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. 2021 Oct 28;12:6230. doi: 10.1038/s41467-021-26446-x

Fig. 8. TE-DCs mediate negative selection against blood-borne antigens.

Fig. 8

a Purified thymic dendritic cells (DCs) from untreated or ovalbumin (Ova) injected wildtype, Cx3cr1gfp/gfp or Cx3cl1–/– mice were co-cultured for 3 days with CD4+CD8+ OT-I thymocytes with or without adding exogenous Ova. The number of viable OT-I cells in each sample was assessed by flow cytometry and compared to control co-cultures of WT DCs without Ova. n = 2 experiments with DC preparations from 4–8 mice/group (n = 4 for Cx3cl1−/− + Ova i.v., Cx3cr1gfp/gfp+ Ova i.v. and Cx3cl1−/− + Ova i.v. + Ova in vitro; n = 5 for WT + Ova i.v., Cx3cr1gfp/gfp + Ova i.v. + Ova in vitro; n = 6 for WT + Ova i.v. + Ova in vitro; n = 8 for WT + PBS i.v. and WT + PBS i.v. + Ova in vitro; one-way ANOVA). b Lethally irradiated CD45.1 or (c) CX3CR1–/– × CD45.1 animals were reconstituted with mixed BM at a 1:1 ratio of b CD45.2 and OT-I × CD45.1/2 or c CD45.1 × CX3CR1–/– and OT-I × CD45.1/2 × CX3CR1–/– BM. Deletion of OT-I T cells was induced 18 days after bone marrow transplantation by injection of 100 µg ovalbumin + 100 µg non-depleting anti-α4 integrin mAb (Ova) or 10 µg SIINFEKL peptide + 100 µg non-depleting anti-α4 integrin mAb (SIINFEKL). Control mice were left untreated (Ctrl). Deletion was assessed by determining the percentage of CD8+ OT-I cells among all CD8+ cells in each animal by flow cytometry. Pooled data with n = 13 (Ctrl and Ova) and n = 11 (SIINFEKL) of two independent experiments is shown (b) or 5 animals (c) per group (one-way ANOVA). d Presentation of data in b and c as the percentage of CD8+ OT-I T cells relative to control animals in WT (open bars) and Cx3cr1gfp/gfp (closed bars) animals. n = 8 for all groups except n = 7 for WT ctrl and Cx3cr1gfp/gfp SIINFEKL. Bars and error bars represent mean ± SEM. a ***P = 0.005, **P = 0.0027 for WT Ova i.v. vs Cx3cl1−/− Ova i.v. and **P = 0.0068 for WT Ova i.v. vs Cx3cr1gfp/gfp Ova i.v.; n.s.: not significant. b *P = 0.0183 (Ctrl vs Ova) and P = 0.0384 (Ova vs. SIINFEKL), ****P < 0.0001. c **P = 0.0033, *P = 0.0141, n.s. not significant. d ***P = 0.0002.