Immunosuppressive regimens and complement inhibitors Recipient ID

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(1)Table S1. Immunosuppressive regimens and complement inhibitors Recipient ID. Infused Islet (IEq/kg). Immunosuppressive regimen. R1. 50,000. tacrolimus, leflunomide, MMF, basiliximab, rituximab. (-). day 6. R2. 20,000. campath, tacrolimus, leflunomide, MMF. (-). day 6. R3. 24,000. ATG, bortezomib, leflunomide, MMF. (-). day 2. R4. 30,000. ATG, bortezomib. (-). day 6. R5. 98,000. anti-CD154, anti-human ICAM-1, rapamycin. CVF. day 225. R6. 96,000. anti-CD154, anti-human ICAM-1, rapamycin. CVF. day 365. R7. 61,000. anti-CD154, anti-human ICAM-1, rapamycin. CVF. day 362. R8. 98,000. anti-CD154, anti-human ICAM-1, rapamycin. control (-). day 150. R9. 105,000. anti-CD154, anti-human ICAM-1, rapamycin,. control (-). day 155. R10. 100,000. anti-CD154, ATG, rapamycin. factor H. day 156. R11. 100,000. anti-CD154, ATG, rapamycin. factor H. elective end at day 256. Complement inhibitor. Functional survival of grafts. ATG, anti-thymocyte globulin; anti-human ICAM-1, anti-human intercellular adhesion molecule 1 antibody; MMF, mycophenolate mofetil.

(2) 120. P=0.0011. P=0.0015. % Level of C3a. 100. Anti-factor B Isotypic control P=0.0007. 80 60. P=0.0367. 40 20. 0 None Ice. None. 10. 40 20 Anti-factor B or isotype (μg). 60. 37C. Figure S1. Effects of anti-human factor B antibody on adult porcine islet cell-induced complement activation. Isolated islet clusters were incubated in 20% human serum at 37°C for 1 h with or without anti-human factor B antibody or its isotype-matched antibody. After incubation, C3a levels in the supernatants were measured by ELISA. The percent level of C3a was calculated based on comparisons with the positive control (without anti-human factor B, incubated at 37°C), designated as 100%, and the negative control (without antihuman factor B, incubated on ice), designated as 0%. The data presented are the cumulative results of two independent experiments performed in duplicate. Each number indicates the Pvalue of the experimental group relative to the corresponding isotypic control..

(3) 62.95 %. 26.47 %. 29.89%. I. 36.74%. 69.54%. 48.46%. 58.64%. 30.11%. 46.13%. 28.83%. 65.83%. 34.90%. 66.68%. 31.23% 60.05%. 64.71%. SSC. D. 0. 200. 400. 600. 800. 1000. 0. 200. 400. 600. 800. FSC None. None. 1000. 0. 200. 400. 600. 800. 40. 10000. 200. 400. 600. 800. 1000. 0. 200. 600. 800. 40. 60. Anti-factor B (μg). Ice. 400. 1000. 0. 200. 400. 600. 800. 60. Isotypic antibody (μg). 1000. 0. 200. 400. 600. 800. 8. 1000. 0. 200. 400. 600. 1000. 16 Factor H (μg). 37C. I (%) 64±5. 27±4. 43±3. 47±3. 36±2. 33±2. 36±1. 34±3. D (%) 23±4. 61±2. 43±6. 38±6. 57±4. 62±5. 59±2. 61±3. 0.0438. 0.0257. P, vs. positive control. 800. Figure S2. Effects of complement inhibitors on the damage of adult porcine islet cells after contact with human serum. Isolated islets in single-cell suspensions were incubated in 20% human serum at 37°C for 1 h with or without anti-human factor B antibody, isotype-matched antibody or human factor H and analyzed by flow cytometry to obtain histograms. A major population of cells in the negative control was gated as I (intact cells), and the major population of cells in the positive control was gated as D (damaged cells). A representative histogram illustrates the relationship between the proportion of cells in the I and D regions with and without the addition of complement inhibitors. The cumulative proportion of cells in the I and D regions from two independent experiments in duplicate is shown. Each number indicates the P-value of the experimental group relative to the positive control..

(4) SDC, Materials and Methods. Antibodies for immunofluorescence assay: fluorescein-conjugated rabbit anti-human IgM and anti-human IgG (DAKO, Glostrup, Denmark), fluorescein-conjugated goat anti-human C3 (MP Biomedical, Santa Ana, CA), and goat anti-human factor B and fluorescein-conjugated rabbit anti-goat IgG (Comtech, Tyler, TX). ELISA kits: OptEIA Human C3a ELISA (BD Biosciences), Bb plus EIA (Quidel), Human C4a ELISA (BD Biosciences), C4d Fragment EIA (Quidel), and Enzygnost® TAT micro ELISA (Siemens Healthcare Diagnostics, Munich, Germany)..

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