Abstract

Checkpoint blockade enhances effector T cell function and has elicited long-term remission in a subset of patients with a broad spectrum of cancers. TIGIT is a checkpoint receptor thought to be involved in mediating T cell exhaustion in tumors; however, the relevance of TIGIT to the dysfunction of natural killer (NK) cells remains poorly understood. Here we found that TIGIT, but not the other checkpoint molecules CTLA-4 and PD-1, was associated with NK cell exhaustion in tumor-bearing mice and patients with colon cancer. Blockade of TIGIT prevented NK cell exhaustion and promoted NK cell–dependent tumor immunity in several tumor-bearing mouse models. Furthermore, blockade of TIGIT resulted in potent tumor-specific T cell immunity in an NK cell–dependent manner, enhanced therapy with antibody to the PD-1 ligand PD-L1 and sustained memory immunity in tumor re-challenge models. This work demonstrates that TIGIT constitutes a previously unappreciated checkpoint in NK cells and that targeting TIGIT alone or in combination with other checkpoint receptors is a promising anti-cancer therapeutic strategy.

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Acknowledgements

We acknowledged funding support from the following sources: Natural Science Foundation of China (#81788101 to Z.T., #31570893 to R.S., and #81501355 to J.B.); Chinese Academy of Science (XDPB030301 to Z.T.); the Ministry of Science & Technology of China (2017ZX10203206003 to R.S., 2017ZX10202203-002-001 to Y.C.); the Science and Technology Innovation Fund of Shenzhen (JCYJ20150521094519472 and JCYJ20150630114942288 to J.B.). We thank Bristol-Myers Squibb for Tigit–/–mice; T. W. Mak (University of Toronto) for the Nfil3+/– mice; S. Su (Shantou University) for GKO mice; L. Bai and Z. Lian (University of Science and Technology of China) for C57BL/6 J Cd4–/– mice; X. Wang (Inner Mongolia University) for Rag2–/– mice; E. Vivier (INSERM) for Ncr1iCre/+ mice; and Z. Fan (Institute of Biophysics, Chinese Academy of Sciences) for Tigitfl/fl mice.

Author information

Affiliations

  1. Hefei National Laboratory for Physical Sciences at Microscale, University of Science and Technology of China, Hefei, Anhui, China

    • Qing Zhang
    • , Haiming Wei
    • , Rui Sun
    •  & Zhigang Tian
  2. Institute of Immunology and The CAS Key Laboratory of Innate Immunity and Chronic Disease, School of Life Sciences and Medical Center, University of Science and Technology of China, Hefei, Anhui, China

    • Qing Zhang
    • , Jiacheng Bi
    • , Xiaodong Zheng
    • , Yongyan Chen
    • , Hui Peng
    • , Haiming Wei
    • , Rui Sun
    •  & Zhigang Tian
  3. Shenzhen Laboratory of Fully Humanized Antibody Engineering, Institute of Biomedicine and Biotechnology, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen, China

    • Jiacheng Bi
  4. the First Affiliated Hospital of Anhui Medical University, Hefei, Anhui, China

    • Hua Wang
    • , Wenyong Wu
    • , Zhengguang Wang
    •  & Qiang Wu

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Contributions

Q.Z., R.S. and Z.T. initiated and designed the research; Q.Z. performed all the experiments and analyzed and interpreted results; Q.Z., J.B. and Z.T. wrote the manuscript; X.Z., Y.C., H.P. and H. Wei contributed to discussions of results; and H. Wang, W.W., Z.W. and Q.W. provided clinical specimens, and clinical and pathological information.

Competing interests

The authors declare no competing interests.

Corresponding authors

Correspondence to Rui Sun or Zhigang Tian.

Integrated supplementary information

  1. Supplementary Figure 1 Phenotypes of tumor-infiltrating lymphocytes.

    Supplementary Figure 1. Phenotypes of tumor-infiltrating lymphocytes. (a) Frequency and MFI of TIGIT+ cells in CD8+ T cells (CD45+CD3+CD56) from peritumoral regions (PT) and intratumoral regions (IT) in patients with CRC (n = 16). (b) Representative flow cytometry plot of TIGIT expression in NK cells from the spleen, liver and lung as described in Fig. 1b. (c) Frequencies of TIGIT+ in CD8+ T cells in B16-pulmonary metastasis model (n = 4 mice per group). (d) Representative flow cytometry plots of tumor-infiltrating NK cells and TIGIT expression on NK cells in four subcutaneous tumor models. (e) Frequencies of TIGIT+ cells in CD8+ T cells as described in Fig. 1c (n = 5 mice per group). (f, g) Frequencies of PD-1+, or CTLA-4+ cells in CD8+ or CD4+ T cells as described in Fig. 1d (n = 5 mice per group). (h) Frequencies of TIGIT+CTLA-4, TIGIT+CTLA-4+, and TIGITCTLA-4+ cells in tumor-infiltrating CD4+ T cells (n = 5 mice per group). (i) Frequencies of CD226, CD96, LAG3, TIM3, Ly49A, Ly49C/I, NKG2A and NKG2D (n = 16, 16, 15, 23, 5, 5, 5, 5 mice) in TIGIT and TIGIT+ tumor-infiltrating NK cells in B16-pulmonary metastasis model. (b-i) Data are representative of at least three independent experiments. ns, not significant (P > 0.05), *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; Error bars are mean ± s. e. m. (c, e, f, g); paired two-tailed t-test (a, i); one-way ANOVA followed by Tukey's (c, h) or Dunnett's (e, f, g) multiple comparisons test.

