Sustained activation and tumor targeting of NKT cells using a CD1d–anti-HER2–scFv fusion protein induce antitumor effects in mice
J. Clin. Invest. Kathrin Stirnemann, et al. 118:994 doi:10.1172/JCI33249 [Go to this article.]

Figure 5
iNKT cells are required for the antitumor effect and are characterized by a sustained activation with TH1 bias. (A) The antitumor activity is lost in the absence of iNKT cells. C57BL/6 or CD1d^–/– mice were grafted on the left flank with 700,000 B16-HER2 cells, and systemic treatment with the αGalCer/sCD1d–anti-HER2 was started 2 days later (40 μg/i.v. injection every 3 days). For each mouse strain, a group was left untreated. Results represent the kinetic of s.c. tumor growth (in mm³) as the mean ± SD of 4 mice per group. (B) Ex vivo IFNγ production by liver iNKT cells after several injections of αGalCer/sCD1d–anti-HER2 fusion. Liver lymphocytes were isolated 20 minutes after the sixth injection of PBS (control), αGalCer, or sCD1d–anti-HER2 protein, and cultured for 1 h. Cells were stained with anti-NK1.1–PE and anti-CD3–FITC, and intracellularly with anti-IFNγ–APC. Graph shows percent of IFNγ-producing iNKT (gated on NK1.1⁺ CD3⁺ cells). (C) Sustained IFNγ production by liver iNKT cells after several i.v. injections as indicated, followed by in vitro rechallenge with the same stimuli as in vivo. Liver lymphocytes were isolated 3 days after the fifth injection and restimulated in vitro for 6 hours. Naive mice were tested with αGalCer or αGalCer/sCD1d–anti-HER2. Cells were stained as described in B and results are expressed as fold increase of IFNγ-producing liver iNKT cells compared with the nonstimulated iNKT fraction derived from the same mouse. White bar, no in vitro stimulation; gray bar, in vitro αGalCer; black bar, in vitro sCD1d–anti-HER2. Results show the mean ± SD of 3 different experiments. *P < 0.03, **P < 0.005.