protocol. RNA preparation for transcriptional analysis. Transcriptional RNA profiling was performed on sorted CD14 + monocytes and CD3 + CD4 + T cells from stored PBMC. Dead cells were excluded using propidium iodide. Total RNA was extracted using the Arcturus p 3
5 PicoPure RNA isolation kit (Applied Biosystems) or RNeasy Micro kit (Qiagen). Any contaminating genomic DNA was removed with DNase I (Qiagen) treatment. RNA quantity was determined using the Nanodrop ND-1000 spectrophotometer (Thermo Scientifc) and quality determined using the RNA 6000 Pico kit (Agilent Technologies) according to the manufacturer s instructions. The data discussed in this publication have been deposited in NCBI's Gene Expression Omnibus, and are accessible through GEO Series accession number GSE p 4
6 Figure S1 100 a 90 *** b **** c *** IL L-6 (pg/ml) *** d % pstat3 + cells Days post SCT Cy/TBI Flu/Mel CD14 + Monocytes CD4 + T cells CD8 + T cells **** *** **** ** **** **** *** **** Controls Untreated IL-6R IL 6R mab mab Controls Untreated IL-6R IL 6R mab mab Controls Untreated IL-6R IL 6R mab mab baseline pstat3 (B) IL 6 induced pstat3 (IL 6) G CSF induced pstat3 (G CSF) Figure S1: IL-6 expression and signalling after allosct. (a) Kinetics of IL-6 expression in plasma of recipients of 10/10 HLA-matched allosct after Cy/TBI or Flu/Mel conditioning and cyclosporin and MTX immune suppression (n = 53), ***P < vs. day -7. (b) Comparison of IL-6 levels in patients 7 days after allosct with Cy/TBI (n = 28) or Flu/Mel (n = 25) conditioning, ****P < Data presented as mean ± SEM. (c) Soluble IL-6R levels in plasma and IL-6R mab concentration in sera at day 30 after allosct, **P = , r 2 = (d) pstat3 expression as a proportion of CD14 + monocytes, CD4 + and CD8 + T cells in control recipients (n = 26) compared to IL- 6R mab-treated recipients (n = 11) 30 days after transplantation. IL-6 and G-CSF recombinant proteins were used as positive and negative controls for IL-6R stimulation. Note neither CD4 nor CD8 T cells respond to G-CSF, consistent with the known low or absent levels of the G-CSFR on T cells. Data analyzed using the Kruskal-Wallis test with Dunn s test for multiple comparisons and medians presented. CD14 + monocytes: ****P < , ***P = , **P = ; CD4 + T cells: ****P < ; CD8 + T cells: ****P < , ***P = p 5
7 Figure S2 a b c In ncidence grade II-I IV acute GVHD Cy/TBI Days post SCT IV acute GVHD Incidence grade II Flu/Mel Days post SCT Number at risk ree Survival Progression Fr Months post SCT Figure S2: Effect of IL-6 inhibition on transplant outcome. Comparison of grade II-IV acute GVHD incidence in recipients of (a) myeloablative (n = 16) and (b) RIC (n = 32) regimes after IL-6R inhibition. (c) Progression free survival after IL-6R inhibition. Analysis was performed using R software (version ). p 6
8 Figure S3 a b c IL-4 (pg/m l) Figure S3: Systemic cytokine levels over time in patients receiving IL-6 inhibition. Comparison of (a) TNF, (b) IL-1β and (c) IL-4 levels in plasma of controls (n = 53) vs. IL-6R mab-treated (n = 48) recipients. IL-6R mab-treated versus controls (i.e. open vs. black stacked bars). Data presented as mean ± SEM. # P = 0.018, increased cytokine levels in controls vs. respective baseline day -7 levels. p 7
9 Figure S4 Figure S4: Immune reconstitution following IL-6R inhibition. Innate and adaptive cell populations were enumerated in peripheral blood from patients ts up to one year post transplant. a t. Comparison of controls o (n =21 25) vs. IL-6R mab treated eated(n =28 47) recipients e *P < 0.05, **P p 8
