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Supplementary Figures

Supplementary Figure 1. NKT ligand-loaded tumour antigen-presenting B cell- and monocyte-based vaccine induces NKT, NK and CD8 T cell responses. (A) The cytokine profiles of liver inkt cells were assessed in B/Mo/Adk35-E6E7/αGC-injected mice at various times. The levels of IL-4 and IFN-γ were detected by flow cytometry. (B) Twelve days after TC-1 WT cell implantation, IFN-γ production by tumour-infiltrating NK cells was assessed in B/Mo-, B/Mo/Adk35-E6E7- and B/Mo/Adk35-E6E7/αGC-injected mice. (C) C57BL/6 mice were vaccinated with the indicated form of B/Mo. One week later, in vivo CTL assays were performed by injecting CFSE-labelled syngeneic targets. CFSE high, peptide pulsed target; CFSE low, peptide unpulsed control. In vitro assays were performed using vaccinated mice splenocytes that were restimulated with the HPV16 E6/E7 peptide mixture (E6: EVYDAFRDL, E7: RAHYNIVTF), and cytotoxicity against TC-1 cells was measured using a standard 51 Cr release assay. The data shown are from at least 2 individual experiments with similar results. The data in A, B and C were analysed by two-way ANOVA with Bonferroni multiple comparison tests. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

Supplementary Figure 2. Memory antitumour responses induced by vaccination. (A) Tumour-free mice from Fig. 1A and naïve mice (n=6) were vaccinated with the indicated cellular vaccine at day 6 after TC-1 tumour s.c. rechallenge (1x10 5 ) on the opposite flank as on day 0. The data shown are from at least 2 individual experiments with similar results.

Supplementary Figure 3. H-2D b, K b or Beta 2 microglobulin knock out tumour cells. (A) The expression levels of H-2D b and H-2K b on TC-1 WT or TC-1 H-2K b,d b KO cells were measured by flow cytometry. (B) The H-2D b expression level in TC-1 WT or TC-1 H-2K b,d b KO-bearing mouse tumour (n=5) sections was detected by immunohistochemistry (H2-D b : brown, tumour: blue, Scale bar: 100μm). (C-E) The tumour size was measured after s.c. implantation of the indicated tumour cells. The data shown are from at least 2 individual experiments with similar results.

Supplementary Figure 4. Efficacy of NK, inkt cell depletion by anti-asialo-gm-1 or anti NK1.1 (PK136). The tumour bearing mice were treated with control IgG, anti-nk1.1 (PK136) or anti-asialo GM-1 to deplete NK cells on day 12 after tumour injection (TC-1). NK cells and NKT cells were detected in the spleen by flow cytometry.

Supplementary Figure 5. Tim-3 and PD-1 expression on natural killer cells in the indicated organs. The kinetic expression levels of Tim-3 and PD-1 on natural killer cells from TC-1 WT or H-2K b,d b KO tumour-bearing mice (n=5) were analysed by flow cytometry.

Supplementary Figure 6. Tim-3 and PD-1 expression on NK cells in vitro. (A) The daily kinetic Tim-3 and PD-1 expression levels on NK cells were analysed after MC38 WT or H- 2K b,d b KO tumour cells were cocultured in vitro with splenic NK cells from C57BL/6 mice. (B) The expression levels of Tim-3 and PD-1 on NK cells were assessed after coculture with TC-1 H-2K b,d b KO cells via transwell or the addition of tumour-conditioned medium. The data were analysed using a two-tailed unpaired Student s t test *P<0.05, **P<0.01, ***P<0.001.

Supplementary Figure 7. Percent, number and function of NK cells in MHC class I- deficient tumours. (A-C) Twelve days after TC-1 WT or H-2K b,d b KO cell implantation, (A) the percent and (B) number of NK cells were analysed in the indicated organs. (C) Lymphocytes from the indicated organs were restimulated in vitro with anti-nk1.1 or control IgG for 30 minutes, and IFN-γ production was then evaluated by flow cytometry. The data shown are from at least 2 individual experiments with similar results. The data in C were analysed using a two-tailed unpaired Student s t test. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

Supplementary Figure 8. ERK activation kinetics and functional analysis of TC-1 WT or H-2K b,d b tumour-infiltrating NK cells. (A) ERK1/2 phosphorylation was detected by western blot analysis after restimulation in vitro with anti-nk1.1 or control IgG antibody for 30 minutes with TC-1 WT or H-2K b,d b KO implantation (5x10 5 )-infiltrating NK cells at different time points (B) The expression of H-2K b, D b, IFN-γ and Granzyme B on tumour cells or intratumoural NK cells from mice bearing TC-1 WT tumours was measured over time.

