Figure 1. Accumulative TCR signaling in naive CD8⁺ T cells is heterogenous during steady-state conditions.

(A) Representative flow cytometry plots of Nur77-GFP fluorescence of naive CD44^LO CD62L^HI CD8⁺ and CD4⁺ cells or CD4⁺ Foxp3-IRES-RFP⁺ T cells. All plots shown are from non-TCR transgenic mice (B) Contour plot (left) shows CD5 and Nur77-GFP expression by total naive polyclonal CD8⁺ T cells. Overlaid histogram (center) depicts GFP fluorescence for GFP^LO and GFP^HI cells. GFP^LO cells are the 10% of cells with the lowest (blue) GFP fluorescence intensity, whereas GFP^HI cells are the 10% of cells with the highest (red) GFP fluorescence intensity. Histogram (right) shows the CD5 expression for GFP^LO and GFP^HI populations. (C) Histograms show Nur77-GFP fluorescence intensities of naive CD8⁺ T cells from WT Nur77-GFP or OT-I-Nur77-GFP-TCRα^−/− mice. The numbers indicate the geometric mean fluorescence intensity (gMFI) calculated for the whole population. (D) Offset histograms show Nur77-GFP expression in naive polyclonal CD8⁺ T cells harvested from the spleen, mesenteric lymph nodes, or Peyer’s Patches. (E) Flow cytometry plots of naive polyclonal CD8⁺ T cells after intravascular labeling of cells in the red pulp by intravenous injection of CD45.2-APC antibody intravenously prior to euthanasia. (F) Histograms show expression of TCRβ and CD8α by polyclonal naive GFP^LO and GFP^HI CD8⁺ T cells. (G) Histograms show the GFP fluorescence intensity of total CD8⁺ T cells (left) or FACS-sorted GFP^LO and GFP^HI cells (middle). A total of 5×10⁵ GFP^LO or GFP^HI (top and bottom 10%) polyclonal CD8⁺ T cells were adoptively transferred into separate WT congenic recipients. Histogram (right) shows GFP fluorescence of transferred T cells seven days post-transfer and gated on naive CD8⁺ T cells and the congenic marker expression. Data represent two (A, C, D, G) to three (B, E, F) independent experiments with n = 2–3 mice.