Chimeric pan HLA I IgG1 was generated by fusion of the heavy and light chain variable regions from

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1 Materials and Methods HLA Antibodies Chimeric pan HLA I IgG1 was generated by fusion of the heavy and light chain variable regions from the murine monoclonal pan HLA class I antibody W6/32 with human constant regions as previously described. Monoclonal fully human HLA class I antibodies were derived from multiparous women, and recognize cross reactive antigen groups of HLA class I. Clone SN230G6 recognizes HLA- A2/B17, clone SN607D8 recognizes A2/A28 (A68, A69), and clone MUL2C6 recognizes an epitope shared by HLA-A1/A3/A11/A24/A38/A80. Allosera containing HLA antibodies were obtained from the UCLA HLA reference serum repository, and have been characterized for HLA specificities by single antigen assay (LabScreen, One Lambda), Luminex PRA (Immucor) and cytotoxic antibody screen. These sera are utilized in proficiency testing for HLA antibody assays. Sera were heat-inactivated at 56 C for 30min to eliminate endogenous complement activity and pre-cleared by centrifugation at 5,000 x g prior to use. Specificities can be found in Table 1. Supplemental Figure 1A shows the binding of allele specific monoclonal IgG1 at 10ng/mL to 1µg/mL to endothelial cells from three different donors by cell-based ELISA. Supplemental Figure 1B demonstrates human IgG binding to HAEC from three different donors, exposed to relevant HLA allosera, as well as chimeric pan HLA I IgG1 at 100ng/mL and monoclonal allele specific IgG1, also at 100ng/mL. Based on these and other preliminary experiments, monoclonal antibodies were used at 100ng/mL except where indicated. Complement Source and Handling Purified C3a and C5a were from Complement Technology, Inc (Tyler, TX). Normal human AB serum complement (Quidel, San Diego, CA, and Innovative Research, Novi, MI) and C1q-depleted sera (Complement Tech) were obtained as sources of human complement. Human serum complement was stored at -80 C, rapidly thawed at 37 C and diluted in basal medium directly to a final concentration of 25% prior to use in experiments. As a control, normal human serum complement was incubated at 56 C for 30min to inactivate complement proteases.

2 Cells and Cell Culture Human aortic endothelial cells (HAEC, donors Y126, X127 and H126) were isolated from the aortic rings of deceased cardiac donors (IRB ) or obtained from ATCC (donor 3F1153), and cultured as previously described. Briefly, HAEC were seeded at confluence on gelatin-coated tissue culture plates in complete medium containing 20% heat-inactivated fetal bovine serum (FBS), antibiotics, heparin and ECGS. All experiments were repeated with endothelial cells between passages 4-9 from 2-3 different donors to rule out responses unique to certain donors. HLA-A and B typing for endothelial cells can be found in Table 2. Endothelial cell monolayers remained intact after exposure to anti-hla antibodies in the presence of intact human complement, as measured by live/dead stain by flow cytometry and microscopy (Supplemental Figure 2). The monocytic cell line Mono Mac 6 (MM6) was generously provided by Dr. Judith Berliner and Dr. Ziegler-Heitbrock, and cultured as described. Primary monocytes were enriched from the peripheral blood of 3 healthy donors by negative selection using the RosetteSep Human Monocyte Enrichment Cocktail (StemCell Technologies). Endothelial P-selectin Cell-Based ELISA Rapid upregulation of P-selectin at the endothelial cell surface was measured by a cell-based ELISA as described. Briefly, confluent HAEC in a 96 well tissue culture plate were stimulated with purified C3a or C5a (1nM-100nM) in M199 supplemented with 5% FBS for 5min. Alternatively, HAEC were treated with negative control or HLA antibodies for 30 min in M199 supplemented with 25% human AB serum as a source of intact complement (C ), C1q-depleted complement, or with heat-inactivated serum (HI-C ) as a control. PMA (200nM, 20min) was used as a positive control for P-selectin induction. Cell surface P-selectin on unpermeabilized cells fixed with 4% paraformaldehyde was probed after blocking with 5% BSA for 30min with sheep anti-human P-selectin at 1µg/mL (R&D

