Ligation of CD8 leads to apoptosis of thymocytes that have not undergone positive selection

Abstract

Thymocytes that are not positively selected are said to undergo "death by neglect." We have found that ligation of CD8, either by antibodies or MHC class I molecules, induces apoptosis of CD4(+)CD8+ double-positive (DP) thymocytes. The susceptibility of thymocytes to CD8-mediated apoptosis is developmentally regulated and confined to a subpopulation of DP thymocytes. Stimulation through CD3 protects thymocytes from CD8-mediated apoptosis. We suggest that during thymocyte development, binding of CD8 to MHC class I molecules without T cell receptor engagement induces apoptosis in immature DP thymocytes. Our data are consistent with a model in which thymocytes that do not survive positive selection undergo "death by instruction" instead of death by neglect.

Fig. 1.

Antibody crosslinking of CD8, but not CD4, induces annexin V binding on a subset of thymocytes. Thymocytes from MHC^–/– mice were treated with antibodies to either CD4, CD8α, or CD8β and goat anti-rat Ig, dexamethasone, DMSO, or were left untreated (as indicated in the top left corner of the plot) for 6 h. Cells were stained with FITC-conjugated annexin V and PI. Annexin V binding is shown on the x axis, and PI staining is shown on the y axis. In each plot, the percentage of cells in the gate is given.

Fig. 2.

Ligation of CD8 with TL or H-2K^b induces apoptosis. Thymocytes from MHC^–/– mice were treated with microspheres coated with wild-type (H-2K^b) or mutant (H-2K^bLys-227) H-2K^b molecules; microspheres coated with TL molecules either in the absence (TL) or presence of CD8-blocking antibody (anti-CD8/TL); biotinylated antibody to CD8a and streptavidin-coated microspheres (CD8-XL); or microspheres coated with IE^k for 30 min. Cells were stained with FITC-annexin V and PI. Annexin V binding is shown on the x axis, and PI staining is shown on the y axis. The number in each plot represents the percentage of cells in the gate.

Fig. 3.

CD8-mediated apoptosis is developmentally regulated. Thymocytes from B6 or MHC^–/– mice were incubated with antibody to CD8 under crosslinking conditions or beads were coated with MHC class I molecules (gray bars) or were left untreated (black bars), and stained with FITC-annexin V and PI. The percentage of annexin⁺ cells was determined by flow cytometry. The error bars represent the SD of at least three mice per group. (A) Thymocytes were treated with antibody to CD8α and goat anti-rat Ig for 6 h. (B) Thymocytes were treated with TL-coated microspheres for 30 min.

Fig. 4.

Thymocytes susceptible to CD8-mediated apoptosis are prepositive selection. DP thymocytes from C57BL/6 mice were sorted based on either CD5, CD69, or CD2 expression. Cells were incubated with antibody to CD8 under crosslinking conditions or beads were coated with MHC class I molecules (gray bars) or were left untreated (black bars) for 30 min. Cells were stained with FITC-annexin V and the percentage of annexin⁺ cells was determined by flow cytometry. The error bars represent the SD of at least three mice per group. (A) Prepositive selection-sorted cells (CD5^lo; CD69^–; and CD2^lo) stimulated with biotinylated antibody to CD8 and streptavidin-coated microspheres. (B) Postpositive selection-sorted cells (CD5^hi; CD69⁺; and CD2^hi) stimulated with biotinylated antibody to CD8 and streptavidin-coated microspheres. (C) Prepositive selection-sorted cells stimulated with TL-coated microspheres. (D) Postpositive selection-sorted cells stimulated with TL-coated microspheres.

Fig. 5.

PMA or CD3 crosslinking reduces CD8-mediated apoptosis. Thymocytes from MHC^–/– mice were treated with biotinylated antibody to CD8 and streptavidin-coated microspheres or were left untreated (nil) for 30 min. Thymocytes were also treated with PMA, biotinylated anti-CD3, or a biotinylated control antibody (R4GA2) with and without CD8-crosslinking. Annexin V binding is shown on the x axis, and PI staining is shown on the y axis. In each plot, the percentage of cells in the gate is given.