Fig. 4: fgTiO2 selectively targets PD-L1+ LysoMac and LysoDC cells.
From: Immunocompetent cell targeting by food-additive titanium dioxide
a–h In situ, single-cell analysis of key immune-cell subtypes of the mouse subepithelial dome. a, b Example images immunofluorescently labelled for B220, CD3 and CD11c (i.e., identifying B-lymphocytes, T-lymphocytes and phagocytic mononuclear cells, respectively) from wild-type (WT) mice fed (a) without (WT) or (b) with (WT + TiO2) fgTiO2 dietary supplementation (0.0625% w/w of diet). fgTiO2 was measured from thresholded reflectance images. Translucent red circle-markers are placed on reflectant foci to aid visualisation. c, d Flow cytometry-type plots showing cell area/cell aspect ratio, (e–g) immunofluorescence distributions and (h) cell counts for B220 + , CD11c+ and CD3+ cells (n = 3 animals). All measures remained similar regardless of fgTiO2 feeding (cell count comparison (h), P = ≥ 0.26, two-sided unpaired samples T-tests). i–l fgTiO2-recipient cells of the subepithelial dome were CD11c+ in ~ 95% of cases. m–o These cells also exhibited a unique autofluorescent signature as previously described for subepithelial dome LysoMac and LysoDC cells. o Again, this was true for almost all fgTiO2 + /CD11c+ cells and, equally, these autofluorescent cells were similarly present in (n) controls without fgTiO2 feeding (further data shown, Supplementary Fig. 8). p–u Montaged CD11c + /fgTiO2+ or CD11c + /fgTiO2- cell populations from the SEDs of mice fed the fgTiO2 supplemented diet visualised in terms of (r, s) autofluorescence signature. fgTiO2 is seen to both selectively and specifically target the autofluorescent cells and (t, u) the diversity of CD4 and MHCII expression confirms that both LysoMac and LysoDCs take up fgTiO2. v–z fgTiO2+ cells of the SED expressed the immunotolerance marker programmed death ligand 1 (PD-L1) whereas fgTiO2+ pigment cells at the base of the Peyer’s patch tended to lose PD-L1 expression (y versus (z), respectively). w PD-L1 expression in the Peyer’s patches was not significantly perturbed by feeding the fgTiO2-supplemented diet (P = 0.34, z = −0.96, two-sided Wilcoxon rank-sum analysis, n = 6 animals). The image-data presented in (i, m, p–u, x–z) were collected from SED tissue sections from n = 3 mice. Scale bars: a, b = 50 μm; i, m, p–u = 10 μm; x = 500 μm; y, z = 10 μm.
