Fig 2: NK cells mediate the killing of tumor cells in vivo by inducing IFN-? and granzyme B secretion in the ID8 TME and in vitro cell-based systems in an indicated regimen-dependent manner. (A and B) Three slides of samples from ID8 tumors treated with the indicated regimens or controls were incubated with antimouse NK1.1 or antimouse IFN-? (A) or antimouse granzyme B (B) and followed by secondary Abs and IF analysis as described previously. The relative expression of mouse NK1.1 and IFN-? (A) or granzyme B (B) among these tumors with treatment regimens are shown in the histograms as indicated, respectively. All IF and DAPI images were displayed in online supplemental figures S7 and S8. Scale bar: 20 µm. (C) Three slides of samples from these ID8 tumors were subjected to TUNEL assays (Promega) for determining cellular apoptosis. Archetypal TUNEL images were shown; all TUNEL images were displayed in online supplemental figure S9. Scale bar: 20 µm. (D) Three slides of samples from these ID8 tumors were subjected to cleaved-PARP1 IF analysis for determining cellular apoptosis. All cleaved-PARP1 IF images were displayed in online supplemental figure S10. Scale bar: 20 µm. (E) Mouse metastatic tumor 4T1-Luc cells (40,000 cells/well) were treated with NK-92 (10,000 cells/well) in the presence of low-dose SN-38 or other pharmacological drugs as indicated or DMSO (control) for 36 hours. After removing NK cells, the survival rates of tumor cells were measured by cell counting. *P<0.022. (F) Similarly, 4T1-Luc tumor cells (20,000 cells/well) were treated with NK-92 (10,000 cells/well) in the presence of higher doses of pharmacological drugs than those used in figure part E. **P<0.0006. Abs, antibodies; DMSO, dimethylsulfoxide; IF, immunofluorescence; IFN-?, interferon-?; NK, natural killer; TME, tumor microenvironment.

Fig 3: Reduced IFN-I production and dendritic cell activation following ZIKVBR infection do not impact antigen-experienced CD8 T cell division.a, b Ifna (a) and Ifnb1 (b) mRNA expression in the spleen were analyzed by RT-qPCR 12 hpi with ZIKVCDN or ZIKVBR, or mock-infection. Data are expressed as fold change over expression in mock-infected mice 12 hpi. Dotted line indicates a fold change of 1. Data are representative of two independent experiments with n = 3 mice per group, and were analyzed by two-tailed, unpaired Student’s t-test. In a p = 0.0047 and in b p = 0.000004. c Total bioactive IFN-I were analyzed in the serum 12 hpi with ZIKVCDN or ZIKVBR using the B16-blue reporter cell line. Dotted line indicates average OD in the serum of mice 12 h post-mock infection. Data are representative of two independent experiments with n = 3 mice per group, and were analyzed by two-tailed, unpaired Student’s t-test, p = 0.000011. d–f Representative flow cytometry plots of CD8αloCD11ahi CD8 T cells from ZIKVCDN- or ZIKVBR-infected mice treated with PBS 2 and 3 dpi (d), and from ZIKVBR-infected mice treated with pI:C 2 and 3 dpi. Frequency (e) and number (f) of CD8αloCD11ahi CD8 T cells in the spleen 7 dpi with ZIKVCDN or ZIKVBR, after treatment with either PBS or pI:C 2 and 3 dpi. Data are representative of two independent experiments with n = 5 mice per group and were analyzed by one-way ANOVA with Tukey’s post-test for multiple comparisons. In e ZIKVCDN + PBS versus ZIKVBR + PBS p = 0.0000001, ZIKVCDN + PBS versus ZIKVBR + pI:C p = 0.0000041, ZIKVBR + PBS versus ZIKVBR + pI:C p = 0.0112. In f ZIKVCDN + PBS versus ZIKVBR + PBS p = 0.000000008, ZIKVCDN + PBS versus ZIKVBR + pI:C p = 0.000000059. g–i Representative histogram (g), frequency (h), and number (i) of BrdU+ CD8αloCD11ahi CD8 T cells in the spleen 7 dpi with ZIKVCDN or ZIKVBR. Line on histogram indicates gating strategy used to identify BrdU+ cells. Data are representative of two independent experiments with n = 3 mice per group, and were analyzed by two-tailed, unpaired Student’s t-test. In i p = 0.0013. j–m Representative histograms and geometric mean fluorescence intensity (gMFI) of CD80 (j, k) and CD86 (l, m) expression on CD3−CD19−NK1.1− CD11c+MHC-II+ splenic DCs 2 dpi with ZIKVCDN or ZIKVBR, or injection with an equivalent dose of UV-ZIKV. Data are representative of two independent experiments with n = 3 mice per group, and were analyzed by one-way ANOVA with Tukey’s post-test of multiple comparisons. In k ZIKVCDN versus UV-ZIKV p = 0.0002 and ZIKVCDN versus ZIKVBR p = 0.0001. In m ZIKVCDN versus UV-ZIKV p = 0.000002 and ZIKVCDN versus ZIKVBR p = 0.000002. All data are shown as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001. Red squares and red lines = ZIKVCDN-infected mice, blue triangles and blue lines = ZIKVBR-infected mice, black squares and black lines = UV-ZIKV-injected mice, gray lines = isotype controls. Source data are provided as a Source Data file.

