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Rae1 and H60 ligands of the NKG2D receptor stimulate tumour immunity

Nature volume 413, pages 165–171 (2001)Cite this article

Abstract

Natural killer (NK) cells attack many tumour cell lines, and are thought to have a critical role in anti-tumour immunity1,2,3,4,5,6,7; however, the interaction between NK cells and tumour targets is poorly understood. The stimulatory lectin-like NKG2D receptor8,9,10,11,12,13 is expressed by NK cells, activated CD8+ T cells and by activated macrophages in mice11. Several distinct cell-surface ligands that are related to class I major histocompatibility complex molecules have been identified11,12,13,14, some of which are expressed at high levels by tumour cells but not by normal cells in adults11,13,15,16. However, no direct evidence links the expression of these ‘induced self’ ligands with tumour cell rejection. Here we demonstrate that ectopic expression of the murine NKG2D ligands Rae1β or H60 in several tumour cell lines results in potent rejection of the tumour cells by syngeneic mice. Rejection is mediated by NK cells and/or CD8+ T cells. The ligand-expressing tumour cells induce potent priming of cytotoxic T cells and sensitization of NK cells in vivo. Mice that are exposed to live or irradiated tumour cells expressing Rae1 or H60 are specifically immune to subsequent challenge with tumour cells that lack NKG2D ligands, suggesting application of the ligands in the design of tumour vaccines.

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Figure 1: EL4 and B16-BL6 tumour cells that express NKG2D ligands are rejected by NK cells in syngeneic mice.
Figure 2: RMA cells expressing NKG2D ligands are rejected by NK cells and CD8 T cells in syngeneic mice.
Figure 3: Vaccination with ligand-expressing tumour cells confers specific immunity to the corresponding ligand-negative tumour cells.
Figure 4: CTL responses primed by irradiated ligand-transduced RMA cells.
Figure 5: RMA cells expressing NKG2D ligands stimulate NK cell recruitment and activation in vivo.

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Acknowledgements

We thank C. W. McMahon, and N. Fernandez for critical reading of the manuscript; N. Fernandez for PK136 antibody; J. Egen for help with the retroviral transfection system; H. Nolla for cell sorting; J. Hsia and C. White for technical assistance; the members of the Raulet laboratory for discussions; and J. P. Allison, M. Fasso, Y. H. Chien and B. Sha for their gift of reagents and cell lines. This work was supported by grants from the National Institute of Health (D.H.R.). A.D. is a Physician Postdoctoral Fellow of the Howard Hughes Medical Institute.

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  1. Department of Molecular and Cell Biology and Cancer Research Laboratory, 485 Life Sciences Addition, University of California, Berkeley, 94720, California, USA

    Andreas Diefenbach, Eric R. Jensen, Amanda M. Jamieson & David H. Raulet

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  1. Andreas Diefenbach
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  2. Eric R. Jensen
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Correspondence to David H. Raulet.

Supplementary information

Figure 1

(GIF 12.9 KB)

Vaccination of B6-Rag1-/- mice with ligand-expressing tumor cells does not confer specific immunity to the respective ligand-negative tumor cells. B6-Rag1-/- mice that had previously rejected ligand-transduced tumor cells (1x10 4 RMA, 1x104 B16-BL6 or 5x106 EL4 transductants) were inoculated subcutaneously with untransduced tumor cells of the same type (RMA: 1x105; B16-BL6: 1x104; EL4: 5x106). Primary exposure occurred 8-12 weeks before challenge.

Figure 2

(GIF 46.5 KB)

CTL responses primed by irradiated ligand-transduced B16-BL6 cells. Mice were vaccinated twice with 5x106 irradiated B16 transductants or PBS. Three weeks later splenocytes were restimulated with irradiated B16-Rae1b cells. a, Lysis of B16-BL6 cells. b, Target cell expression of Rae1b resulted in enhanced CTL lysis. c, d, Complement mediated-depletion of CD8 cells but not NK cells abrogates activity of effector cells from B16-Rae1b vaccinated mice. e, Elevated percentage of B16-specific IFN-g producing effector CD8+CD3+ T cells in mice primed with ligand-transduced B16 cells. Priming cells indicated at bottom. f, Expression of Rae1b by target cells enhances IFN-g response. The enhancement was completely blocked by anti-NKG2D antibody. g, h, Enhanced lysis of target cells expressing Rae1b is blocked by anti-NKG2D antibody. i, CTL priming occurs in the absence of NK cells. Mice were depleted of NK1.1+ or CD8+ cells prior to and during vaccination with tumor cell transductants. Effector cells were tested for lytic activity and IFN-g production versus B16-Rae1b target cells. j, CTL from B16-Rae1b vaccinated mice specifically recognize B16 cells and remain tolerant of syngeneic T cell blasts. Effector cells from B16-Rae1b vaccinated mice were tested for lysis of the indicated tumor cells as well as Con A blasts from syngeneic mice. Data are representative of three experiments.

Figure 3

(GIF 40.7 KB)

Induction of NK cell activation by RMA cells expressing NKG2D ligands does not require the presence of T and/or B cells, as shown in B6-Rag1-/- mice. NK cells were induced in B6-Rag1-/- mice as in Fig. 4 of the main paper with RMA transductants or RMA/S cells. a, b, Effector cells were conventional NK cells. c, d, Induction of NK cells in B6-Rag1-/- mice by ligand-transduced cells was blocked by injection of a non-depleting anti-NKG2D antiserum just prior to tumor cell inoculation. Lysis of YAC-1 tumor cells and production of IFN-g after stimulation with YAC-1 target cells were assayed. Data are representative of two experiments.

Figure 4

(GIF 11.8 KB)

B16 or EL4 cells expressing NKG2D ligands stimulate NK cell recruitment and activation in vivo. Groups of five B6 mice were injected intraperitoneally with 5x106 irradiated tumor cell transductants or PBS. Peritoneal wash cells were recovered two days later. Compared to ligand-negative cells, ligand-transduced tumor cells stimulated elevated percentages of NK (NK1.1+CD3-) cells (a, d), which exhibited enhanced capacity to lyse YAC-1 target cells (b, e), and more of which produced IFN-g when stimulated with YAC-1 target cells (c, f). Effector cells were destroyed by complement lysis with anti-NK1.1 but not anti-CD8 antibody (b, e).

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Diefenbach, A., Jensen, E., Jamieson, A. et al. Rae1 and H60 ligands of the NKG2D receptor stimulate tumour immunity. Nature 413, 165–171 (2001). https://doi.org/10.1038/35093109

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