Figure 3.
Figure 3. The HERV-K18.1 SAg is necessary and sufficient for thymocyte deletion affecting primarily Vβ7CD4 cells. With the deletion assay, the capacity of the wt and mutant HERV-K18 SAg to negatively select deletion-sensitive human Vβ7 thymocytes was addressed. (A) Coculture of HERV-K18 SAg transfectants with freshly isolated thymocytes decreased percentages and absolute numbers of immature thymocytes (Supplemental Figure 1). Thymocytes were cultured with increasing numbers of vector- and SAg-transfected APCs (A20) and were subsequently analyzed for CD4/CD8 expression after the exclusion of dead cells. The percentages of DN, DP, SPCD4, and SPCD8 thymocytes are indicated in each quadrant. Respective reductions in absolute thymocyte numbers for 5 independent deletion assays are shown in Supplemental Figure 1. (B) Negative thymocyte selection was SAg dependent because it was present in independently generated SAg transfectants but was lost on SAg mutation. Thymocytes were cultured in the presence of vector-transfected A20 cells (horizontal bars), of A20 cells expressing the HERV-K18.1 SAg frameshift mutant (Δ53; ♦), of 2 distinct batches of HERV-K18.1 wt SAg-transfected A20 cells (K18.1 SAg1a+b; ▴), and of an independently generated second wt SAg transfectant (K18.1 SAg2; ▪). mt indicates mutant. (C) HERV-K18 SAg–induced negative selection affects primarily Vβ7CD4 thymocytes, when the extent of deletion (Figure 3C) and the specificity of deletion (Figure 3D) are jointly taken into account. Vβ use of DP and SP thymocytes from 5 donors was determined after 48-hour culture with vector and HERV-K18.1 SAg transfectants. Reductions obtained for SAg, compared with the vector control, are indicated in percentages for immature DP and mature SP thymocytes, and statistically significant results are annotated (t test, *P < .05; **P < .01). ♦ represent the mean ± 1 standard deviation of values obtained from 5 donors. (D) Absolute values of panel C are shown, and significant results are annotated (t test, *P < .05; **P < .01). Open and filled symbols represent individual values; horizontal bars represent the mean.

The HERV-K18.1 SAg is necessary and sufficient for thymocyte deletion affecting primarily Vβ7CD4 cells. With the deletion assay, the capacity of the wt and mutant HERV-K18 SAg to negatively select deletion-sensitive human Vβ7 thymocytes was addressed. (A) Coculture of HERV-K18 SAg transfectants with freshly isolated thymocytes decreased percentages and absolute numbers of immature thymocytes (Supplemental Figure 1). Thymocytes were cultured with increasing numbers of vector- and SAg-transfected APCs (A20) and were subsequently analyzed for CD4/CD8 expression after the exclusion of dead cells. The percentages of DN, DP, SPCD4, and SPCD8 thymocytes are indicated in each quadrant. Respective reductions in absolute thymocyte numbers for 5 independent deletion assays are shown in Supplemental Figure 1. (B) Negative thymocyte selection was SAg dependent because it was present in independently generated SAg transfectants but was lost on SAg mutation. Thymocytes were cultured in the presence of vector-transfected A20 cells (horizontal bars), of A20 cells expressing the HERV-K18.1 SAg frameshift mutant (Δ53; ♦), of 2 distinct batches of HERV-K18.1 wt SAg-transfected A20 cells (K18.1 SAg1a+b; ▴), and of an independently generated second wt SAg transfectant (K18.1 SAg2; ▪). mt indicates mutant. (C) HERV-K18 SAg–induced negative selection affects primarily Vβ7CD4 thymocytes, when the extent of deletion (Figure 3C) and the specificity of deletion (Figure 3D) are jointly taken into account. Vβ use of DP and SP thymocytes from 5 donors was determined after 48-hour culture with vector and HERV-K18.1 SAg transfectants. Reductions obtained for SAg, compared with the vector control, are indicated in percentages for immature DP and mature SP thymocytes, and statistically significant results are annotated (t test, *P < .05; **P < .01). ♦ represent the mean ± 1 standard deviation of values obtained from 5 donors. (D) Absolute values of panel C are shown, and significant results are annotated (t test, *P < .05; **P < .01). Open and filled symbols represent individual values; horizontal bars represent the mean.

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