Figure 3. Creation of a Mouse Model with a Three-Amino-Acid Mutation in the Cytoplasmic Domain of PD-L1.

(A) Design of the guide RNA (underlined sequence) for CRISPR-Cas9 mutagenesis. The lowercase red letters indicate nucleotide changes leading to the amino acid replacements indicated. A recognition sequence for Pst1 (CTGCAG) was also introduced. The box indicates the PAM (protospacer adjacent motif) sequence.

(B) Total number of cDC1s and cDC2s in WT and Pdl1^CyMt mice as in Figure 1A. n = 3 mice each in data shown, repeated three times with similar results.

(C) Number of FITC+ DCs migrated to the dLN as in Figure 2C. n = 4–5 mice in data shown, data combined from two independent experiments.

(D) PD-L1 expression (by mean fluorescence intensity [MFI]) on FITC+ DCs in the dLN.

(E) CD80 expression (MFI) on FITC+ DCs in the dLN.

(F) Representative flow plots of transferred WT and Pdl1^CyMt BMDCs before transfer and in the dLN (after transfer) as in Figure 2G.

(G) Quantification of (F) showing the ratio of Pdl1^CyMt BMDCs to WT BMDCs as in Figure 2H.

(H) Number of WT and Pdl1^CyMt BMDCs in the dLN 24 h after transfer.

For (B–E), data are shown from axillary and brachial LNs combined. For (D) and (E), n = 3 mice in data shown, repeated two times with similar results. For (F–H), data are shown from popliteal LNs, n = 6 mice in data shown; data are combined from three different experiments. Statistical analysis was done using a one-way ANOVA (B–D), an unpaired Student’s t test (G), or a paired Student’sttest (H). *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. Error bars indicate standard error of the mean. See also Figures S3 and S4 and Table S1.