Supplementary MaterialsSupplemental data jci-128-96061-s001. than on tumor cells, has an essential function in checkpoint blockade therapy, providing an understanding into the systems of the therapy. = 3 per group). (E) PD-L1 appearance in MC38.WT, MC38.PD-L1C/C, A20.WT, and A20.PD-L1C/C cells was measured by flow cytometry. To stimulate PD-L1 appearance, cells had been treated with 500 U/ml IFN- every day and night. (F and G) C57BL/6 mice (= 5 or 6) had been inoculated with 1 106 MC38.MC38 or WT.PD-L1C/C cells. After tumors had been established, mice had been treated with 200 g antiCPD-L1 on times 7, 10, and 13. Tumor development (F) and success curve (G) are proven. (H and I) BALB/c mice (= 5) had been inoculated with 3 106 A20.WT or A20.PD-L1C/C cells. Mice were treated with 200 g antiCPD-L1 on days 10 and 13. Tumor growth (H) and survival curve (I) are shown. (JCL) Tissues were collected from MC38.PD-L1C/C tumor-bearing mice. Mean fluorescent intensities of PD-L1 staining in spleen (J), dLN (K), and tumor (L) are shown (= 3). Data show mean SEM and are representative of at Rabbit Polyclonal to KLF least 2 independent experiments. Statistical analysis was performed using an unpaired Students 2-tailed test. Even though PD-L1 in tumor cells could positively correlate with overall patient response to PD-1/PD-L1 blockade, it is hard to determine essential or dominant functions of PD-L1 on tumor versus host cells through current preclinical and clinical studies. To investigate the role of tumor-expressed PD-L1, we knocked out PD-L1 in tumor cells by clustered, regularly interspaced, short palindromic repeatsCassociated nuclease Cas9 (CRISPR/Cas9) technology. Knockout tumor cells lacked PD-L1 expression, as measured by circulation cytometry (Physique 1E). IFNs are strong inducers of PD-L1 (19). When stimulated by IFN-, WT MC38 (MC38.WT) cells upregulated PD-L1 expression while PD-L1Cknockout MC38 (MC38.PD-L1C/C) cells remained unfavorable, indicating a complete ablation of gene expression (Figure 1E). When inoculated into the WT host, MC38.PD-L1C/C tumors grew similarly to WT tumor (Figure 1F). Surprisingly, order Alvocidib response of MC38.PD-L1C/C tumor to PD-L1 blockade therapy was as good as that of WT tumor order Alvocidib (Figure 1, F and G). Comparable results were observed using PD-L1Cdeficient A20 tumor (Physique order Alvocidib 1, E, H, and I). Both PD-L1Cdeficient MC38 and A20 tumors also responded to PD-1 blockade therapy well (Supplemental Physique 2, A and B). To find out whether there are differences in host PD-L1 expression between MC38.WT and MC38.PD-L1C/C tumors, tissues were collected and PD-L1 expression was evaluated by flow cytometry. Interestingly, while tumor cells completely lost PD-L1 expression, the levels of PD-L1 in myeloid cells from MC38.PD-L1C/C tumor-bearing mice were similar to their counterparts in WT tumor-bearing mice (Supplemental Figure 1E and Figure 1, JCL). Collectively, these data suggest that PD-L1 on tumor cells is not essential for the response to PD-L1 blockade in these models. It is possible that myeloid cellCexpressed PD-L1 is sufficient to limit immune responses, and thus myeloid cells may mediate the response to checkpoint blockade therapy. AntiCPD-L1 Ab targets to tumor tissue regardless of the status of tumor-expressed PD-L1. Lack of PD-L1 expression on a biopsy specimen cannot exclude PD-L1 expression in different areas of tumor tissues or subsequent expression after sampling. Additionally, the lack of.