Activated mast cells promote differentiation of B cells into effector cells

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Supplementary,information, Activated mast cells promote differentiation of B cells into effector cells Anna-Karin E. Palm 1, Gianni Garcia Faroldi 2, Marcus Lundberg 1, Gunnar Pejler 3, 2 and Sandra Kleinau 1* 1 Uppsala University, Department of Cell and Molecular Biology, Uppsala, Sweden 2 Swedish University of Agricultural Sciences, Department of Anatomy, Physiology and Biochemistry, Uppsala, Sweden 3 Uppsala University, Department of Medical Biochemistry and Microbiology, Uppsala, Sweden # #

Supplementary Table S1. Quantification of the cytokine array presented in Fig. 2a. Values are mean expression intensity ± standard deviation of duplicates for every spot on the cytokine array, as quantified using the ImageJ software. Mediators indicated in bold represent those that are profoundly up-regulated in co-cultures of MCs and B cells vs. their expression in cultures of MCs or B cells alone. Mediator Naïve BCs BCR-act. BCs Act. MCs BCR-act. BCs + act. MCs AXL 2.38±.69 3.1±.68 1.35±.65 2.49±.7 BLC 14.1±1.36 14.83±1.69 8.85±.89 14.42±3.3 CD3L 3.62±.48 9.85±.5 1.63±.74 6.±.1 CD3 3.2±.49 4.67±1.48 2.1±.36 3.89±.92 CD4.81±.3 1.69±.42.45±.9 2.±1.9 CRG-2.87±.42 6.84±.39 1.3±.6 6.7±2.12 CTACK 6.49±.77 1.6±1.96 6.47±1.5 13.54±6.96 CXCL16 2.34±.61 6.13±1.9 3.18±.48 12.79±.28 Eotaxin 5.1±.28 8.69±.86 4.45±.23 5.69±1.64 Eotaxin-2 1.68±1.45 1.61±.12 117.39±5.44 121.79±9.19 Fas Ligand 33.41±7.6 39.93±1.11 33.42±.63 4.15±6.4 Fractalkine 4.46±.56 4.78±.68 6.29±3.32 6.36±2.67 G-CSF 2.46±.16 3.22±.46 2.15±.64 4.59±2.5 GM-CSF 1.82±.86 1.93±.54 29.48±8.24 25.87±.1 IFNγ 1.34±.23 4.65±.27 1.72±.54 4.49±.45 IGFBP-3 15.13±1.45 17.38±.47 1.89±.52 17.77±5.26 IGFBP-5 12.34±2.87 15.51±.66 11.21±.6 2.85±.39 IGFBP-6 2.9±.1 3.79±.15 1.52±.1 3.29±.57 IL-1α 24.56±2.16 35.38±.62 22.19±2.69 34.32±.8 IL-1β 3.61±.3 6.12±.6 2.95±. 11.86±3.98 IL-2 2.8±.38 35.11±.4 3.44±.9 34.71±5.8 IL-3 1.55±. 5.44±.38 35.58±1.44 37.2±5.2 IL-3β 6.2±.23 9.24±.71 13.39±1.15 2.77±3.2 IL-4 12.68±2.37 12.29±2.32 11.41±2.63 14.9±3.39 IL-5 2.61±.4 5.6±.29 7.36±.32 13.74±1.65 IL-6 1.28±.9 1.98±.52 199.61±3.81 21.45±1.1 IL-9 1.15±1.52 42.39±3.87 11.19±.32 38.95±2.75 IL-1 5.25±.39 6.84±.85.81±.24 6.26±.1 IL-12p4/p7 1.44±.93 12.98±3.31 2.92±1.2 11.63±.3 IL-12p7 12.45±.45 17.75±4.1 1.5±.54 15.68±1.67 IL-13 3.64±.43 7.22±1.57 8.25±1.4 12.24±2.5 IL-17.4±.16 1.43±.3.19±.13.56±.11 KC 2.6±.85 4.4±.97 1.26±.3 3.98±.4 LeptinR 19.15±1.67 25.42±4.66 16.64±2.42 24.57±.6 Leptin 9.31±1.16 17.77±3.2 7.34±.98 23.15±9.49 LIX 55.6±4.28 58.35±9.12 54.9±6.43 64.59±1.2 L-Selectin 17.3±2.78 2.1±6.94 8.54±2.75 52.3±1.76 Lymphotactin 51.78±1.19 56.21±.97 5.84±7.6 58.33±.53 MCP1 23.42±1.67 29.18±2.6 87.62±5.43 13.32±2.95