  2. Supplementary Figure 2 The TIGIT ligand CD155 is abundant in human and murine tumors.

    (a) Representative images of immunolabeling for CD155 and CD112 in human tumor tissues (paraffin sections from 20 tumor patients with colorectal cancer or lung cancer). Scale bars are 100 μm. (b) The expression of CD155 and CD112 on tumor cells we used in the murine tumor models. (a, b) The experiment was repeated independently for four times with similar results.

  3. Supplementary Figure 3 Conditional knockout of TIGIT on NK cells prevents the exhaustion of TILs.

    (a) Representative histograms of TIGIT expression on NK and T cells from Tigitfl/fl- Ncr1iCre/+, Tigitfl/fl- Ncr1+/+, Tigit–/– and WT mice. Grey shadow represent isotype staining. The experiment was repeated independently for three times with similar results. (b, c) Tigitfl/fl- Ncr1iCre/+ and Tigitfl/fl- Ncr1+/+ mice were intravenously injected with 1.5 × 105 B16/F10 cells (n = 4). TILs were analyzed 15-d post challenge. (b) Expression of CD226 and CD96 in tumor-infiltrating NK cells. (c) Expression of PD-1 and Tim3 in tumor-infiltrating CD8+ T cells. (b-c) Data are representative of three independent experiments. ns, not significant (P > 0.05), *P < 0.05, **P < 0.01; Error bars are mean ± s. e. m. (b, c); unpaired two-tailed t-test (b, c).

  4. Supplementary Figure 4 TIGIT blockade alone inhibits tumor growth and reverses the exhaustion of tumor-infiltrating CD8+ T cells.

    (a) Expression of CD107a, TNF, and IFN-γ in tumor-infiltrating CD8+ T cells in CT26-tumor bearing mice (n = 5 mice per group). (b, c) B16 melanoma model. Mice were subcutaneously injected with 5 × 104 B16/F10 cells, and treated with anti-TIGIT mAb and control IgG for 3 times (once every 3 d). (b) Tumor size over time (anti-TIGIT, IgG, PBS, n = 6, 5, 4 mice). (c) Survival of mice (anti-TIGIT, IgG, PBS, n = 7, 7, 6 mice). Data are representative of three (a) or two (b, c) independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001; Error bars are mean ± s. e. m. (a, b); unpaired two-tailed t-test (a); multiple t tests (b); Mantel-Cox test (c).

  5. Supplementary Figure 5 pLIVE-mFlt3L injection accumulates DCs and NK cells, and promotes the activation of NK cells.

    (a-c), Mice were hydro-dynamically injected with 10 μg pLIVE-mFlt3L or pLIVE-NULL. Serum was collected at indicated time post injection. The peripheral blood was collected from the tail vein 2 weeks after the injection. Splenic and Hepatic cells were isolated 4 weeks after the injection. (a) Soluble Flt3L level in serum (n = 4 mice per group) and the frequency of DCs in PBMC (pLIVE-NULL, pLIVE-Flt3L, n = 4, 7 mice). (b) The frequency and absolute numbers of DCs cells in spleen and liver (n = 3 mice per group). (c) The frequency and absolute numbers of NK cells and CD69+ NK cells in spleen and liver (n = 5 mice per group). (d) The frequency of NK cells, CD69+ NK cells and absolute number of CD69+ NK cells in lung from B16 pulmonary metastasis model. Mice were treated as described in Fig. 3n (n = 4 mice per group). (a-d) Data are representative of three independent experiments. ns, not significant (P > 0.05), *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; Error bars are mean ± s. e. m. (a-d); Two-way ANOVA followed by Sidak's multiple comparisons test (a left); unpaired two-tailed t-test (a right, b, c); one-way ANOVA followed by Tukey's multiple comparisons test (d).

  6. Supplementary Figure 6 TIGIT blockade inhibits tumor growth in Rag2–/–and NOD-SCID mice.

    (a, c) Tumor size over time (Rag2–/–, IgG, anti-TIGIT, n = 6, 6; NOD-SCID, IgG, anti-TIGIT, n = 16, 18). (b, d) Expression of IFN-γ (n = 5 mice per group), TNF (n = 5 mice per group) and CD226 (n = 6 mice per group) in tumor-infiltrating NK cells. (a-d) Data are representative of two independent experiments. ns, not significant (P > 0.05), *P < 0.05, **P < 0.01; Error bars are mean ±s. e. m. (a-d); multiple t tests (a, c); unpaired two-tailed t-test (b, d).

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    Supplementary Figures 1-6 and Supplementary Table 1

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https://doi.org/10.1038/s41590-018-0132-0

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