Fig 2: a-melittin-NPs induce the generation of T-cell immunity in the liver of tumor-bearing mice. Groups of mice (n = 4 mice per group) were injected intrasplenically with 2 × 105 B16F10 cells in 100 µl of PBS and treated as shown in Fig. 3a. Hepatic lymphocytes were collected 6 days after injection by in vivo perfusion and in vitro digestion to assess the changes in leukocytes infiltration in the liver. Quantification was performed by flow cytometry. a Absolute numbers of innate and adaptive immune cells in the liver after different treatments. b Representative immunofluorescence images from the liver for NK1.1 (upper panel) and CD8 (lower panel). White dotted lines indicate metastatic lesions and red indicates the NK1.1+ or CD8+ cells. Scale bar, 20 µm. c, d The percentages c and absolute numbers d of NK cells according to their developmental phenotype. e The percentages (upper panel) and absolute numbers (lower panel) of NK cells according the expression of CD69, Ki-67, and granzyme B. f, g The percentages (upper panel) and absolute numbers (lower panel) of CD4+ T cells f and CD8+ T cells g according their expression of ICOS, Ki-67, granzyme B, and Tim3. Error bars indicate SEM. n.s. not significant; ***P < 0.001; **P < 0.01; *P < 0.05; by one-way ANOVA followed by Bonferroni’s post hoc test

Fig 3: The combination of VV plus a-PD-L1 treatment enhances infiltration of effector T cells and reduces Treg cells and exhausted CD8+ T cells in the TME.B6 mice were intraperitoneally inoculated with 5 × 105 MC38-luc and treated with VV and/or a-PD-L1 as described. Single cells were made from primary tumours collected from tumour-bearing mice at day 5 post first treatment, blocked with a-CD16/32 Ab and then stained with antibodies against CD45, CD8, CD4, PD-1, ICOS, PD-1, CTLA-4, TIM-3, LAG-3, TIGIT and Foxp3 to determine the quantities of CD8+ T cells (a), CD8+ T-cell activation (b–d), CD8+ T-cell exhaustion (e–h), Treg cells (i), CD8+/CD4+Foxp3+ T cells (j) and CD4+Foxp3- T cells (k,l) in the TME. Of note, the anti-PD-L1 antibody clone 10F.9G2 was used for therapy while clone MHI5 was used for subsequent phenotypic analysis. Data were analysed using Student's t-test (*P<0.05; **P<0.01; ***P<0.001).

Fig 4: Increased activation and proliferation of LN iNKT cells.(A) Plots show differentially expressed genes for pairwise comparisons from iNKT cells from the depicted tissues. A fold change cut-off of 1.5 and adjusted p-value cut off of 0.01 were applied to colour code differentially expressed genes on the plot (red). The numbers of differentially expressed genes are indicated in the graphs. (B) Heat map showing RNAseq analyses of selected transcripts significantly changed in iNKT cells from thymus, mLN or iLN versus spleen. n = 4 samples. (C) Top, Representative flow-cytometry histograms showing GFP expression in iNKT cells (grey) and T cells (blue) from the depicted tissues of Nur77GFP mice. iNKT cells from the tissues of Nur77GFP- mice are shown as control (empty profile). Bottom, representative flow-cytometry plots showing CD25, ICOS and CCR8 expression in iNKT cells from the tissues of WT mice (grey) and control (blue). (D–E) Ki-67 expression in iNKT cells and T cells from the depicted tissues of WT mice. Ki-67 MFI for iNKT cells (E) is related to T cells from each tissue. Bars represent mean ± SEM. *p<0.05, **p<0.01, two-tailed unpaired t-test. n = 5 mice from two independent experiments. (F) Quantification of EdU incorporation for iNKT cells from the depicted tissues after 48 hr of EdU administration in vivo. Bars represent mean ± SEM. **p<0.01, ****p<0.0001 two-tailed unpaired t-test. n = 6 mice from three independent experiments. (G) Representative flow-cytometry plot showing EdU incorporation in iNKT cells (left) and frequency of Vß7 or Vß8.1/8.2-expressing EdU+ and EdU- iNKT cells (right). n = 4 mice. (H) Heat map representation of the frequency of TRBV amongst low-abundance (one copy) or more abundant (>2 copies) CDR3ß sequences obtained from iLN and mLN as indicated. Data obtained from RNAseq and pooled from four samples per tissue.Figure 3—source data 1.Increased activation and proliferation of LN iNKT cells.

Fig 5: The Deficiency of TCF1 and LEF1 Leads to a Severe Reduction of aTregs(A) Expression-expression plot of transcriptomes of WT Tregs isolated from Foxp3YFP-Cre/wtTcf7wt/wtLef1wt/wt mice and dKO Tregs from Foxp3YFP-Cre/wtTcf7fl/flLef1fl/fl mice. Superimposed is the bulk Treg gene signature. Numbers depict the Treg signature genes over- or under-represented in dKO versus WT samples.(B) GSEA analysis of the “hallmark” gene sets deposited in MSigDB. Both p values and FDR q values were shown. NES, normalized enrichment score.(C) Volcano plots showing the comparisons between WT and dKO Tregs. The T cell activation gene signature (red) was superimposed. The numbers of T cell activation signature genes that were over- or under-represented in each comparison and the p value (by ?2 test) were shown.(D) The proliferation of WT and dKO Tregs in Spleen, pLNs, and mLNs measured by Ki67 staining.(E) Row-normalized heatmap of the expression of bulk Treg signature genes across r-, a-, and e-Treg subtypes (left). Superimposing each sub-cluster defined on the left to the FC plot comparing dKO versus WT Tregs (right).(F) The percentages of Treg subsets in dKO and WT littermate controls using the gating strategy defined in Figure S5E.(G) Representative fluorescence-activated cell sorting (FACS) plots showing the gating strategy and the compositions of Treg pool based on the expression of Helios, ICOS, and CD62L in WT and dKO mice (top). The percentages and numbers of each Treg subsets in Helios+ Treg compartment (bottom).(H) The effect of retroviral transduction of Tcf7 on Treg differentiation in vitro. Experimental settings (top) and statistical data of Treg subsets in control (pMIG) and Tcf7-transduction groups (bottom).The data are representative of one experiment with n = 2 replicates for each Treg subsets (A–C and E) or n = 2 (F–H), n = 3 (D) independent experiments. n = 3 mice in each group. Shown are mean ± SEM. p values in (C) were calculated using the chi-square (?2) test. p values in (D) and (F)–(H) were calculated using the two-tailed unpaired Student’s t test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, n.s., non-significant.See also Figure S5.