Extended Data Fig. 3: The LPS antibody detects intracellular replicating S.Tm in vivo.

(a) An antibody directed at LPS could detect living bacteria or LPS particles that are ingested by phagocytes or displayed on the surface of antigen presenting cells. To test the specificity of LPS antibody for living bacteria, we first examined LPS particles in cells grown in vitro. RAW264.7 murine macrophages were infected with S.Tm-GFP or heat killed S.Tm-GFP (MOI = 100) and fixed for imaging 24 hpi. Cells were immunolabeled with anti-LPS antibody and stained with DAPI. Maximum intensity projections of confocal images spanning the entire z-dimension of the cell are shown. Dotted lines show the cell borders. Right panels are higher magnification images of the white boxed regions in left panels. Experiments were repeated in triplicate. These data show that the antibody directed at LPS could detect living bacteria and LPS particles ingested by phagocytes in vitro. We then sought to determine if the antibody to LPS also detected LPS particles on the surface of antigen presenting cells (b) or intracellularly in vivo (c). (b) C57BL/6 mice infected with 1x10⁹ S.Tm by i.g. route and splenocytes were collected from these samples and subjected to CyTOF with the following modifications: Mouse splenocytes were either permeabilized or unpermeablized with Perm-S buffer (see Methods) and then immunolabeled with anti-CD45-Y⁸⁹ and anti-S.Tm LPS-Dy¹⁶³ antibodies (top). Contour plots from each samples is shown (top plots) and quantification of the number of LPS^pos cells within total CD45^pos splenocytes collected from C57BL/6 mice from three independent experiments is shown (bottom). Error bars show: Mean +/− SEM, Statistics: multiple T-tests. Detergent permeabilization was required to detect LPS^pos signal in infected CD45^pos splenocytes, indicating that LPS-Dy¹⁶³ does not primarily detect LPS particles displayed on the surface of antigen presenting cells in vivo. (c) We next tested if the LPS-Dy¹⁶³ detected LPS particles within immune cells. C57BL/6 mice infected intraperitoneally (i.p.) with PBS (n=3), or 1x10⁴ S.Tm (n=3) or heat killed S.Tm (n=3) and stained with anti-S.Tm LPS-Dy¹⁶³ and anti-CD45-Y⁸⁹ antibodies. Data was analyzed by CyTOF. Representative contour plots are shown to the left and quantification of LPS signal in CD45^pos splenocytes from three experiments is shown to the right. Graph error bars show Mean +/− SEM. Statistics show one-way ANOVA with Dunnett’s multiple comparison. p<0.05 compared to uninfected control is shown. We did not detect LPS^pos signal in CD45^pos splenocytes isolated from mice infected with heat killed S.Tm by the i.p route of infection consistent with CyTOF detection of living intracellular bacteria and not LPS particles in vivo. (d) We tested whether the CyTOF procedure accurately portrayed the levels of intracellular replicating S.Tm in vivo. C57BL/6 mice were either uninfected or infected i.g. with 1x10⁹ S.Tm for 4 days. Splenocytes were collected from each mouse and analyzed for total CFUs per spleen and processes for CyTOF. Mass cytometry contour plots as in (d) of splenocytes collected from an uninfected mouse or from three individual i.g. infected mice are shown. CFU/g of bacteria in the spleen of each mouse is shown above the plots. (n=1 mouse per CFU value, 1x10⁹ S.Tm bacteria/infection). These data show an increase of LPS^pos staining with an increased CFU/g S.Tm. (e) We sought to confirm that the LPS^pos signal in our large immune cell analysis correlated with bacterial burden so we analyzed the wild-type S.Tm samples from our large cohort analysis (Extended Data Table 1) for correlation between %LPS^pos cells and CFU recovered from mouse splenocytes infected i.g. with S.Tm. The total number of LPS^pos CD45^pos signal detected in spleen tissue increased linearly with the total number of living bacteria (CFU/g of spleen tissue) recovered from these animals. Linear regression analysis performed on GraphPad Prism R² = 0.967, F(1,32), p<0.0001. These data confirm that total LPS^pos staining correlates with the increasing levels of S.Tm infection in vivo.