Fig. 2.

TiO₂implants induce T cell exhaustion by recruiting immunosuppressive immune cells. (a) Immunohistochemical analysis of tissues surrounding injected TiO₂ nanoparticles (NPs) on day 3. Scale bar, 100 μm. "UNTX" indicates untreated control. (b) The proportion of leukocytes (CD45⁺) in tissues surrounding injected TiO₂ NPs on day 3. (c) CD11b expression based on immunohistochemistry analysis. (d) Proportion of monocytes/macrophages (CD11b⁺CD14⁺), (e) DCs (CD11b⁺CD11c⁺), and (f) myeloid-derived suppressor cells (CD11b⁺Gr-1⁺) in tissue surrounding injected TiO₂ NPs based on by flow cytometry. (g) Blood routine analysis for mice with and without TiO₂ NP injection. (h) Immunohistochemical analysis of PD-1 expression in tissues on day 1 (scale bar, 100 μm) and (i) corresponding quantitation. (j-m) TiO₂ NP-induced PD-1 expression in (j) leukocytes, (k) monocytes/macrophages, (l) MDSCs, and (m) DCs. (n-q) Analysis of PD-1 (n) and the T cell exhaustion markers TIM-3 (o), LAG-3 (p) and CTLA-4 (q) in CD3⁺ T cells after TiO₂ NP treatment. (r) Confocal image of PD-1 expression on macrophages. (s) Flow cytometry image and quantitative analysis of PD-1 on RAW 264.7 cells after co-culture with TiO₂ NPs. (t) Western blot analysis of PD-1 expression in RAW 264.7 cells (GAPDH was used as the reference), and (u) corresponding quantitation of PD-1. (v) PCR analysis of PD-1 mRNA expression. (w) NF-κB mRNA expression by RAW 264.7 cells. (x) Flow cytometry chart of PD-1, and (y) quantitation of PD-1. (z) PCR analysis of PD-1 mRNA expression on RAW 264.7 cells after different treatments. Values indicate mean ± SEM (n=3–6). *P<0.05, **P<0.01, ***P<0.005, ****P<0.001 by Student`s t-test and one-way ANOVA using the Tukey post-test.