Fig. 6: SIGN-R1-mediated C3 activation and opsonization of S. pneumoniae enhances the uptake and decomposition of the bacteria by SIGN-R1⁺ cells in vitro and in vivo.

a Live S. pneumoniae type 14 (Pn14) was incubated with 5% NHS at 37 °C for 30 min in the presence of 10 μg bovine serum albumin, transferrin (Tfe), or purified SIGN-R1, immunostained for C1q, and analyzed by FACS. b As in a, but 10 μg Tfe or C1q was used, and organisms were immunostained for C1q, iC3b, or membrane attack complex (MAC). c As in a, but organisms were immunoblotted for C3. d In total, 1 × 10⁸ mitomycin C-treated and PKH26-labeled S. pneumoniae (PKH26-MitC-Pn14) were incubated with CHO transfectants in the presence of 10 μg C1q or 5% NHS. The binding of organisms to cells was assessed by FACS. e DCEK_SIGN transfectants were incubated with MitC-Pn14 (1 × 10⁶, 15 h, 37 °C) without or with mouse sera (NMS or C3- or C1q-depleted mouse sera), and cells were immunostained for SIGN-R1 (green) or CPS14 (red), followed by microscopic analysis. Representative cells are shown in the boxes. f CFSE-MitC-Pn14 (1 × 10⁸, green) were injected intravenously into control, SIGN-R1-KO, or C3-KO mice for 1 h and spleen sections were immunostained for SIGN-R1 (blue) and CPS14 (red). Representative areas in splenic MZs are highlighted in the boxes (middle row). Organisms captured in the red pulp (arrowheads) are highlighted (bottom row). Scale bars e, 20 µm; f, 50 µm