Fig 2: Qualitative characteristics of lung macrophages of Alk and Alk+Mad2 mice (A) Lung tumors and non-tumor areas were processed and stained for CD45, CD64, CD11c, and CD11b. Representative flow plots show alive CD45+ cells out of all lymphocytes after exclusion of debris and doublets; CD64+ cells in the alive CD45+ cells; CD11b+ interstitial macrophages from the CD64+CD45+AquaZombie– cells (up) and MHCIIhigh and MHCIIlow subpopulation of CD11b+ interstitial macrophages (down). (B) Percentage of interstitial macrophages gated as CD11b+ and CD11c- population from CD45+ and CD64+ population in tumors (Alk; n = 4, Alk+Mad2; n = 7) and in non-tumor area (Alk; n = 10, Alk+Mad2; n = 10). (C) Percentage of MHCIIhigh and MHCIIlow population in CD11b+ and CD11c- cells in tumors (Alk; n = 4, Alk+Mad2; n = 7) and non-tumor areas (Alk; n = 10 Alk+Mad2; n = 10). * p < 0.05; Two-tailed t-test.

Fig 3: Characterization of Alk and Alk+Mad2 mice 6 weeks after tumor initiation. (A) Percentage of tumor area/total lung area on H&E-stained cross-sections of Alk and Alk+Mad2 mice. (B) Number of nodules per mm2 in Alk and Alk+Mad2 mice. (C) Number of alveolar macrophages in the bronchoalveolar lavage fluid of Alk, Alk+Mad2, and Mad2 mice. (D) Percentage of interstitial macrophages gated as CD11b+ and CD11c- population from CD45+ and CD64+ population in the non-tumor area of Alk, Alk+Mad2, and Mad2 groups. (E) ELISA detection of IL-6 in the BALF of Alk, Alk+Mad2, and Mad2 mice. (F) Percentage of CD8+ cells in the non-tumor area of Alk, Alk+Mad2, and Mad2 lung tissues (immunohistochemical analyses, left panel) and percentage of CD8+ cells gated from CD3+ population in the non-tumor area (FACS analyses, right panel). (G) Representative flow plots of lung non-tumor areas, stained for CD45, NKp46, CD3, and percent of NKp46 cells from CD45+ cells in Alk, Alk+Mad2, and Mad2 mice. Two-tailed t-test in (A,B) and one-way ANOVA test for (C–E,G). Kruskal–Wallis, ANOVA in (F). In (A,B) Alk; n = 9 and Alk+Mad2; n = 9 mice. In (C–G) Alk; n = 6, Alk+Mad2; n = 6, Mad2; n = 5. ns, not significant; * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Fig 4: 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 CD8aloCD11ahi 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 CD8aloCD11ahi 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+ CD8aloCD11ahi 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 5: ANGPTL2 stimulates dendritic cell-mediated immune responses. (A) In vitro cross-presentation of OVA proteins by LPS-stimulated or LPS-unstimulated bone-marrow-derived dendritic cells (BMDCs) derived from WT or Angptl2-/- mice using the CD8+ T-cell hybridoma B3Z. ß-galactosidase activity in B3Z cells incubated with unstimulated BMDCs untreated with OVA proteins was set at 1. Data are means ± SD; n = 4 per group. (ns) Not significant (P > 0.05), one-way ANOVA test followed by Tukey's multiple comparison test. (B) Comparison of levels of Il12p40 transcripts in LPS-stimulated or LPS-unstimulated BMDCs derived from WT or Angptl2-/- mice. Levels in unstimulated WT BMDCs were set to 1. Data are means ± SD; n = 3 per group. (ns) Not significant (P > 0.05), one-way ANOVA test followed by Tukey's multiple comparison test. (C) In vitro cross-presentation of OVA proteins by WT BMDCs stimulated with or without recombinant ANGPTL2 (rANGPTL2) protein using B3Z cells. ß-galactosidase activity in B3Z cells incubated with unstimulated BMDCs untreated with OVA proteins was set to 1. Data are means ± SD; n = 4 per group. (ns) Not significant (P > 0.05); (***) P < 0.001, one-way ANOVA test followed by Tukey's multiple comparison test. (D) Comparison of levels of Il12p40 transcripts in WT BMDCs treated with or without rANGPTL2. Levels in ANGPTL2-untreated BMDCs were set to 1. Data are means ± SD; n = 3 per group. (***) P < 0.001, unpaired t-test. (E) Frequency of CD11c+MHC class II (MHC II)hi cells in WT BMDCs treated with or without rANGPTL2, as assessed by FACS analysis. Frequency is calculated as a percentage of total CD11c+ cells. Data are means ± SD; n = 3 per group. (***) P < 0.001, unpaired t-test. (F) Relative expression levels (median fluorescence intensity, MFI) of costimulatory molecules (CD86, CD80, and CD40) in WT BMDCs treated with or without rANGPTL2, as assessed by FACS analysis. Levels in ANGPTL2-untreated BMDCs were set to 1. Data are means ± SD; n = 3 per group. (***) P < 0.001, unpaired t-test. (G) Schematic illustrating the experimental design of dendritic cell immunization of WT male mice. rA2, recombinant ANGPTL2 protein; SINFEKL, H-2Kb-restricted OVA class I epitope. (H) Comparison of tumor volumes in C57BL/6 male mice bearing B16-OVA cells transferred with untreated dendritic cells (DC) or ANGPTL2-treated (DC + rA2 + SIINFEKL) or untreated (DC + SIINFEKL) DCs pulsed with SIINFEKL. Data are means ± SEM; n = 6 tumors per group. (ns) Not significant (P > 0.05); (**) P < 0.01, two-way ANOVA test comparing each group with the PBS group. (I) Kaplan–Meier tumor-free survival curves of mice bearing B16-OVA cells transferred with untreated dendritic cells (DC) or ANGPTL2-treated (DC + rA2 + SIINFEKL) or untreated (DC + SIINFEKL) DCs pulsed with SIINFEKL (n = 5 mice for PBS and DC groups or n = 4 mice for DC + SIINFEKL and DC + rA2 + SIINFEKL groups). (ns) Not significant (P > 0.05); (*) P < 0.05, log-rank test comparing each group with the PBS group.