Gal-1 opposed partial agonist-driven positive selection of conventional CD8⁺ T cells. (A) CD8 versus CD4 expression profiles of thymocytes from wild-type (left) and gal-1^−/− (right) female H-Y TCR transgenic mice. Percentage of cells in each subset is denoted in its respective quadrant, and the number of CD8⁺ SP thymocytes is in parentheses. (B) Flow cytometric histograms of CD3 (left) and HSA (right) staining of DP (top) and CD8⁺ SP (bottom) thymocytes from wild-type (shaded) and gal-1^−/− (thin line) mice. Note change in y-axis between histograms. (C) The total number of CD8⁺ SP thymocytes from wild-type (n = 4) and gal-1^−/− (n = 4) female H-Y TCR mice. Each point represents one mouse and was determined by multiplying the percentage of CD8⁺ SP thymocytes by the total number of thymocytes from each individual mouse throughout 4 independent experiments. The horizontal bar represents the mean. (D) Flow histograms of CD8 staining of splenocytes from wild-type (left) and gal-1^−/− (right) female H-Y TCR mice. The numbers correspond to the percentage and total number of cells in the marker M1, which were set based on an isotype control and represent the CD8⁺ population. All dot plot profiles and histograms are representative of 4 independent experiments. (E) The total number of CD8⁺ T cells from spleens of wild-type (n = 4) and gal-1^−/− (n = 4) female H-Y TCR mice. Each point represents one mouse, and the total number of CD8⁺ T cells was determined as described in panel C over the course of 4 independent experiments. (F) The total number of CD8⁺ SP thymocytes (left) and splenic CD8⁺ T cells (right) from wild-type (n = 4) and gal-1^−/− (n = 5 for thymus, n = 4 for spleen) OT-1 TCR transgenic mice. Each point represents one mouse and was determined as described in panel C over 4 independent experiments. (G) Histograms of CD25 staining of unstimulated (left) and anti-CD3/anti-CD28–stimulated (right) enriched CD8⁺ T cells (80%-90% purity) from wild-type (shaded) and gal-1^−/− (thin line) OT-1 TCR mice. Flow profiles are representative of 2 independent experiments. (H) Percent of CD8⁺ SP thymocytes from C57Bl/6 fetal thymii cultured (FTOC) in the presence ( and ■) or absence (□) of gal-1 inhibitor. (I) CD4:CD8 thymocyte ratio from a single FTOC experiment treated with or without gal-1 inhibitor. FTOC results are representative of 3 independent experiments. For panels C and E, statistical significance was determined by 2-tailed paired Student t tests. For panel F, one-tailed unpaired Student t tests were performed for OT-1 studies because these experiments were done to corroborate the female H-Y studies in which a hypothesis was previously established. P values are listed in panels.