Supplementary Figure 9. The PD-1/PDL1 axis transmits a negative signal on exhausted NK cells. (A) The expression level of PDL-1on MC38 H-2K b, D b KO was measured by flow cytometry. (B) MC38 H-2K b, D b KO tumour cells (1x10 5 ) were implanted into mice, which were then treated with anti-pd-1 (300 μg) or control IgG twice weekly. A depleting antibody (2.43) for CD8+ T cell depletion was injected i.p. twice weekly. The data shown are from at least 2 individual experiments with similar results. The data in B was analysed by two-way ANOVA with Bonferroni multiple comparison tests. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

Supplementary Figure 10. HeLa β2m KO cells induce dysfunction of human NK cells. (A- B) Tim-3 + or Tim-3 - human NK cells cocultured with HeLa β2m KO cells for 3 days were restimulated in vitro with human ULBP-2, and IFN-γ and granzyme B production and T-bet and Eomes expression were assessed by flow cytometry. The data shown are from at least 2 individual experiments with similar results.

Supplementary Figure 11. Exhausted NK cells induced in MHC class I knockout tumourbearing mice are reversed by NKT-dependent activation. (A-B) Eleven days after TC-1 WT or H-2K b,d b KO cell implantation, each tumour-bearing (A) C57BL/6 or (B) Jα281 KO mouse was vaccinated with B/Mo or B/Mo/αGC (1x10 6 ). In addition, 12 hours later, lymphocytes from the indicated organs were stimulated with control IgG or anti-nk1.1 for 30 minutes, and IFN-γ production by NK cells was analysed by flow cytometry. The data were analysed using a two-tailed unpaired Student s t test. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

Supplementary Figure 12. NKT cells induce various cytokines in tumour-bearing mice. (A) The cytokine profile in the serum of B/Mo/αGC-injected WT, TC-1WT, TC-1 H2-K b D b KO or WT&H-2K b,d b KO mixed tumour-bearing mice at various times. The levels of IFN-γ, TNF-α and IL-4 were detected by ELISA. (B) Twelve days after TC-1 WT or H-2K b,d b KO implantation, intratumoural lymphocytes were stimulated in vitro with TNF-α (20 ng/ml) and IL-4 (20 ng/ml) and then restimulated with anti-nk1.1. The data were analysed using a twotailed unpaired Student s t test. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

Supplementary Figure 13. IL-21 drives the functional reversal of Tim-3 + PD-1 + NK cells. Nine days after TC-1 H-2K b,d b KO implantation, anti-ifn-γ (500 μg/mouse) or anti-il-21r (300 μg/mouse) or both were injected via i.p., and two days later B/Mo/αGC (1x10 6 cells/mouse) was injected i.v. with anti-ifn-γ (500 μg/mouse) or anti-il-21r (300 μg/mouse) or both. In addition, after an additional 12 hours, infiltrating lymphocytes were restimulated in vitro with anti-nk1.1, and the production of perforin and granzyme B was assessed by flow cytometry. The data were analysed using a two-tailed unpaired Student s t test. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001

Supplementary Figure 14. Intratumoural injection of IL-21 induces functional reversal of exhausted Tim-3 + PD-1 + NK cells in Rag / mice. (A-E) MC38 WT- or H-2K b,d b KObearing Rag 1 -/- mice (n=5) were treated with IL-21 (10 μg/mouse) by intratumoural injection every 3 days from days 9 to 15. (A) Tumour growth was measured using a metric calliper 2-3 times per week. (B) The number of NK cells was analysed as ( = % of CD45.2 NKp46 cells x No.of tumor infiltrating lymphocyte Tumor weight (g) ). (C-E) Tumour-infiltrating lymphocytes were restimulated using anti-nk1.1, and (C) the expression of Tim-3 and PD-1 and (D-E) the production of IFN-γ and CD107a were analysed in the indicated cell populations. The data were analysed using a two-tailed unpaired Student s t test. *P<0.05, **P<0.01, ***P<0.001.

Supplementary Figure 15. IL-21 elicits Foxo1 phosphorylation and T-bet upregulation in mice and humans. (A) Intracellular staining of pfoxo1 and T-bet in Tim-3 + PD-1 + and Tim- 3 - PD-1 - NK cells from MC38 H-2K b,d b KO-bearing Rag 1 -/- mice that were treated with IL-21 (10 μg/mouse) by intratumoural injection every 3 days. (B) Intracellular staining of pfoxo1 and T-bet in the indicated NK cells stimulated with IL-21 (50 ng/ml) in vitro for 1 hour. The data were analysed using a two-tailed unpaired Student s t test. *P<0.05, **P<0.01, ***P<0.001.

Supplementary Figure 16. In vitro cytotoxicity of intratumoural NK cells from cancer patients. Isolated Tim-3 + PD-1 + or Tim-3 PD-1 NK cells were incubated overnight in the presence or absence of ril-21 (20 ng/ml) and cocultured with 51 Cr-labeled K562 cells as target cells. The data were analysed by two-way ANOVA with Bonferroni multiple comparisons tests. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001

Supplementary Figure 17. Original western blot image for Figure 5C and Supplementary Figure 8A.

Supplementary Table 1. Antibody information for flow cytometry and western blot.

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