Alexander Puck, Stefan Hopf, Madhura Modak, Otto Majdic, Petra Cejka,. Stephan Bl¨uml, Klaus Schmetterer, Catharina Arnold-Schrauf, Jens G. Gerwien,.
These are not the final page numbers
European Journal of Immunology Supporting Information for DOI 10.1002/eji.201646405 Alexander Puck, Stefan Hopf, Madhura Modak, Otto Majdic, Petra Cejka, Stephan Bluml, Klaus Schmetterer, Catharina Arnold-Schrauf, Jens G. Gerwien, ¨ Klaus S. Frederiksen, Elisabeth Thell, Judith Leitner, Peter Steinberger, Regina Aigner, Maria Seyerl-Jiresch, Gerhard J. Zlabinger and Johannes Stockl ¨ The soluble cytoplasmic tail of CD45 (ct-CD45) in human plasma contributes to keep T cells in a quiescent state
C 2016 The Authors. European Journal of Immunology published by WILEY-VCH Verlag
GmbH & Co. KGaA, Weinheim.
www.eji-journal.eu
Supporting Information Tables Supporting Information Table 1. Expression of selected regulators of T cell activation in the presence of ct-CD45 determined via microarray analysis. Numbers denote log2 fold changes relative to untreated cells.
Symbol CBLB EGR2 EGR3 DGKA TOB1 ITCH RNF128 SIRT1 KLF2 SLFN12 SLFN5 SLFN11 SLFN13
CD3/CD28/ct-CD45 12 h -0,1 -0,7 -0,6 0,3 0,2 0,0 0,0 0,1 0,7 0,4 0,2 0,6 0,1
CD3/CD28/ct-CD45 24 h -0,1 -0,8 -0,4 0,1 0,0 -0,6 0,0 0,3 1,2 0,9 0,1 -0,7 0,0
Supporting Information Table 2. Primers used for qPCR. Target GAPDH_F GAPDH_R CDK2_F CDK2_R CDK4_F CDK4_R CyclinD2_F CyclinD2_R CyclinD3_F CyclinD3_R CyclinE1_F CyclinE1_R p27kip_F p27kip_R
Primer sequence CGACCACTTTGTCAAGCTCA AGGGGAGATTCAGTGTGGTG TTGTCAAGCTGCTGGATGTC TTTAAGGTCTCGGTGGAGGA TTCTGGTGACAAGTGGTGGA CTGGTCGGCTTCAGAGTTTC CTGGGGAAGTTGAAGTGGAA ATCATCGACGGTGGGTACAT AGACCTTTTTGGCCCTCTGT CCTGAGTGCAGCTTCGATCT AATGCGAGCAATTCTTCTGG CTGGTGCAACTTTGGAGGAT CCGGCTAACTCTGAGGACAC AGAAGAATCGTCGGTTGCAG
CD3/CD63/ct-CD45 12 h 0,0 -1,8 -1,9 0,1 0,3 0,3 -0,1 0,0 0,8 0,5 0,1 0,3 -0,3
CD3/CD63/ct-CD45 24 h 0,0 -1,9 -1,0 0,4 0,2 0,0 -0,1 0,1 0,9 0,4 0,4 0,1 0,1
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Supporting Information Figure 1. Detection and immunoprecipitation of ct-CD45. (A) Schematic depiction of the ct-CD45 domain composition and of the binding sites of the monoclonal antibodies used for the ELISA (above). Detection of recombinant ct-CD45 and ct-CD45-Ig as well as of truncated ct-CD45-Ig constructs, comprising only domain D1 or D2 via the ELISA system (below). Numbers in brackets indicate the region in the total CD45 amino acid (aa) sequence that is contained within the respective construct. (B) Comparison of ct-CD45 levels via ELISA in pooled plasma and pooled serum derived from adult and umbilical cord blood. (C) Analysis of ctCD45 levels via ELISA in plasma treated with bead-coupled 8-301 mAb (ct-CD45 depl) or VIT200 mAb (ctrl depl) as a control. (D) Immunoprecipitation of recombinant or native ct-CD45 from plasma via bead-coupled 8-301 mAb. Beads were then stained using E19-G and secondary fluorochrome-conjugated antibody and analyzed via flow cytometry, gating on single beads.
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Supporting Information Figure 2. ct-CD45 levels do not correlate with age or sex. ct-CD45 levels were measured via ELISA in adult plasma and analyzed with respect to (A) age and (B) sex of the donors (male, n=8, mean age=46.1 ; female n=8, mean age=45.5). (A) Data was analyzed using linear regression and Pearson‘s r . r, Pearson correlation coefficient or (B) Mann-Whitney U test. P values: *, p < 0.05, not significant (ns) p > 0.05.
