File S1 - Failure To Detect Functional Neutrophil B Helper Cells in the Human Spleen

Includes Figures S1–S4 and Tables S1–S3. Figure S1. Splenic marginal zone B cells are able to differentiate into plasmablasts in response to CpG/IL2, but not in response to blood or spleen neutrophils. FACS plot of CD27/CD38 double staining of CD20pos B cells cultured for 7 days with indicated stimuli. Numbers indicate percentage of the total B cell population. Figure S2. Measurement of neutrophil reactive oxygen species in response to different stimuli. Production of reactive oxygen species by neutrophils from spleen and blood. RFU: relative fluorescence units, which are a derivative of H2O2 production. Zymosan 1 mg/ml; STZ: serum-treated zymosan 1 mg/ml; PMA: Phorbol 12-Myristate 13-Acetate 100 ng/ml; fMetLeuPhe 1 µM; PAF: platelet-activating factor 1 µM. Spleen n = 2, blood n = 3. Error bars represent standard deviation. Figure S3. Purity analysis of different neutrophil isolates. a. FSC/SSC plot and May-Grünwald/Giemsa stained cytospins of different neutrophil isolates. Neutrophils (upper gate) and lymfocytes (lower gate) are gated according to canonical FSC/SSC pattern. Numbers indicate percentages of total events. Original magnification of cytospins 540x. Data are representative of four independent experiments. b. Characterisation of the lymfocyte population contaminating the EasySep-isolated spleen neutrophils. Numbers indicate percentages of the lymfocyte population. Figure S4. Ficoll density gradient centrifugation prior to EasySep isolation does not remove the contaminating B cell population from EasySep-isolated splenic neutrophils. FSC/SSC pattern of splenic neutrophils separated either directly from splenocytes with the Human Neutrophil Enrichment kit (left), or separated from splenocytes with a Histopaque-1077 gradient followed by purification with the Human Neutrophil Enrichment kit (right). Numbers indicate percentage of total events. Data are representative of 2 independent experiments. Figure S5. Expression patterns of splenic neutrophils do not differ from their circulating counterpart. a.Gating strategy for neutrophils (upper gate) and monocytes (lower gate) by canonical FSC/SSC pattern in blood and spleen. The monocyte gate may include small percentages of other cells, especially in spleen, and serves only to show a positive control for the antibodies used. b.Staining of HLA-DR, CD86, CD95 and CD40L of splenic neutrophils as gated in Figure 2 and splenic monocytes as gated in Figure S5a. Data are representative of 11 independent experiments. Black lines: Staining with monoclonal antibody. Gray shading: isotype control. c. Staining of HLA-DR, CD86, CD95 and CD40L of blood neutrophils as gated in Figure 2 and blood monocytes as gated in Figure S5a. Data are representative of 11 independent experiments. Black lines: Staining with monoclonal antibody. Gray shading: isotype control. d.Staining of CD40L in human CD40L expressing fibroblast cell line. Black lines: Staining with monoclonal antibody. Gray shading: isotype control. e. CD15/CD16 double staining of blood and splenic neutrophils, as gated in Figure 2. Antibodies used: CD15: clone HI98, FITC. CD16: clone 3G8, APC. Data shown are of blood and splenic neutrophils from a single donor, and representative of two other experiments with unpaired samples. Figure S6. Incubation with collagenase buffer does not influence expression profile of neutrophils. Expression levels of HLA-DR, CD40L, CD86 and CD95 in neutrophils and monocytes from unseparated splenocyte suspensions, gated as in Figure S5a. Splenocytes suspensions were obtained either by perfusion with ‘collagenase buffer’ followed by 30 minute incubation at 37°C, or by perfusion with PBS alone with direct further processing. Data summarize 2 independent experiments, open triangles represent 1 donor, closed circles represent the other donor. Δ MFI: median fluorescence intensity minus median fluorescence intensity of appropiate isotype control. Table S1. Origin and characteristics of tissue samples. Table S2. Contents of collagenase buffer. Table S3. Antibodies used in flow cytometry. (PDF)