Extended Data Fig. 9: LAG3 deletion has moderate impacts on proliferation, but phenotypically skews cells to an effector, rather than restrained phenotype.
From: Autoreactive CD8+ T cells are restrained by an exhaustion-like program that is maintained by LAG3
The consequences of LAG3 deletion were evaluated by flow cytometry to phenotype intra-islet CD8 + T cells for survival, proliferation, and IR/exhaustion related marker expression. (a-e) Flow cytometry was performed on 8-week-old female Lag3∆TM and Cre Controls taking cells from ndLN, pLN and islets. Data points derived from islets having <40 tetramer+ cells were excluded. Tetramer staining in lymph nodes was negligible and never exceeded 40 Tetramer+ cells. (a) BrdU was injected intraperitoneally 12 hours prior to harvest, and percent BrdU, Ki67, cleaved Caspase 3 (p = .0496), and BCL2 were assessed by flow cytometry (2 independent experiments, n = 6-8 per genotype). (b) CD8 + T cells were labeled with cell trace violet, sorted into 96 well round bottom plate containing 0.05 ug/mL αCD3/CD28, and 200 U/mL IL-2 in cRPMI, and cultured for 60 hours and analyzed by flow cytometry (2 independent experiments, n = 6 per genotype). (c) IRs/markers of restraint (TIGIT, TCF1, PD1, and TOX) expression were quantified on total and tetramer positive CD8 + T cells (3 independent experiments, n = 13-15 per genotype Tetramer: TIGIT, PD1, TOX, p = .0473, 0.0473, 0.0096). (d) Percent expression of effector molecules CD44 (3 independent experiments, n = 13-15 per genotype ndLN, pLN, Islets p = .0016, 0.0007 .0037) and KLRG1 (1 independent experiment, n = 4-5 per genotype p = .036). (e) Percent expression of PD1 and TOX (Nrpv7 + p = .0259, InsB+ p = .0204), CD39 and Eomes (p = .03), double positive populations, markers of exhaustion, were monitored on bulk and tetramer positive ndLN, pLN, and intra-islet CD8 + T cells (3 independent experiments, n = 13-15 per genotype and 2 independent experiments, n = 5-6 per genotype, respectively). (a-e) Each data point corresponds to a single mouse. A two-sided nonparametric Mann-Whitney statistical test was preformed where P = * < 0.05, ** < 0.01, *** < 0.001, **** < 0.0001. Unlabeled indicates not statistically significant. Graphs portray the median.