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Supporting Information Figure 3. Titration of ct-CD45-Ig. Human T cells were stimulated via plate-bound CD3 or CD3/CD63 antibodies in the presence of graded amounts of ct-CD45-Ig. Concentrations indicated were used for coating of protein to 96 well plates. Proliferation was analyzed via thymidine incorporation. cpm, counts per minute. Data show mean ± SD and are representative of at least two independent experiments.
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Supporting Information Figure 4. Effects of ct-CD45 on the proliferation of T cell subsets and cord blood T cells. T cells were stimulated via plate-bound CD3, CD3/ CD63 or CD3/CD28 antibodies in the presence of medium alone, ct-CD45-Ig or ctrl Ig. Proliferation of (A) adult CD3+CD4+, (B) adult CD3+CD8+ and (C) cord blood CD3+ T cells was analyzed via thymidine incorporation. cpm, counts per minute. unstim, unstimulated control. (D) Bright field micrograph of CD3/CD63-stimulated T cells that were activated for 3 days in the presence or absence of ct-CD45-Ig. Scale bar, 25 µm. (A-C) Data show mean ± SD and are representative of at least two independent experiments.
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Supporting Information Figure 5. Effects of ct-CD45-Ig on the proliferation of freshly isolated CD3+ T cells. T cells were stimulated via plate-bound CD3 or CD3/ CD63 antibodies in the presence of medium alone, ct-CD45-Ig or ctrl Ig. cpm, counts per minute. unstim, unstimulated control. Data show mean ± SD and are representative of at least three independent experiments.
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Supporting Information Figure 6. Expression of SLFN12, KLF2 and EGR3 6 hours after T cell activation in the presence of ct-CD45. Human CD3+ T cells were stimulated for 6 hours via plate-bound CD3/CD63 antibodies in the presence of medium alone, ct-CD45-Ig or ctrl Ig. mRNA levels of the indicated genes were analyzed via qPCR relative to CD3E. Data show mean ± SD and are representative of at least two independent experiments.
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Supporting Information Figure 7. Regulation of SLFN12/KLF2 during T cell activation and SLFN12 overexpression experiments. (A) T cells were stimulated with plate-bound CD3, CD3/CD63 or CD3/CD28 antibodies over a 24 hours time course. Expression levels of SLFN12 and KLF2 were analyzed via qPCR using CD3E as a reference gene. (B) T cells were activated for 48 hours with Human T-Activator CD3/CD28 Dynabeads and recombinant human IL-2 (150 U/ml) for 2-3 days and were retrovirally transduced with SLFN12 and control vectors. Cells were then expanded in IL-2 (75 U/ml). SLFN12 mRNA expression was determined via qPCR on day 10 after gene transduction. (C) SLFN12 mRNA expression in Jurkat cells transduced with SLFN12 or empty control vector on day 3 after gene transduction. (D) FSC/SSC analysis of Jurkat cells on day 3 post transduction. (E) FSC/SSC analysis of SLFN12-transduced Hela Ohio cells on day 3 after gene transduction. (D, E) Numbers given indicate percentages of live cells in the gate. (A-C) Data are expressed as mean ± SD and (A-E) are representative of at least 3 independent experiments.
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Supporting Information Figure 8. ct-CD45 levels do not correlate with clinical parameters of RA patients. ct-CD45 was measured via ELISA in RA patient serum and was correlated with (A) c-reactive protein (CRP) and (B) rheuma factor (RF) levels and with the disease activity scores (C) CDAI and (D) DAS28. Data was analyzed using linear regression and Pearson‘s r . r, Pearson correlation coefficient. P values: *, p < 0.05, not significant (ns), p > 0.05.
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Supporting Information Figure 9. ct-CD45 levels are reduced in patients diagnosed with systemic lupus erythematosus (SLE). ct-CD45 was measured via ELISA in plasma derived from SLE patients (n=8) and compared to matched controls (n=9). Mean ± SEM are given. P values: *, p < 0.05. Mann-Whitney U test.
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Supporting Information Figure 10. ct-CD45 levels do not correlate with leukocyte numbers of SLE patients. Ct-CD45 levels were measured via ELISA in sera of SLE patients and correlated with leukocyte numbers determined at the time of the blood draw. Linear regression and Pearson’s r were calculated. r, Pearson correlation coefficient. P values: *, p < 0.05, not significant (ns), p > 0.05.
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Supporting Information Figure 11. Differential Effects of immunosuppressive treatment on ct-CD45 levels of RA and SLE patients. Ct-CD45 levels were analyzed via ELISA in sera obtained from (A) RA patients receiving treatment that includes methotrexate (MTX) or that received treatment other than MTX (w/o MTX) and (B) SLE patients that were either treated with chloroquine or azathioprine. Mean ± SEM are given. Mann-Whitney U test. P values: *, p < 0.05.
