Extended Data Fig. 1: Validation of the effect of REGNASE-1 deletion on CD8+ T cell accumulation in tumour immunity using the in vivo dual transfer system.
From: Targeting REGNASE-1 programs long-lived effector T cells for cancer therapy
a, Diagram of the in vivo dual transfer system. OT-I cells transduced with sgRNA viral vectors expressing distinct fluorescent proteins were mixed and transferred into the same tumour-bearing hosts, in which further analyses were performed. b, Gating strategy for sgRNA-transduced OT-I cell analysis. c, d, OT-I cells transduced with non-targeting control sgRNA (mCherry+) were mixed at a 1:1 ratio with cells either transduced with control sgRNA (ametrine+) (c (n = 2), d (n = 5), left, top) or two different sgRNAs targeting Regnase-1 (Regnase-1 sgRNA, ametrine+, c (n = 4), left, bottom; or Regnase-1 sgRNA no. 2, ametrine+, d (n = 5), left, bottom), and transferred into tumour-bearing hosts. Mice were analysed at 7 days after adoptive transfer for the proportion of OT-I cells in CD8α+ cells (c, d, left), and the quantification of relative OT-I cell percentages in CD8α+ cells (normalized to input) in the spleen and TILs (c, d, right). Numbers in plots indicate the frequencies of OT-I cells. e, OT-I cells transduced with control sgRNA (ametrine+) were mixed at a 1:1 ratio with cells transduced with Regnase-1 sgRNA (mCherry+) and transferred into tumour-bearing hosts (n = 5). Mice were analysed at 7 days after adoptive transfer for the proportion of OT-I cells in CD8α+ cells (left), and the quantification of relative OT-I cell percentage in CD8α+ cells (normalized to input) in the spleen and TILs (right). Numbers in plots indicate the frequencies of OT-I cells. f, Insertion and deletion (indel) mutations after CRISPR targeted disruption in OT-I cells transduced with either control sgRNA or Regnase-1 sgRNA, via deep sequencing analysis of indels generated at the exonic target site of the Regnase-1 gene, including 97.3% of indel events in Regnase-1-sgRNA-transduced cells isolated from tumours compared to 1.3% in control-sgRNA-transduced cells. Mean ± s.e.m. (c–e). ***P < 0.001. Two-tailed unpaired Student’s t-test (d, e). Data are representative of two independent experiments (e).
