Fig. 2.

Immune checkpoint proteins are dynamically dysregulated in HO lesions. a Immunofluorescence staining showed the expression of CD28 and PD1 in WT mice at 1, 2 and 4 weeks p.i. In Nse-BMP4 mice, immunostaining revealed a distinct expression pattern of stimulatory ICs (CD27, CD28, CD40, CD278) in HO lesions at 1, 2, and 4 weeks p.i. b–d Quantitation of the percentages of cells expressing IC proteins in Wt and Nse-BMP4 mice at 1, 2, and 4 weeks p.i. (n = 4 per group per time point). In WT mice with injury, the percentages of cells expressing ICs were generally low. In contrast, in Nse-BMP4 mice, the stimulatory ICs, including CD27, CD28, CD278, and CD40, and inhibitory ICs, including Tim3, CD152, PD1, and PD-L1, were differentially dysregulated. Note that stimulatory ICs generally were abnormally increased at early stages and then decreased, while some inhibitory ICs remained increased at late stages. *P < 0.02 compared to wild-type control at all time points. ^#P < 0.05 compared to 1 week time point in the Nse-BMP4 group. Statistics were performed using a repeated-measures ANOVA with Bonferroni’s post hoc test. Scale bar = 200 μm