br B F metastatic model The mice were sacrificed
B16-F10 metastatic model. The mice were sacrificed on day 14 there-after, and the lungs were checked for tumor nodules (Figure 4E). An average of 257 and 242 nodules were found in the PBS and DTT groups, respectively, whereas an average of 92 and 79 nodules were found in the DPDL1E group and mAb group, respectively. Further-more, the number of large nodules (more than 3 mm in diameter) were significantly lower in the DPDL1E group of mice. In a parallel experiments using mAb, we found no significant difference between the DPDL1E group and mAb group in terms of the total number of nodules and the numbers of large nodules (Figure 4F-G), indicating that DPDL1E vaccination achieved a similar level of protection as mAbs.
DPDL1E Inhibits Tumor Growth in a Therapeutic Mouse Melanoma Model
To evaluate the therapeutic efficacy of the DPDL1E vaccine, B16-F10
tumor-bearing C57BL/6 mice were established as described previ-ously.22,23 When the tumors were palpable, the mice were treated with DPDL1E or the DTT vaccine. For comparison, another group of mice was treated with a mAb (Figure 5A). Compared with the iso-type or DTT control groups, both the DPDL1E vaccine and PD-L1 mAb inhibited tumor growth (Figure 5B). The mean survival time of each group was 16.25 days (DTT group), 16.25 days (isotype group), 19 days (DPDL1E group), and 20.25 days (PD-L1 mAb group) (Figure 5C). More strikingly, tumor growth was markedly in-hibited in a subset of mice in both the DPDL1E vaccine- and PD-L1 mAb-treated groups (Figure S1B).
To verify that anti-PD-L1 CORM-3 were the major contributors, we purified antibodies from the immunized mice and transferred them into unimmunized tumor-bearing mice (Figure 5D). Compared with antibodies from the control groups, antibodies from DPDL1E immunized mice inhibited tumor growth (Figure 5E) and prolonged survival (Figure 5F). These data suggest that PD-L1-specific anti-bodies play a key role in tumor control.
To test whether a combination of DPDL1E and PD-L1 mAb could benefit therapy, we immunized tumor-bearing mice following the schedule in Figure 5A. Combination of the DPDL1E vaccine and PD-L1 mAb synergistically inhibited tumor growth (Figure 5G) and prolonged the survival time (Figure 5H). These data indicate that the anti-PD-L1 by vaccine approach is promising as an alterna-tive or combinatorial cancer therapy.
DPDL1E Vaccination Increases the Ratio of CD8+
T Cells/Myeloid-Derived Suppressor Cells (MDSCs) in
Tumors and Peripheral Blood
Because the level of tumor-infiltrating lymphocytes (TILs) and the
ratio of CD8+ T cells/MDSCs have been shown to be associated with anti-tumor immunity,22,23 we measured the level of CD8+
T cells in the B16-F10 melanoma model using an immunohistochem-istry (IHC) assay. Compared with the DTT-treated group, DPDL1E vaccination increased the level of CD8+ T cell infiltration in both pre-ventive (Figures 6A and S3A) and therapeutic mouse melanoma models (Figures 6B and S3B). In the therapeutic setting, the levels
Figure 3. The CTL Response Induced by DPDL1E Vaccination
(A) Lymphocytes from spleens of DPDL1E- and DTT-immunized mice were used as effector cells. PD-L1-positive expressed B16-F10 cells were used as target cells. Cytotoxicity was assessed with an LDH release assay. Statistically significant differences were determined using Student’s t test. (B) Lymphocytes isolated from DTT- and DPDL1E-immunized mice were stimulated with His-PD-L1 recombinant protein or Con A for 72 h. Cell proliferation was measured with the CCK-8 method. (C) The con-centrations of TNF-a, IFN-g, and IL-2 in supernatant after 72 h stimulation. The cytokines were detected using an ELISA kit. Anti-CD3/CD28 beads (0.5 mg/mL) were used as a positive control. (D) Proliferation of T cells was determined using a CFSE-based assay in vitro. CD8+ and CD4+ T cells were gated and analyzed using FCM. Both CD8+ and CD4+ T cells proliferated more than the DTT control group after His-PD-L1 stimulation. Statistically significant differences were calculated by Student’s t test. NS, not significant; *p < 0.05; **p < 0.01.