3 Systems, Minneapolis, MN) followed by donkey anti-sheep IgG-HRP (R&D), and detected by colorimetric assay (TMB substrate, Sigma, St. Louis, MO) quantified on microplate reader (650 nm, Molecular Devices, Sunnyvale, CA). As a parallel control, endothelial cells were permeabilized with Triton X 100 (0.1%) for 15min during the blocking step, before probing for P-selectin. Results are reported as mean optical density (OD) in replicate wells with range, or fold increase in mean OD normalized to stained, untreated HAEC. ELISA for Complement Split Products C3a and C5a in supernatants were quantified by ELISA (Quidel), according to manufacturer s instructions. HAEC were incubated with HLA antibodies for 15min, then M199 with 25% human serum complement was added for 30min. Supernatant was removed and immediately frozen on dry ice, then transferred for storage at -80 C. To detect C3a, supernatants of M199 with 25% human serum complement were diluted 1:1250 for a final serum concentration of 1:5000 per manufacturer recommendations. To detect C5a, supernatants were diluted 1:12.5 for a final serum concentration of 1:50. Results are expressed as mean OD of colorimetric change of TMB substrate. Static Monocyte Adhesion Assay Adherence of monocytic cells to endothelium under static conditions was measured as described. Briefly, CFSE-labeled MM6 were incubated with C3a- or C5a-stimulated HAEC for 20min. Alternatively, MM6 or primary human monocytes were resuspended in RPMI supplemented with heatinactivated or intact 25% human serum complement, and added to stimulated HAEC for 45min. Nonadherent monocytes were removed by washing. Fluorescence microscopy fields (4x objective, 5-10 fields imaged and counted per condition) of CFSE-labeled monocytes bound to endothelial monolayers were acquired on a Nikon Ti Eclipse. To block monocyte adhesion receptors, MM6 were pretreated with 10µ/mL neutralizing antibody to the αl integrin CD11a (LFA-1), αm integrin CD11b (Mac-1), or β1 integrin (VLA-4) (all migg1, from Biolegend, San Diego, CA) at 10μg/mL.

4 Complement Inhibitors TNT003, a monoclonal mouse anti-human C1s, was made as previously described. TNT003 and a non-specific isotype control antibody (Control, IgG2a, #BE0085, BioXCell, West Lebanon NH), were cleaved to F(ab ) 2 fragments (F(ab ) 2 Prep Kit, #44988, Thermo Scientific, Rockford IL), to prevent any non-specific Fc-mediated effects of the monoclonal inhibitors. To inhibit activation of the classical complement pathway, human serum complement was preincubated with the F(ab ) 2 of TNT003 or control at 1-25μg/mL for 15min, prior to use in experiments. TNT003 was maintained in medium for the duration of the experiment. Anti-human C5 murine IgG1 was purchased from Quidel. Statistical Analyses Groups were first compared using two-way ANOVA with repeated measures, followed by Tukey s or Bonferroni s multiple comparisons test to measure statistical differences between groups. ns, p>0.1; # p<0.1; *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<

5 Supplemental Figure S1 A B 1

6 A. Binding of human IgG to endothelial cells was measured by cell-based ELISA. HAEC were cultured to confluence in a 96-well tissue culture plate and exposed to monoclonal HLA-A3/A11 IgG1 at the indicated concentrations, or relevant HLA allosera, for 20 minutes. HLA-A3/A11 IgG1 recognizes HLA-A1 and HLA-A11 on HAEC Y126, HLA-A11 on HAEC 3F1153, and HLA-A3 on HAEC H126. Then, supernatants were removed, cells were fixed with paraformaldehyde, and cell surface human IgG bound to the cells was probed with anti-human IgG H+L-HRP. Bound IgG was detected with TMB substrate. The mean OD in duplicate wells is presented, which each scatter dot representing one of three different HAEC. B. Binding of human IgG to endothelial cells was measured by cell-based ELISA. HAEC were cultured to confluence in a 96-well tissue culture plate and exposed to pan HLA I IgG1 or relevant monoclonal allele specific IgG1 at 100ng/mL, or relevant HLA allosera, for 20 minutes. Cell surface bound human IgG was probed as in (A). 2

