Fig. 3.

Enhanced effector function of Grail ^−/− CD8⁺ T cells in vitro and in vivo. a CD45.1⁺ congenic mice were inoculated with EG-7 tumours and, CD45.2⁺ OT-I or Grail ^−/− OT-I cells were transferred 5 days later. Eight days after OT-1 transfer, TILs were isolated and stimulated with OVA peptide stained for IFN-γ and granzyme B (GzmB), and analysed in CD45.2⁺CD8⁺ gate. The bar graph shows mean ± SEM as well as the percentage from individual mice per group (n = 6 mice from two experiments). b Mixed bone marrow chimeric mice were inoculated with EG-7 tumour cells. TILs were stimulated as in a and analysed for IFN-γ and GzmB production in CD45.2⁻CD8⁺ (WT) or CD45.2⁺CD8⁺ (Grail ^−/−) gate. The bar graph shows mean ± SEM as well as the percentage from individual mice per group (n = 10 mice from two experiments). c FACS-sorted naive CD8⁺ T cells were activated with anti-CD3 alone or together with anti-CD28. Three days later, expression of IFN-γ and GzmB was analysed by intracellular staining. N = 3 independent experiments. d Naive OT-I and Grail ^−/− OT-I cells were activated with OVA peptide and irradiated WT APCs. Two days later, expression of IFN-γ and GzmB was analysed by intracellular staining. N = 3 independent experiments. e Two days after activation with OT-I peptide, WT and Grail ^−/− CD8⁺ T cells were incubated with 1 µg/ml OVA peptide-pulsed EL-4 cells labelled with DDAO-SE for 2 h. The cells were fixed, permeabilized and stained with anti-cleaved caspase-3 mAb. N = 3 independent experiments. f Spleen cells from OT-I or Grail ^−/− OT-I mice were adoptively transferred into C57BL/6J mice (n = 3 mice per group) followed by s.c. vaccination with OVA peptide and anti-CD40. Imiquimod cream was applied on the vaccination site. In addition, mice received 100,000 IU rhIL-2 by i.p. Three days after vaccination, mice were injected with a 1:1 mix of target cells loaded with 1 µg/ml peptide and labelled with 5 µmol/l CFSE or unloaded and labelled with 0.5 µmol/l CFSE. Four hours later, splenocytes from the recipients were analysed by flow cytometry **p < 0.01, ***p < 0.001 as determined using a Student’s t-test