Recruitment and activation of natural killer (NK) cells in vivo determined by the target cell phenotype. An adaptive component of NK cell-mediated responses

Abstract

Natural killer (NK) cells can spontaneously lyse certain virally infected and transformed cells. However, early in immune responses NK cells are further activated and recruited to tissue sites where they perform effector functions. This process is dependent on cytokines, but it is unclear if it is regulated by NK cell recognition of susceptible target cells. We show here that infiltration of activated NK cells into the peritoneal cavity in response to tumor cells is controlled by the tumor major histocompatibility complex (MHC) class I phenotype. Tumor cells lacking appropriate MHC class I expression induced NK cell infiltration, cytotoxic activation, and induction of transcription of interferon gamma in NK cells. The induction of these responses was inhibited by restoration of tumor cell MHC class I expression. The NK cells responding to MHC class I-deficient tumor cells were approximately 10 times as active as endogenous NK cells on a per cell basis. Although these effector cells showed a typical NK specificity in that they preferentially killed MHC class I-deficient cells, this specificity was even more distinct during induction of the intraperitoneal response. Observations are discussed in relation to a possible adaptive component of the NK response, i.e., recruitment/activation in response to challenges that only NK cells are able to neutralize.

Figure 1

Intraperitoneal NK cell activity induced by RMA-S lymphoma cells. (a) Syngeneic B6 mice were inoculated with irradiated RMA-S cells, and the PECs were tested after 1–4 d against NK-susceptible, ⁵¹Cr-labeled YAC-1 target cells in a standard cytotoxicity assay. (b) Comparison of YAC-1 killing present in the peritoneal cavity and the spleen 3 d after intraperitoneal inoculation with irradiated RMA-S cells. (c) The cytotoxic PECs induced by RMA-S are asialo-GM₁ ⁺. PECs induced by RMA-S injection were stained with either anti-CD8 or antiasialo-GM₁ antiserum, subjected to complement-mediated depletion, and subsequently tested in a ⁵¹Cr-release assay against YAC-1 target cells.

Figure 4

Strong increase in cytotoxic activity of NK1.1⁺ cells induced by intraperitoneal RMA-S inoculation. PECs from RMA-S–injected B6 (d–f) or control mice (a–c) were analyzed by FACS^® (b and e) and tested for cytotoxic activity against ⁵¹Cr-labeled YAC-1 target cells (a and d) 3 d after tumor cell inoculation. A significant increase in NK1.1⁺ cell numbers is induced by RMA-S. In the right panels, PECs from control (c) or RMA-S–inoculated (f) mice were FACS^® sorted into NK1.1⁺ and NK1.1⁻ populations and tested for cytotoxic activity against YAC-1 target cells. A strong activation of cytotoxic activity, as measured per NK1.1⁺ cell, is induced by RMA-S.

Figure 2

The induction of an intraperitoneal NK cell response is inhibited by restoration of tumor MHC class I expression. Irradiated tumor cells were inoculated intraperitoneally into syngeneic B6 mice, and PEC cells were tested on day 3 in a ⁵¹Cr-release assay against the target cells indicated. Stimulator cells used to inoculate syngeneic (H-2^b) C57Bl/6 mice: (a) PBS control; (b) RMA; (c) RMA-S; and (d) RMA-S.mtp2. Each diagram represents a mean of at least five experiments.

Figure 3

Regulation of NK cell activation in C.B-17 SCID mice by H-2D^d molecules. Irradiated (H-2^b) tumor cells were injected intraperitoneally into allogeneic C.B-17 (H-2^d) SCID mice, and PECs were tested on day 3 in a ⁵¹Cr-release assay against target cells indicated. Stimulator cells used to inoculate allogeneic (H-2^d) C.B-17 SCID mice: (a) BSS control; (b) RMA; and (c) RMA-D^d. The MHC class I–specific induction of the response occurs independently of T and B cells.

Figure 6

Induction of IFN-γ but not IL-12 in NK1.1⁺ PECs by RMA-S cells. PECs from mice inoculated with either RMA-S or RMA-S.Ham-2 were FACS^® sorted into NK1.1⁺ and NK1.1⁻ populations and analyzed for IFN-γ (a) and IL-12 (p40) transcripts (b). Competitive PCR was used for the quantitation of transcripts (see Materials and Methods), and the intensity of the bands is displayed as percent compared with a β-actin standard. In c, the serum levels of IFN-α and IFN-β were measured using DELFIA (see Materials and Methods). Induction of IFN-γ transcription is observed in intraperitoneal NK1.1⁺ cells but not in NK1.1⁻ cells and is dependent on tumor MHC class I expression.

Figure 5

Cytotoxic activation of intraperitoneal NK1.1⁺ cells depends on the MHC class I expression of the tumor cells. PECs from untreated B6 (a) or tumor cell–injected B6 mice (b) were sorted by FACS^® into NK1.1⁺ and NK1.1⁻ populations and tested for cytotoxic activity against YAC-1 target cells. Note that the cytotoxic activity from all mice is restricted to the NK1.1⁺ population. (a) The cytotoxic activity of endogenous NK1.1⁺ cells from spleen and peritoneal cavity. NK1.1⁺ cells from the peritoneal cavity show comparatively low cytotoxic activity. (b) NK1.1⁺ cells from RMA-S–injected mice have a drastically induced cytotoxic activity compared with endogenous NK1.1⁺ cells from the intraperitoneal cavity and spleen (compare to Fig. 5 a). Activation of NK1.1⁺ cells is inhibited by TAP transfection of RMA-S. (c) Cell size of NK1.1⁺ cells in untreated (black line) or RMA-S–injected mice (gray line) versus poly I:C–induced NK1.1⁺ cells (stippled line). The mean FSC for spleen lymphocytes and RBCs in this experiment was 409 and 216, respectively. No difference in cell size was observed between RMA-S–activated and endogenous NK1.1⁺ cells.

Figure 7

Secretion of IFN-γ protein by RMA-S–induced PECs. Secretion of IFN-γ protein from untreated (unfilled bars) and RMA-S–induced (filled bars) PECs was measured by sandwich ELISA. 2 × 10⁵ RMA-S cells or 50 IU of IL-12 was added for 20 h before measuring secretion. In the presence of medium only, the release of IFN-γ was <10 pg/ml. Secretion of IFN-γ protein is induced, but this requires the presence of either RMA-S or IL-12.