TIGIT expression in naive T cells is accelerated by coculture with si-tolerant T cells at an early-stimulation stage. TIGIT expression in naive T cells is accelerated by coculture with si-tolerant T cells at an early-stimulation stage. (A) Spleen cells prepared from OVA-TCR-Tg mice receiving in vivo priming with TSST-1 or PBS were restimulated with OVA in vitro (left panel). CD4+CD25+ T cells of activated T cells or si-tolerant T cells were separated by cell sorter (right panel). Numbers indicate the relative percentages of cells in each boxed area. (B) Heat map shows representative inhibitory surface molecule genes differentially expressed in activated T cells (Activate T) and si-tolerant T cells (Si-tolerant T). Total RNA extracts from si-tolerant or activated T cells were each obtained from two experiments and pooled to have sufficient amounts for microarray analysis. (C) FACS analysis of representative inhibitory molecules on CD4+ T cells insi-tolerant T cells (Si-tolerant T, red line) or in activated T cells (Activate T, blue line) after OVA stimulation. Control and Naive represent staining control (dotted line) and unstimulated naive T cells (black line), respectively. Histograms shown are representative of two individual experiments. (D) Left, schematic model for induction of tolerant state into naive T cells for generating the next new si-tolerant T cells. OVA-TCR-Tg spleen cells were stimulated with OVA (0.1 mg/ml) in conventional culture or in coculture with si-tolerant T cells from OVA-GFP mice. Middle, Time course of TIGIT expression in naive T cells that were cultured with OVA (OVA stimulation, red line) in the absence or presence of si-tolerant cells. Upper panels show activated naive T cells in conventional single culture (Activate T). Middle panels show activated naive T cells in coculture with si-tolerant cells (Coculture T). Lower panels show the previously induced si-tolerant T cells that were used for coculture (Si-tolerant T). The black line represents unstimulated naive T cells. The dotted line represents staining control. Histograms show representative three (12 h) or four (24 h) individual mice. Right, Graphs show mean of TIGIT expression of three (12 h) or four (24 h) mice. Each dot represents an individual mouse. MFI, mean fluorescence intensity. (E) Tigit mRNA expression in naive T cells that were stimulated with OVA for 24 h in single culture or in coculture with si-tolerant T cells. Tolerant represents the previously induced si-tolerant T cells that were used for coculture. (F) Tigit mRNA (left) or surface (right) expression in activated naive T cells cocultured with TIGIT-expressed transfectants. BALB/c spleen CD4+ T cells were cocultured with TIGIT-expressing P815 cells (blue), mock transfectants (red), or T cells alone (green) on anti-CD3–coated plates for 24 h. The gray-shaded histogram represents unstimulated naive T cells. The dotted line represents staining control. Data are representative of three independent experiments (E and F) with three mice per experiment. The graph is shown as mean ± SEM of three mice (E and F). *p < 0.05, **p < 0.01, ***p < 0.001, Tukey test (D–F). N.S., not significant. Naoko Negishi et al. ImmunoHorizons 2018;2:338-348 Copyright © 2018 The Authors