TLT-1 injection in immunocompetent mice suppresses CD8 T cells in vivo. (A) The subcutaneous B16F10 tumor model experiment and recTLT-1 administration strategy shown as a schematic. Age- and gender-matched control mice were injected subcutaneously with the tumor vehicle (HBSS). After 1 wk of tumor growth, one group of mice (n = 8) were injected with recTLT-1 once per week, while the other group (Veh, n = 8) received the vehicle. (B–D) The peripheral blood T cells (TCRb + ve) were analyzed from whole blood for circulating CD8⁺ fraction and for intracellular IFNγ levels after recTLT-1 or vehicle treatments (injections). The peripheral blood mononuclear cells from blood were stained for TCRb-FITC (T cell marker), CD8-Alexa-405, and IFNγ-PE or its isotype control-PE. The TCRb + cells were gated for CD8⁺ fractions as shown in B, and these CD8 T cells were subgated for IFNγ-positive cells in C for all three groups after treatment. The representative contour plots in B and the quantitations (C and D) are shown. Statistical significance tested by Mann–Whitney test. (E and F) The mice (control, tumor [VEH], or tumor [TLT-1]) were sacrificed, and harvested spleens were observed to be of different sizes as shown by whole spleen representative images (E) and weight (n = 4 per group; F). The scale bar equals 10 mm. Statistical significance tested by Mann–Whitney test. (G and H) The spleen cells were activated with anti-CD3/CD28 (H, top panel) or left unstimulated (H, bottom panel). The IFNγ-producing CD8⁺ T cells (%; G) and CD4⁺ T cells from splenic cultures were quantified, and contour plots for CD8 T cells are shown with outliers (n = 3–4 per group), while the CD4 T cell data are presented in Fig. S2. The FC gates show the percentage of cells, and quantitation are presented as scatter graphs with mean ± SEM and analyzed by non-parametric Mann–Whitney test or Kruskal–Wallis test as appropriate. Significant differences are indicated with asterisks (*, P < 0.05; **, P < 0.01). The data represent a minimum of three independent experiments.