Fig. 6.

Regulation of Grail expression by TGF-β in CD8⁺ TILs. a FACS-sorted naive CD8⁺ T cells were activated with plate-bound anti-CD3/anti-CD28 in the presence or absence of TGF-β, followed by western blot to detect Grail levels. β-actin was used as a loading control. b WT mice were injected with rIgG or anti-TGF-β blocking antibodies on day 5, 7 and 9 after EG-7 inoculation and monitored daily for tumour growth. On day 17, tumours were isolated from each group and weight determined as in Fig. 1. (n = 3 independent experiments with five mice per group). c, d CD8⁺ T cells from spleens and TILs were FACS-sorted and restimulated with plate-bound anti-CD3 for 4 h. The mRNA expression of Grail and Cbl-b (c) and IL-21R (d) were detected by RT-PCR analysis. Results for target genes are presented after normalising to β-actin and shown as mean ± SEM. The results shown are representative of three independent experiments. *p < 0.05, **p < 0.01 as determined using a Student’s t-test