Rae1 and H60 ligands of the NKG2D receptor stimulate tumour immunity

Abstract

Natural killer (NK) cells attack many tumour cell lines, and are thought to have a critical role in anti-tumour immunity; however, the interaction between NK cells and tumour targets is poorly understood. The stimulatory lectin-like NKG2D receptor is expressed by NK cells, activated CD8+ T cells and by activated macrophages in mice. Several distinct cell-surface ligands that are related to class I major histocompatibility complex molecules have been identified, some of which are expressed at high levels by tumour cells but not by normal cells in adults. 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 Rae1beta 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.

Figure 1

EL4 and B16-BL6 tumor cells expressing NKG2D ligands are rejected by NK cells in syngeneic mice. C57BL/6 or B6-Rag1^−/− mice were treated with the indicated antibodies before inoculation with the indicated number of EL4 (a, b) or B16-BL6 (c-e) tumor cell transductants. Tumor growth was monitored thereafter. Data are representative of at least three experiments. f, g, B6 mice were injected i.v. with 3×10⁵ B16-BL6 transductants. Lung metastases were counted two weeks after the tumor challenge (f) or the lung lobes from representative mice in a separate experiment were examined three weeks after tumor challenge (g).

Figure 2

RMA cells expressing NKG2D ligands are rejected by NK cells and CD8 T cells in syngeneic mice. B6 (a) or B6-Rag1^−/− (b) mice were pretreated with the indicated antibodies and inoculated subcutaneously with the indicated numbers of RMA transductants or RMA/S cells. In instances where tumors grew in some but not all mice, the fractions are indicated in the panels. Data are representative of four experiments. c, Levels of NKG2D ligands determined by staining with fluorescently labeled tetrameric NKG2D. Tumor cell transductants, the YAC-1 lymphoma and the Sa1N fibrosarcoma are compared. d, The anti tumor response depends on Rae1β levels. Survival of B6 mice that had been inoculated with 1×10⁵ RMA cells, or RMA cells expressing high (Rae1^hi ) or intermediate (Rae1^int ) levels of Rae1β. Terminally moribund mice were sacrificed. Data are representative of two experiments.

Figure 3

Vaccination with ligand-expressing tumor cells confers specific immunity to the corresponding ligand-negative tumor cells. a, B6 mice that had previously rejected ligand-transduced tumor cells (5×10⁶ EL4, 1×10⁴ B16-BL6 or 1×10⁴ RMA transductants) were inoculated subcutaneously with control-transduced tumor cells of the same type (EL4: 5×10⁶ ; B16-BL6: 1×10⁴ ; RMA: 1×10⁵). Primary exposure occurred 8-12 weeks before challenge. b, B6 mice that had been vaccinated 12 weeks earlier with each ligand-transduced tumor cell type were injected with the same or different ligand-negative cell lines. The primary and secondary tumor doses were as in (a). c, Depletion of CD8⁺ cells during the primary challenge prevents development of immunity. Rechallenge with control-transduced tumor cells occurred 8-12 weeks after vaccination. Naïve B6 mice were challenged in parallel. d, B6 mice were injected with PBS or were vaccinated with 5×10⁶ irradiated or 1×10⁴ living transduced or untransduced B16-BL6 tumor cells. Mice were challenged 10 days later with 10⁴ untransduced B16-BL6 cells in the opposite flank. Data are representative of two experiments.

Figure 4

CTL responses primed by irradiated ligand-transduced RMA cells. Mice were vaccinated with 5×10⁶ irradiated RMA transductants or PBS. Two weeks later splenocytes were restimulated with irradiated RMA-Rae1β cells. a, Lysis of RMA cells. b, Target cell expression of Rae1 resulted in enhanced CTL lysis. c, d, Complement mediated-depletion of CD8 cells but not NK cells abrogates activity of effector cells from RMA-Rae1β vaccinated mice. e, Elevated percentage of RMA-specific IFN-γ producing effector CD8⁺ CD3⁺ T cells in mice primed with ligand-transduced RMA cells. Priming cells indicated at bottom. f, Expression of Rae1 by target cells enhances IFN-γ response. The enhancement was completely blocked by anti-NKG2D antibody. g, h, Enhanced lysis of target cells expressing Rae1β 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-γ production versus RMA target cells. j, CTL from RMA-Rae1β vaccinated mice specifically recognize RMA cells and remain tolerant of syngeneic T cell blasts. Effector cells from RMA-Rae1β 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 5

RMA cells expressing NKG2D ligands stimulate NK cell recruitment and activation in vivo. Groups of five B6 mice were injected intraperitoneally with 5×10⁶ irradiated RMA transductants, RMA/S cells or PBS. Peritoneal wash cells were recovered two days later. Compared to RMA cells, ligand-transduced RMA cells stimulated elevated percentages of NK (NK1.1⁺ CD3⁻ ) cells (a), which exhibited enhanced capacity to lyse YAC-1 target cells (b), and more of which produced IFN-γ when stimulated with YAC-1 target cells (c). Effector cells were destroyed by complement lysis with anti-NK1.1 but not anti-CD8 antibody (b), and pretreatment of mice with anti-NK1.1 antibody but not with anti-CD8 antibody prevented induction of cytotoxic activity (d). e, NK cell induction by ligand-transduced cells was blocked by injection of a non-depleting anti-NKG2D antiserum, but not by a control antiserum, just prior to tumor cell inoculation. The response to RMA/S cells was unaffected. Lysis of YAC-1 tumor cells and production of IFN-γ after stimulation with YAC-1 target cells were assayed. Data are representative of two experiments.