MCP5 12.68±1.37 19.2±2.59 15.28±.12 36.22±2.98 M-CSF 25.48±.24 3.99±.49 21.33±4.94 31.95±3.18 MIG 32.2±.16 31.71±1.64 26.78±.29 34.32±.42 MIP-1α 4.69±1.61 15.11±.67 21.14±1.3 41.33±2.43 MIP-1γ 24.56±1.54 54.13±3.9 128.16±1.38 162.76±5.5 MIP-2 34.51±1.53 42.37±2.45 37.38±1.25 48.62±1.28 MIP-3β 45.78±.67 54.3±1.92 43.64±.92 53.59±3.89 MIP-3α 9.39±.8 15.63±2.55 6.57±.18 13.±.1 PF-4 21.91±1.95 25.7±1.3 16.2±1.5 2.34±6.6 P-Selectin 29.39±.61 35.17±2.37 26.17±1.39 32.22±6.82 RANTES 13.46±.77 15.64±1.9 13.27±4.49 22.92±13.29 SCF 4.46±1.25 6.45±.16 3.37±.81 15.39±6.81 SDF-1 62.6±1.15 65.68±.93 49.38±4.17 59.15±5.88 TARC 6.47±.73 17.78±1.58 4.83±.24 16.2±2.65 TCA-3 22.74±.19 127.42±.59 8.9±5.16 143.16±2.97 TECK 3.92±.42 11.3±1.59 2.3±.84 13.38±.78 TIMP-1 14.93±1.93 2.7±1.24 9.11±2.62 23.87±.35 TNFα 4.14±.1 5.73±1.21 1.65±.3 5.36±.67 stnfri 2.35±.39 3.79±.6 2.91±.55 11.38±1.1 stnfrii 7.89±.21 11.92±2.99 6.15±.26 24.85±.37 TPO 32.63±1.95 71.6±4.41 27.21±.34 56.34±4.4 VCAM-1 6.2±.2 1.1±1.46 1.3±.35 19.44±1.51 VEGF 1.33±1.78 17.73±4.47 8.58±1.61 23.46±6.34

Supplementary figure S1 B cells 22.9 % SSC B22 Supplementary Figure S1. Gating strategy for B cells. Cells from the MC:B cell cocultures were stained for surface markers and the viability dye 7-AAD and analyzed by flow cytometry. B cells were defined as 7- AAD - B22 high. SSC = side scatter

1 5 Supplementary figure S2 CD43 - splenocytes 14.9 % 1 5 Post-sort purity check % 1 5 Post-sort purity check 87.8 % 1 4 1 4 1 4 1 3 1 2 66 % 1 3 1 2 92 % 1 3 1 2 1.2 % CD1d 1 2 1 3 1 4 1 5 1 2 1 3 1 4 1 5 1 2 1 3 1 4 1 5 CD23 Supplementary Figure S2. Sorting strategy for MZ and. Splenocytes were first enriched for B cells using MACS, and subsequently stained for CD1d and CD23 and sorted into marginal zone (MZ) and follicular (FO) B cells using FACS. were defined as CD1d high CD23 low and as CD1d low CD23 high (left). Sorting purities were routinely around 9% for both (middle) and MZ B cells (right)., follicular B cells;, marginal zone B cells.

Supplementary figure S3 CD43 - splenocytes Post-sort purity check IgM + B cells 8 6 15 IgM + 86.7 % IgM + 99.6 % 1 4 cell count 2 IgM 1 2 1 3 1 4 1 5 5 1 2 1 3 1 4 1 5 Supplementary Figure S3. Sorting strategy for IgM + B cells. Splenocytes were first enriched for B cells using MACS, and subsequently stained for IgM. The IgM + fraction was routinely sorted with a purity of >98%.

Supplementary figure S4 A.4.3 β-hexosaminidase release after 4 hours OD 45.2.1. + naïve B cells rest MC + act B cells naïve B cells act B cells MC + A23187 B.4.3 β-hexosaminidase release after 24 hours OD 45.2.1. + naïve B cells rest MC + act B cells naïve B cells act B cells MC + A23187 C 8 IL-6 gene expression fold change 6 4 2 + naïve B cells rest MC + act B cells naïve B cells act B cells MC + A23187 Supplementary Figure S4. Coculture of MCs and B cells does not provoke MC activation. Non-activated MCs () were cocultured with naïve or BCR-activated B cells (act B cells) and MC activation was monitored as the release of β-hexosaminidase (A, 4 hours incubation; and B, 24 hours incubation) and the expression of the IL-6 gene as determined by real time quantitative RT-PCR (C, 4 hours incubation). As negative controls,, naïve or act B cells were cultured alone. As positive control for MC activation, MCs were incubated with 1 µm calcium ionophore A23187. Note that stimulation of MCs by calcium ionophore produces a robust β-hexosaminidase release and profound IL-6 gene upregulation, whereas coculture of MCs with B cells does not result in MC activation. Results are expressed as mean + SEM and represent 2 independent experiments in duplicates. MC, mast cell; BCR, B cell receptor

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