CR1 protein, human

Wild type C57BL/6J mice aged 8–12 weeks were purchased from the National Laboratory Animal Center (Taipei, Taiwan). All animals were housed in the Tzu-Chi University Animal Center in a specific-pathogen-free, temperature and lighting controlled environment with free access to filtered water and food. The experimental procedures were approved by the Animal Care and Use Committee of Tzu-Chi University (approval ID: 101019). Fluorescence-labeled anti-IgM, anti-IgD, anti-B220, anti-CD43, anti-CD21/CD35 and anti-CD23 Igs that used in the flow cytometry B lymphocyte analyses, were purchased from (eBioscience, San Diego, CA and BD Biosciences, Franklin Lakes, NJ). Anti-IgM and anti-IgD were used to identify subsets as described. Anti-CD21/CD35 and anti-CD23 Igs were used to identify spleen B cell subsets following previously described methods. Following previously described methods^{33 (link),34 (link),40 (link)–43 (link)}, we analyze spleen transitional T1, T2, and follicular and marginal zone, mature B cells, bone marrow mature (IgD⁺IgM^-, IgD⁺IgM⁺) and immature (IgD^-IgM⁺) B cells, and pre-pro-B (CD43⁺B220⁺), pre-B and pro-B (CD43^-B220⁺) cells. A flow cytometer (FACSCalibur; BD Biosciences, San Jose, CA, USA) was used in this study as described^{86 (link)–88 (link)}. C57BL/6J mice were immunized with rGST, rNS1, rDR4 and rTACI for 2 immunization cycles (50 µg immunogen/mouse/immunization cycle) in 1 wk intervals and then the bone marrow and spleen lymphocytes were analyzed 7 d later using aforementioned B cell markers. To analyze the potential suppressive effect of prior immunizations of NS1 on later induction of neutralizing antibody, C57BL/6J mice were first immunized with rGST, rNS1, rDR4 and rTACI by 2 immunization cycles (50 µg immunogen/mouse/immunization cycle) and then immunized with 2 additional immunization cycles of DENV rEIII in 1 wk intervals. The anti-EIII titer and the DENV-neutralizing property of these polyclonal antibody fractions were then analyzed.

Spleens and peritoneal exudate cells (PECs) from naïve or WNV-infected mice at the indicated time points were harvested and dissociated into single cell suspensions as previously described [72 (link)]. After erythrocytes lysis, splenocytes and PECs from peritoneal lavage were filtered and processed for staining for flow cytometry. Cells were incubated with Aqua Live-Dead Fixable Dead Cells staining kit (Molecular Probes, Life Technologies, Waltham, MA) protected from light for 20 min at 4°C. Subsequently, cells were incubated with anti-CD16/CD32 blocking Ab (2.4G2) for 10 min at RT and then stained with various Ab mixtures at 4°C. Cells were stained with mAbs conjugated to FITC, PE, allophycocyanin, eFluor450, allophycocyanin-eFluor780, PerCPCy5.5, PE-Cy7, AlexaFluor647, BUV395, BV605, BV421, BV711 and BV650. For analysis of splenic B cell subsets (gating strategy in S1A Fig) according to Gilitay et al. [55 ], and PECs B cells according to Giordano et al. [72 (link),73 (link)], seven- or eight-colors flow cytometry was performed using combinations of mAbs against B220 (RA3-6B2), CD38 (90), GL7 (GL7), CD24 (M1/69) and CD138 (281–2) from BD Horizon/Biosciences (San Jose, CA, USA); IgM (II/41), CD69 (H1.2F3) and CD5 (53–7.3) from eBioscience, (San Diego, CA, USA); CD21/CD35 (7E9), IgD (11-26c.2a), CD93 (AA4.1) and CD23 (B3B4) from BioLegend (San Diego, CA, USA). For analysis of other myeloid cell subsets (gating strategy in S2 Fig), nine- to eleven-colors flow cytometry was performed using combinations of mAbs against: CD19 (1D3), CD11b (M1/70), CD11c (N418) from eBioscience (San Diego, CA, USA); B220 (RA3-6B2) and Ly6C (AL-21) from BD Horizon/Biosciences (San Jose, CA, USA); CD3 (17A2), NK1.1 (PK136), CD8α (53–6.7), Ly6G (1A8), F4/80 (BM8) and CD169 (3D6.112) from BioLegend (San Diego, CA, USA). Splenic myeloid cell subsets were defined as described previously [72 (link)] with some modifications (gating strategy in S3 Fig). After gating out B cells and T cells (CD19^-CD3^-) cell populations were phenotyped as follows: NK cells, NK1.1^hiCD11b^int; neutrophils (Nph), CD11b^hiLy6G^hiLy6C^intSSC^int-NK1.1^-; eosinophils (Eosph), CD11b^hiSSC^hiLy6G^loLy6C^intNK1.1^-; Ly6C^hi monocytes (Ly6C^hi MO), CD11b^hiLy6C^hiCD11c^-SSC^-Ly6G^-NK1.1^-; Ly6C^hi dendritic cells (Ly6C^hi DC, CD11b^hiLy6C^hiCD11c^hi SSC^-Ly6G^-NK1.1^-; Ly6C^lo MO, CD11b^intCD11c^-Ly6C^loSSC^-Ly6G^-NK1.1^-; plasmacytoid DCs (pDC), CD11b^-CD11c^loB220⁺Ly6G^-NK1.1^-; CD8⁺ cDCs, CD11c^hiCD8⁺B220^-Ly6G^-NK1.1^-; CD8^- cDCs, CD11c^hiCD8^-B220^-Ly6G^-NK1.1^-; red pulp macrophages (RPM), F4/80⁺SSC^hiCD11b⁺CD11c^loLy6G^-NK1.1^-; marginal zone macrophages (MZM), CD169⁺F4/80^-CD11b⁺CD11c^loLy6G^-NK1.1^-).
For analysis of T cell populations, seven- to eight-colors flow cytometry was performed using combinations of mAbs against: CD3 (145-2C11), CD4 (RM4-5), CD62L (MEL-14) from eBioscience (San Diego, CA, USA); CD8α (53–6.7), CD44 (IM7), interferon (IFN)γ (XMG1.2) and TNFα (MP6-XT22) from BioLegend (San Diego, CA, USA). T cells subsets were defined as previously described [74 (link)]: CD4⁺ T cells, CD3⁺CD4⁺CD8^-; CD8⁺ T cells, CD3⁺CD4^-CD8⁺; CD4⁺ T naïve (CD4 Tn), CD3⁺CD4⁺ CD8^-CD62L⁺CD44^lo-); CD4⁺ T effector (CD4 Teff), CD3⁺CD4⁺ CD8^-CD62L^-CD44^hi; CD4⁺ T central memory (CD4 Tcm), CD3⁺CD4⁺ CD8^-CD62L⁺CD44⁺; CD8⁺ T naïve (CD8 Tn), CD3⁺CD4^-CD8⁺CD62L⁺CD44^lo-); CD8⁺ T effector (CD8 Tef), CD3⁺CD4^-CD8⁺CD62L^-CD44^hi; CD8⁺ T central memory (CD8 Tcm), CD3⁺CD4^-CD8⁺CD62L⁺CD44⁺.
For intracellular staining cells were stained with mAbs for surface markers, fixed and permeabilized using 0.1% saponin in staining buffer (2% FBS in PBS) followed by anti-IFNγ or anti-TNFα staining for 20 min at RT. Cells were processed on an LSRII FACScan analyzer (Becton Dickinson, Franklin Lakes, NJ, USA) using FACSDiva software and data analysis was performed with FlowJo software.

