Fig 2: BXCL701 treatment enhances anti-PD1 efficacy in mT3-2D tumors. (A) Schematic representation of in vivo experimental design testing combination treatment of BXCL701 and anti-PD1 vs single agents alone and PBS controls in mT3-2D tumors. (B) DPP activity measured in the serum of PBS, anti-PD1, BXCL701 or anti-PD1 and BXCL701 (‘Combo’) treated mice (n=5 per group, each dot representing pooled serum from three mice, **p<0.01, ***p<0.001, ****p<0.0001 as determined by ANOVA followed by Tukey’s multiple comparison’s test, any pairwise comparisons not shown were not significant). (C) DPP activity measured in the tumor lysates of PBS, anti-PD1, BXCL701 and combo-treated mice (n=3 per group,*p<0,05 as determined by ANOVA followed by Tukey’s multiple comparison’s test, any pairwise comparisons not shown were not significant). (D) Average mT3-2D tumor growth curves in C57BL/6 mice (n=15 per group) treated with PBS, anti-PD1, BXCL701 and combo. Tumor growth was monitored weekly. (E) Tumor volumes for PBS, anti-PD1, BXCL701 and combo-treated mT3-2D tumors at the end of treatment (week 4). (***p<0.001, ns=nonsignificant as determined by ANOVA followed by Tukey’s multiple comparison’s test). (F) Flow cytometry data representing CD4+, CD8+ and NK1.1+ cells as percent of live cells in tumors collected from PBS andcombo-treated mice (n=3 per group, each dot representing three to four tumors from individual mice, *p<0.05, **p<0.01, as determined by unpaired two-tailed t-test). (G) Flow cytometry data representing percentage of live cells that are CXCR3+CD4+, CXCR3+CD8+ and CXCR3+NK1.1+ cells in tumors from PBS, anti-PD-1, BXCL701 and combo treated mice (n=3 per group, each dot representing three to four tumors from individual mice, *p<0.05, **p<0.01, ***p<0.001, ns=nonsignificant as determined by one-way ANOVA followed by Tukey’s multiple comparison test). (H) Flow cytometry data representing percentage of live cells that are CD4 +IFN?+Tbet+ Th1 cells in tumors from PBS, anti-PD-1, BXCL701 and combo treated mice (n=3 per group, each dot representing three to four tumors from individual mice, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, ns=nonsignificant as determined by one-way ANOVA followed by Tukey’s multiple comparison test). ANOVA, analysis of variance; DPP, dipeptidyl peptidase; PBS, phosphate-buffered saline.

Fig 3: DHA feeding affected NK cell frequency, maturation and Ly49H+ NK cell expansion during the response to MCMV infection. A Flow cytometric analysis and enumeration of NK cells (CD45+CD3-CD19- NK1.1+NKp46+) in the BM, spleens, pLNs and livers of control versus DHA-fed mice. B Flow cytometric analysis and cumulative frequencies of subpopulations of NK cell (CD3-CD19-NK1.1+NKp46+) subsets based on CD11b and CD27 expression in the BM, spleens, pLNs and livers of control versus DHA-fed mice. C Flow cytometric analysis of the KLRG1+ subsets of NK cells (CD3-CD19-NK1.1+NKp46+) in the BM, spleens, pLNs and livers of control versus DHA-fed mice at day 7 postinfection. D Flow cytometric analysis and enumeration of Ly49H+ subsets of NK cells (CD3-CD19-NK1.1+NKp46+) in the BM, spleens, pLNs and livers of control versus DHA-fed mice at day 7 postinfection. E Cumulative frequencies of NK cell (CD3-CD19-NK1.1+NKp46+) subsets found in the spleens, and livers of control versus DHA-fed mice at day 21 postinfection. For each experiment, n = 4 to 9 pooled from 2–3 independent experiments (A–E). Each symbol represents an individual mouse, and the blue dots and red square represent control and DHA-enriched diet-fed mice, respectively. Error bars represent interquartile ranges; *p < 0.05; **p < 0.01; ***p < 0.001

Fig 4: Chi3l1 deletion in T cell inhibits pulmonary metastasis with increase of Th1 and CTL. a Genotyping PCR to confirm CD4 specific deletion of Chi3l1 in the mice. b Representative lung image from B16F10 melanoma injected WT and CD4-Chi3l1 KO mice. Scale bar, 2 mm c Number of pleural colonies in each lung was counted. Data are mean ± SEM of three sets of independent experiments and each dot in graphs represent an individual mouse. d Histology of lung sections by H&E staining. Scale bar, 200 m (e, f) IFN? and/or TNFa producing CD4 T cells and CD8 T cells in the lung was analyzed by intracellular cytokine staining. % of IFN?+, % of TNFa+, and % of TNFa+IFN?+ was represented as scattered graph. g, h IFN? and/or TNFa producing NK cells, and non-lymphocytic populations (NK1.1-CD4-CD8-) in the lung was analyzed by intracellular cytokine staining. % of IFN?+, % of TNFa+, and % of TNFa+IFN?+ was represented as scattered graph. i mRNA expression of genes related to cytotoxicity and Th1 effector functions was analyzed by quantitative RT-PCR. Each gene expression level was normalized to ß-actin. Data are mean ± SD of three sets of independent experiments and each dot in graphs represent an individual mouse. n.s., not significant; *p < 0.05, **p < 0.01, ***p < 0.001 (two-tailed Student’s t-test)

Fig 5: Intratumoral cDC1 Accumulation Depends on NK Cells, Related to Figure 2(A) Quantification of intratumoral NK cells over time. Each circle represents data for one single BRAFV600E tumor from a group of 4-6 tumors per type and time point.(B) Flow cytometric analysis of intratumoral lymphocytes in a Ptgs1/Ptgs2-/- BRAFV600E tumor. Data are representative of three independent experiments.(C) Frequency distribution showing the distance of cDC1 to NK1.1+ cells within an immunofluorescence image of a Ptgs1/Ptgs2-/- BRAFV600E tumor.(D) Quantification of intratumoral NK cells after NK cell depletion in the indicated mice given control or Ptgs1/Ptgs2-/- BRAFV600E tumors.(E) Correlation of total cDC1 numbers and tumor mass in Ptgs1/Ptgs2-/- BRAFV600E tumors in WT mice or WT mice that were depleted of NK cells prior to tumor cell inoculation.(F) Visualization of CD103+ cDC1 localization after surface reconstruction from immunofluorescence images for Ptgs1/Ptgs2-/- BRAFV600E tumors 4 days after transplantation into WT mice, WT mice depleted of NK cells or Rag2-/-Il2rg-/-mice. Scale bar 100µm. Images are representative of individual tumors from 5-7 mice. Arrows indicate multicellular clusters of cDC1, tumor margins are indicated by dashed lines.(G) Quantification of intratumoral cDC1 in immunofluorescence images of Ptgs1/Ptgs2-/- BRAFV600E tumors transplanted into WT mice, WT mice that were depleted of NK cells prior to tumor cell inoculation or Rag2-/-Il2rg-/-mice. Each circle represents data for one individual tumor.(H) Distance analyses based on surface reconstruction shown in (F).(I) Quantification of total CD11c+MHCII+ cells after treatment as in (D).(J) Quantification of intratumoral CD8+ T cells after NK cell depletion.(K) Quantification of intratumoral CD4+ T cells after NK cell depletion.Analyses shown in B-K were performed 4 days after tumor cell inoculation. Data shown in A, D, E, G and I-K are pooled from at least two independent experiments and represented as mean of all mice in each group ± SEM n.s., non-significant, *p < 0.05, **p < 0.01, ***p < 0.001.