In vivo natural killer cell activities revealed by natural killer cell-deficient mice

Abstract

Studies of natural killer (NK) cell function in vivo have been challenging primarily due to the lack of animal models in which NK cells are genetically and selectively deficient. Here, we describe a transgenic mouse with defective natural killing and selective deficiency in NK1.1(+) CD3(-) cells. Despite functionally normal B, T, and NK/T cells, transgenic mice displayed impaired acute in vivo rejection of tumor cells. Adoptive transfer experiments confirmed that NK1.1(+) CD3(-) cells were responsible for acute tumor rejection, establishing the relationship of NK1.1(+) CD3(-) cells to NK cells. Additional studies provided evidence that (i) NK cells play an important role in suppressing tumor metastasis and outgrowth; (ii) NK cells are major producers of IFNgamma in response to bacterial endotoxin but not to interleukin-12, and; (iii) NK cells are not essential for humoral responses to T cell-independent type 2 antigen or the generalized Shwartzman reaction, both of which were previously proposed to involve NK cells.

Figure 1

In vitro natural killing is defective in a Ly49A Tg mouse strain. (A) Spleen cells from WT and Ly49A Tg mice were stained with anti-Ly49A, anti-NK1.1, and anti-CD3. Profiles for Ly49A expression on cells gated on CD3⁻ or CD3⁺ populations are shown. The numbers represent the percentage of cells within the quadrant among all viable cells. (B) Splenocytes from WT (□) and Ly49A Tg (○) mice were used in 4-hr ⁵¹Cr-release cytotoxicity assays against YAC-1, RMA-S, and B16 targets at varying effector/target (E:T) ratios as indicated. Results are expressed as the mean percentage specific lysis ± SD of triplicate wells.

Figure 2

Impaired acute in vivo rejection of tumor cells due to selective reduction of peripheral NK1.1⁺ CD3⁻ cells. (A) Selective reduction of peripheral NK1.1⁺ CD3⁻ cells in Tg mice. Cells prepared from indicated tissues were stained with anti-NK1.1 and anti-CD3. The numbers represent the percentage of cells within the quadrant among all viable cells. (B) Mice were inoculated i.v. with ¹²⁵I-Udr-labeled 3 × 10⁵ YAC-1 tumor cells. After 4 hr, the residual lung radioactivity was measured. Results are expressed as the mean % residual radioactivity ± SD from three to five mice per group. (C) Reconstituted tumor clearance by scid splenocytes. Scid mice that were pretreated with anti-NK1.1 or untreated were injected i.p. with poly-I:C on day −1. Two hours after i.v. infusion of PBS or 8 × 10⁶ splenocytes isolated from anti-NK1.1-treated or untreated scid mice, the recipient mice were inoculated i.v. with ¹²⁵I-Udr-labeled 3 × 10⁴ RMA-S tumor cells. After 6 hr, the residual lung radioactivity was measured. Results are expressed as the mean percentage residual radioactivity ± SD from three mice per group. Where indicated for B and C, anti-NK1.1-treated mice received an i.p. injection of 200 μg of anti-NK1.1 mAb 2 days before tumor inoculation.

Figure 3

Control of experimental lung metastases and tumor outgrowth is impaired in Tg mice. (A) Mice were injected i.v. with 3 × 10⁴ B16 cells. The numbers of macroscopic tumor metastases in the lungs were counted on day 14. Anti-NK1.1-treated mice received three i.p. injections (200 μg/injection) of anti-NK1.1 on days −2, +2, and +7. Results are represented as the average number ± SD from four mice per group. (B) Varying numbers of RMA-S cells as indicated were inoculated s.c. into the flanks of WT (seven mice per group, □), Tg (six to seven mice per group, ○) and anti-NK1.1-treated WT (three to five mice per group, ⋄) mice, respectively. Anti-NK1.1-treated mice received five i.p. injections (200 μg/injection) of anti-NK1.1 on days −4, −2, +7, +14, and +21. Palpable tumors were scored twice weekly.

Figure 4

IFNγ production in response to LPS but not to IL-12 is impaired in Tg mice. (A) Mice were injected i.p. with 200 μg LPS. Sera were collected after 7 hr and assayed for IFNγ by ELISA. Results are expressed as the average ± SD from five to seven mice per group. (B) Mice were injected i.p. with 1 μg IL-12, and sera were collected after 24 hr. Results are expressed as the average ± SD from five mice per group.

Figure 5

Normal Ab responses to TI type 2 antigen in Tg mice. WT (□) and Tg (○) mice were immunized i.p. with 10 μg of TNP-Ficoll. Sera were collected at the indicated dates, diluted 500-fold, and TNP-specific IgM (A) and IgG3 (B) were measured by ELISA. Results are expressed as the average optical density ± SD from three mice per group.