Cryostat sections (7 μm) made from OCT (TissueTek) embedded dLNs were fixed with 2% PFA for 20 min, and then washed and blocked in blocking buffer (PBS with 1% BSA, 0.3% Triton-100, Fc-blocker and 5% rat serum and mouse serum). Sections were then stained in blocking buffer with biotin-labelled anti-mouse IgD (clone 11-26c, eBioscience) and PE-labelled anti-mouse BCL6 antibodies (clone K112-91, BD Biosciences), and subsequently stained with AF488-conjugated streptavidin (Thermo Fisher Scientific). Fluorescent images were captured using a 4× objective on a fluorescence microscope (Keyence).
For imaging the distribution of TLR7-NP in the dLNs, AF647-labelled TLR7-NP was s.c. injected into mice (n = 2) at the tail base. dLNs were harvested 48 h later. Tissue sections were stained with anti-mouse IgD_Al488 (clone 11-26c, SouthernBiotech), CD4_BV421 (clone GK1.5, BioLegend) and CD35_biotin (clone 8C12, BD Biosciences). Streptavidin_A555 (Invitrogen, catalogue number S32355, 1:100) was used to detect biotin. Images were captured using a 20× objective on a fluorescence microscope (Leica, DMi8).

Following stimulation with TNFα in the absence and presence of HA, the neutrophils were washed, suspended in PBS/2% FBS/5 mM EDTA and stained 45 min on ice with PE-conjugated mouse anti-human CD11b mAb (1:50; Biolegend; #560914) and/or Alexa Fluor^® 647-conjugated mouse anti-human CD35 mAb (1:40; Biolegend; #565329). A minimum of 20,000 events in the neutrophil gate, determined by FCS and SSC, were collected on an Attune NxT flow cytometer.

A total of 400 × 10⁶ platelets, purified as described in Section 4.1, were suspended in 1 mL of Tyrode’s buffer and stimulated with 10 or 20 µM of LL-37 (Tocris Bioscience, Bristol, UK) for 20 min at 37 °C in a 5% CO₂ atmosphere (Incubator NUAIRE, model NU-5700, Plymouth, MN, USA). After platelet simulation, the cells were obtained by centrifugation at 480 g for 10 min and the supernatant was stored at −70 °C. Subsequently, platelets were stained using mouse mAb directed against the following human molecules: anti-CD41-PE-Cy5 (Section 4.1), anti-HLA-ABC-APC (clone: G46-2.6), anti-CD86-FITC (2331 (FUN-1)), anti-CD282-APC (clone: 11G7), anti-CD32-APC (clone: FLI8. 26), anti-CD35-FITC (clone: E11) (BD Biosciences, USA), anti-PAC-1-FITC (clone: PAC-1), an-ti-CD62P-FITC (clone: AK4), anti-LOX-1-PE (clone: 15C4), anti-CD80-FITC (clone: 2D10) (Biolegend, San Diego, CA, USA), anti-CD284-APC (clone: HTA125) (Invitrogen, Waltham, MA, USA), anti-PAR1-PE (clone: 731115) (R & D System, Minneapolis, MN, USA), and anti-Dectin1-PE (clone: REA515) (Miltenyi Biotec, San Francisco, CA, USA), and using their respective mouse isotype mAbs: PE-Cy5 IgG1 κ Isotype (Section 4.1), APC IgG1 κ (clone: MOPC-21), APC IgG2b κ (clone: 27-35), FITC IgG1 κ Isotype (clone: MOPC-21), PE IgG1 κ (clone: X40), FITC IgM, κ (clone: MM-30) (Biolegend, San Diego, CA, USA), and PE IgG2b (clone: 133303) (R and D System, USA).