Figure 1.
CD274 expression in muscle, kidney and liver in control (D-7) and conditioned mice (D0).
Female BALB/c mice were conditioned using a Bu-Cy regimen. Samples from control mice (D-7) and conditioned mice (D0) were collected, mRNA was prepared and qPCR was performed. CD274 expression was calculated as a ratio to the house keeping gene (β actin; ACTB). The results are presented as mean ± SE. * significant difference (p<0.05).
Figure 2.
Dynamic expression of CD274 mRNA before and after bone marrow transplantation (syngeneic and allogeneic).
Female BALB/c mice were conditioned using a Bu-Cy regimen and transplanted with BM and SP cells from allogeneic or syngeneic donors. Samples from control mice (D-7, before treatment), conditioned mice (D0) and allogeneic- and syngeneic-transplanted mice at different intervals (D+1, D+3, D+5, D+7 and D+21) from the muscle, kidney and liver were collected, mRNA was prepared and qPCR was performed. Normalized (PD-L1) expression was calculated relative to ACTB. The results are presented as mean ± SE. a. Muscle b. Kidney c. Liver *Statistically different (p≤0.05) from the control value (no treatment with Bu-Cy or transplantation, D-7) as determined by the ANOVA test. ¤ Statistically different (p≤0.05) from the syngeneic-transplanted mice as determined by the ANOVA test.
Figure 3.
Comparison of mRNA expression fold changes.
Fold changes of mRNA expression post allogeneic transplantation relative to control. a. Fold changes of mRNA for muscle and kidney (D+5) and liver (D+7) relative to controls (D-7). The values are presented as mean ± SE. b. Fold changes (D+7) post allogeneic transplantation, compared to microarray data of mRNA fold changes at the same day presented as mean ± SE for PCR results and mean for microarray results (since pooled samples were used). **Significant (p<0.01) ***Significant (p<0.001).
Figure 4.
PD-L1 expression at the protein level as determined by western blot.
Female BALB/c mice were conditioned using a Bu-Cy regimen and transplanted with bone marrow and spleen cells in an allogeneic or syngeneic setting. Samples were taken from control mice (D-7), conditioned mice (D0) and allogeneic- and syngeneic-transplanted mice at different time points (D+5, D+7). Muscle, kidney and liver were collected, and the lysate was prepared and used for western blot.
Figure 5.
Expression of PD-L1 by immunohistochemistry.
Female BALB/c mice were conditioned using a Bu-Cy regimen and transplanted with bone marrow and spleen cells in an allogeneic or syngeneic setting. Samples from control mice (D-7) and from mice five days after allogeneic transplantation (D+5allo) were collected from the muscle, kidney and liver and stained with Anti-B7-H1 Ab. Immunohistochemistry staining for PD-L1 was positive (arrow) after allogeneic transplantation compared to controls (D-7) in all tissues. The strongest PD-L1 staining was obtained in the endothelial cells. Magnification 40X was used in all slides.
Figure 6.
Kinetics of the production of inflammatory cytokines IFNγ and TNFα after chemotherapy based conditioning and transplantation with syngeneic or allogeneic bone marrow and splenic cells.
Female BALB/c mice were conditioned using a Bu-Cy regimen and transplanted with bone marrow and spleen cells in an allogeneic or syngeneic setting (see Methods and materials). Blood samples were collected at different time points prior to and after transplantation (D-7, D0, D+5 and D+7), and serum was separated. Thereafter, serum levels of IFNγ and TNFα were measured employing ELISA techniques. *Statistically different (p≤0.05) from the control value (no treatment with Bu-Cy or transplantation, D-7) as determined by the ANOVA test. ¤ Statistically different (p≤0.05) from the syngeneic-transplanted mice as determined by the ANOVA test.