7 Supplemental Figure S2 3

8 C 4

9 D 5

10 A, B. Four HAEC with disparate HLA expression were incubated with a pooled polyclonal serum (PS) containing HLA antibodies for multiple specificities; this PS was chosen to maximize ligation of HLA on the cell surface by HLA antibodies. After a 30 minute incubation, the unbound HLA antibodies were removed by washing, followed by addition of 25% normal human serum complement at room temperature for 30 min. After this incubation, cells were stained for HLA antibody and complement activation followed by staining with Sytox Blue to assess viability. Cell viability was not altered in the presence of HLA antibody (IgG+) and subsequent human complement activation (C4d+), as demonstrated by the lack of change in the frequency of Sytox Blue positive cells. C. 4x microscopy fields illustrate endothelial morphology in the presence of human serum complement, untreated (upper panel) or after exposure to HLA antibodies (lower panel). D. 10x microscopy fields illustrate CFSE-labeled monocyte adhesion (FITC channel, green spots) to intact endothelial monolayers (bright field). Representative fields are shown for 3F1153 HAEC stimulated with NS or alloserum M

11 Supplemental Figure S3 HAEC were left untreated or stimulated for 20 minutes with negative control IgG1 (control IgG), monoclonal HLA I IgG1 antibodies at the indicated concentrations or PMA as a positive control. Heat-inactivated complement (open circles) or intact complement (closed circles) was added at the beginning of stimulation to a final concentration of 25%. Monolayers were fixed and cell surface P-selectin was probed on unpermeabilized cells by cell-based ELISA. In parallel, untreated wells were permeabilized with Triton X 100 prior to probing for P-selectin. Scatter dots illustrate the results from multiple independent measurements of cell surface P- selectin by HLA I IgG1, in the presence of heat-inactivated (open circles) or intact complement (closed black circles). n=5 for control IgG 100ng/mL and PMA 7

12 200nM, n=8 for anti-hla I IgG1 at each concentration. HI C was compared to intact C for each stimulation using two way ANOVA followed by Bonferroni s multiple comparisons test. * p<0.05 comparing HI C to intact C. 8

13 Supplemental Figure S4 A B HAEC seeded at confluence in a multi-well plate were incubated with negative human serum (NS diluted 1:3) containing no HLA antibodies, or a broadly reactive pooled human alloserum containing relevant HLA antibodies (PS6 diluted 1:3), for 15min at 37 C. Serum was removed, and 25% human complement (heat-inactivated, open circle; intact complement, closed circle) diluted in M199 was added and incubated at 37 C for 45min. Supernatants were collected and C3a (grey circle) and C5a (black circle) were measured by ELISA. Results are expressed as mean OD of anaphylatoxin present in duplicate wells, each scatter dot represents a different endothelial cell. Line indicates the median. 9

14 Supplemental Figure S5 HAEC were left untreated or stimulated with chimeric HLA I IgG1 at the indicated concentrations for 20 minutes, C3a 10nM or C5a 10nM alone for 5 minutes, or HLA I IgG1 for 20 minutes in combination with C3a or C5a for the final 5 minutes of stimulation. Representative experiment showing induction of cell surface P- selectin on HAEC from donor 3F1153, stimulated with C3a or C5a, with (black circles) or without (open circles) anti-hla I IgG1. The mean optical density (OD) of cell surface P-selectin +/- range is given for duplicate wells. Dashed line indicates basal P-selectin cell surface expression on untreated endothelial cells. 10