10 Figure S5 a controls IL-6R mab b c IFNγ 10 3 IFNγ IL-17A IL-17A TNF 10 3 TNF IL IL-4 Figure S5. CD4 T cell cytokine secretion in patients receiving IL-6R inhibition. (a) Representative FACS plots and (b-c) frequency of IFN, TNF, IL-17A and IL-4 expressing CD4 + PBMC at day +14 post allosct from controls (n = 13) vs. IL-6R mab-treated (n = 11) recipients who had engrafted such that analysis was technically feasible. p 9
11 Monocytes a Controls vs. IL-6R mab-treated p< b controls IL-6R mab c Selected gene expression in monocytes (IL-6R mab vs. controls) Figure S6 Gene Symbol Fold change P value Function Decreased Interleukin 8 IL Chemokine (CXCL8) Interleukin 1, beta IL Proinflammatory cytokine Nuclear factor, interleukin 3 regulated NFIL Transcription factor Syntaxin 11 STX E 05 Negative regulator of phagocytosis Kruppel like factor 4 KLF E 06 Regulator of monocyte differentiation Suppressor of cytokine signaling 3 SOCS Regulator of cytokine signaling L Selectin SELL Cell adhesion Increased Interleukin 6 receptor IL6R < 1e 07 Membrane receptor Tumor necrosisfactor factor, induced protein 8 like 1 TNFAIP8L E 05 Immune homeostasis Interferon regulatory factor 5 IRF Interferon signaling Figure S6: Monocyte function in patients receiving IL-6R inhibition. (a) Volcano plot highlighting differential gene expression in sort-purified CD14 + monocytes 30 days after allosct from controls (n = 21) and IL-6R mab-treated (n = 26) patients without grade II-IV acute GVHD. The y-axis represents p-value of differential expression and the x-axis represents foldchange of expression levels. Blue points above the line represent those genes differentially expressed with P < (b) Heat map of microarray analysis using Human HT12v4 Expression BeadChips (Illumina, CA). Presentation of 302 differentially expressed genes at P < identified by unpaired t-test with multivariate permutation correction. (c) Selected immunologically relevant genes differentially regulated ltdinmonocytes from patients t receiving IL-6 inhibition. Note in particular the predicted increase in IL-6R and reduction in SOCS3 mrna in the absence of IL-6 induced STAT3 phosphorylation. p 10
12 Table S1: MiSeq targeted gene list for CD4 T cell analysis Cytokines Cytokine receptors Chemokines and receptors Transcription factors Effector molecules Housekeeping genes IL1A IL1R1 CCR1 TBX21 GZMA GAPDH IL1B IL2RA CCR10 GATA3 GZMB HPRT1 IL2 IL4R CCR2 RORC AHR IL3 IL6R CCR3 FOXP3 ARID5A IL4 IL21R CCR4 BCL6 PRF1 IL4 IL23R CCR5 EOMES TXLNA IL5 IFNAR1 CCR6 SOCS1 IL6 IFNGR1 CCR7 SOCS2 IL8 IFNGR2 CCR8 SOCS3 IL9 IL10RA CCR9 SOCS4 IL10 IL10RB CXCR3 SOCS5 IL13 IL12RB1 CXCR4 SOCS6 IL17A IL12RB2 CXCR5 SOCS7 IL17B IL27RA CXCR6 IL17C IL28RA IL17F TGFBR1 IL18 TGFBR2 IL21 TGFBR3 IL22 TNFRSF1A IL25 TNFRSF1B IL28A IL28B IL29 TNF LTA IFNG IFNA1 IFNA2 IFNA4 IFNA5 IFNA7 IFNA8 IFNA10 IFNA13 IFNA14 IFNA16 IFNA17 IFNA21 IFNB1 p 11
13 Figure S7 a * SOCS3 *** CD4 T cells b assay 1 assay 2 Figure S7. CD4 T cell function in patients receiving IL-6R inhibition. (a) Differentially expressed mrna (SOCS3 and IL-18) in sort purified CD4 + T cells 30 days after allosct from controls (n = 21) and IL-6R mab-treated (n = 27) patients without grade II-IV acute GVHD. The full list of genes interrogated by MiSeq (Illumina, CA) is shown in appendix p 11. Assay 1 and assay 2 refers to different primer combinations for each gene. *P < 0.05, ***P < (b) Unsupervised hierarchical clustering of mrna expression in CD4 T cells fails to segregate untreated and IL-6R mab (TCZ) groups in the samples outlined in (a). Expression is shown for genes (see appendix p 11) that are known to contribute to T cell differentiation and function after allosct. p 12
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