Figure 5. aPD-1 and aLAG-3 ICB with sequential chemotherapy improves the antitumor immune response. (A) Experiment timeline. AB1-HA or CT26 tumor bearing animals were treated with 5FU, GEM or PBS when tumors reached 20–25 mm2 in size. aPD-1 and aLAG-3 ICB were administered 3 days after the second dose of chemotherapy (+6), the same time point as T-cell phenotyping experiments. (B) Survival curves of AB1-HA tumor bearing animals treated with GEM or 5FU chemotherapy in combination with aPD-1 and aLAG-3 ICB. Sample sizes were n=10 per treatment group from two pooled experiments. (C) Survival curves of AB1-HA tumor-bearing animals treated with aPD-1 or aLAG-3 in combination with GEM (n=5 per treatment group, one experiment). (D) Survival curves of CT26 tumor bearing animals treated with GEM or 5FU chemotherapy in combination with aPD-1 and aLAG3 ICB. Sample sizes were n=10 per treatment group from two pooled experiments except GEM (n=2) and GEM+aPD-1+aLAG-3 (n=4). (E) Survival curves of AE17 tumor bearing animals treated with GEM chemotherapy in combination with aPD-1 and aLAG-3 (n=5 per treatment group, one experiment). (F) Experiment timeline. DLN and tumors were harvested 2 days after the full chemo-immunotherapy schedule. (G) Dot plots representing frequencies of TIGIT−PD-1−LAG-3−CTLA-4− CD8+ T cells (4 IhR−) in AB1-HA (left) or CT26 (right) tumors. (H) Dot plots displaying frequencies of CD11c+MHC-II+ dendritic cells in DLNs from AB1-HA (left) and CT26 (right). (I–J) Dot plots showing proportion of conventional dendritic cells (CD11c+MHC-II+): cDC1 (CD11b−XCR1+) and cDC2 (CD11b+XCR1−) in AB1-HA (left) and CT26 (right) tumors (I) and DLNs (J). Data represented as mean±SD. Survival experiments: Mantel-Cox survival test. Flow cytometry experiments: n=4–5/group. Ordinary two-way analysis of variance with Tukey’s multiple comparisons test was used to compare between treatment groups. *p<0.05, **p<0.01, ***p<0.001,****p≤0.0001. aLAG-3, anti-LAG-3; aPD-1, anti-PD-1; cDC, conventional dendritic cell; DC, dendritic cell; DLN, draining lymph nodes; GEM, gemcitabine; ICB, immune checkpoint blockade; TIGIT: T cell immunoreceptor with immunoglobulin and ITIM domain; LAG-3, lymphocyte activating gene-3; CTLA-4: cytotoxic T lymphocyte antigen 4; MHC, major histocompatibility complex; PBS, phosphate-buffered saline; PD-1, programmed cell death protein 1; TUM, tumors.