Fig 4: Low-dose SN-38 significantly increases the animal survival rate, regulates PD-L1 and FOXO3 expression in mouse ID-8 tumor tissues, and engages mouse CD49b/NK1.1/NKp46-positive NK cells infiltrating the ID-8 TME in a syngeneic mouse ID8 tumor model. Mouse ID-8 cells (2×105 cells/injection) were injected into the peritoneal cavity of female C57BL6 mice (n=9/group). (A) The scheme of the therapeutic model is shown. Twenty days after the inoculation of ID-8 cells, the mice were given an intraperitoneal (i.p.) injection (0.1 mL) of SN-38 (20 µg/kg body weight (BW)/mouse) or metformin (20 mg/kg BW/mouse) or an equal amount of DMSO (control) three times per week at even intervals for several weeks (up to 18 weeks). (B) Animal survival rates of the different groups were shown as Kaplan-Meier survival curves, which were calculated by the Kaplan-Meier method. The median survival days were shown. The significance was compared using the log-rank test. P values between the SN-38 group versus the DMSO group and between the metformin group versus the DMSO group were 0.0093 and 0.0328, respectively. (C) Three slides of tumor samples from mice treated with DMSO or SN-38 were incubated with anti-PD-L1 or anti-FOXO3 Abs and followed by Alexa Fluor 488-conjugated or 594-conjugated secondary Abs and IF analysis as described previously. Four archetypal IF images were displayed; all IF images and DAPI (the nuclear staining) images were shown in online supplemental figure S1). (D and E) The relative expression of PD-L1 and FOXO3 between tumors treated with DMSO and SN-38 in vivo are shown in the histograms as indicated, respectively. Scale bar: 20 µm. (F and G) Three slides of samples from ID8 tumors treated with DMSO or SN-38 were incubated with antimouse CD49b Ab (F) or NK1.1 Ab (G) and followed by fluorescent secondary Abs and IF analysis. The IF images and DAPI images were shown in online supplemental figure S2. (H and I) Total lysates of tumor samples were analyzed by immunoblotting with the indicated anti-NKp46 and anti-NKp44 Abs. (J) Similarly, tumor samples were incubated with antimouse IFN-? Ab and secondary Ab. GAPDH represents loading controls. Abs, antibodies; DMSO, dimethylsulfoxide; IF, immunofluorescence; IFN-?, interferon-?; NK, natural killer; PD-L1, programmed death-ligand 1; TME, tumor microenvironment.

Fig 5: C3a receptor antagonism results in tumor growth delay of Pan02 syngeneic mouse model in C57BL/6 mice and is mediated by increased NK cell infiltration in the TME. A, After Pan02 tumor cells implanted subcutaneously in C57BL/6 mice reached a volume of approximately 100 mm3, treatment was started with either daily intraperitoneal injection of vehicle (4% DMSO in PBS; control group, n = 6) or with daily intraperitoneal injection of the small-molecule C3aR antagonist SB290157 (SB290157 group, 5 mg/kg, n = 6) for 22 days and tumor size was monitored during and after treatment until endpoint (tumor ulceration or volume >1,000 mm3). The mean time to reaching 300 mm3 in the control group was 11.2 days compared with 23 days in the SB902157 treatment group (****, P < 0.0001). B, Flow cytometry analysis of control versus C3aR antagonist treated Pan02 tumor-bearing mice. Mice were randomized in two treatment groups (control vs. C3aR antagonist, n = 5 per group) and were treated daily for six treatments and euthanized the day after the last treatment. C3aR antagonism results in an increased NK cells (NK1.1+; as a percentage of live cells) in the TME (**, P = 0.0032). Representative gating of NK cells shown (*, Proportion of NK cells as a percentage of live cells). C, Representative IF staining of control and C3aR antagonist–treated mice (n = 3 per treatment group), showing an increase of NK cells (NK1.1-FITC) in the TME of C3aR antagonist-treated mice compared with vehicle-treated mice. D, Flow cytometry analysis of control versus C3aR antagonist–treated Pan02 tumor-bearing mice. Except a decrease in macrophages (Gr1-CD11b+F4/80+) in the C3aR antagonist–treated tumors (**, P = 0.0055), no statistically significant (ns) difference in B cells (B220+), CD4+ T cells, CD8+ T cells, and MDSCs (Gr1+CD11b+) is noted between the two treatment groups. E, Left: NK and CD8+ T cells depletion experiment scheme. After randomization into the treatment arms, NK cells or CD8+ T cells depletion was achieved by the administration of 100 µg of antibody (anti-NK1.1, or anti-CD8) intraperitoneal on days 0, 4, 8, and 12. Mouse IgG1 was used as isotype control. Mice undergoing NK and CD8+ T cells depletion and receiving IgG1 isotype control antibody were treated with daily intraperitoneal injection of SB290157 (5 mg/kg) from day 0 to day 13. Mice in the control arm were treated with daily vehicle (4% DMSO in PBS). Right: Depletion of NK cells abrogates the effect of the C3aR antagonist, resulting in tumor growth rate similar to the control group, whereas with CD8+ T cells depletion, C3aR antagonist retains its effect on tumor growth delay. Statistical significance determined using t test.