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. Author manuscript; available in PMC: 2020 May 12.
Published in final edited form as: J Immunol. 2008 Nov 15;181(10):6975–6983. doi: 10.4049/jimmunol.181.10.6975

FIGURE 6.

FIGURE 6.

Expression of E8I-CD4 augments TCR signaling and improves the homeostasis of MHC-II-restricted CD8 T cells. A, Intracellular calcium flux in E8I-CD4 CD8 lineage T cells. Single-cell suspensions of LN T cells from WT and E8I-CD4 mice were loaded with Indo-1 dye, washed, and stained with fluorochrome-conjugated Abs to CD4 (RM4.5), CD8a (53–6.72), and 5 μg/ml of biotinylated anti-TCRβ (H57–59) with or without 1 mg/ml of biotinylated anti-CD4 (GK1.5). TCR signaling was initiated in stained cells by cross-linking with avidin after 30 s (arrowheads) of being loaded onto the flow cytometer at 37°C. Data are representative of two independent experiments. B, E8I-CD4 transgene expression on MHC-II-restricted CD8 lineage T cells generated in 8DP4 experimental mice. To assess E8I-CD4 transgene expression in MHC-II-specific CD8 T cells when such cells were generated during thymic selection, we introduced the E8I-CD4 transgene into 8DP4 experimental mice, which were on a CD40β2m0 background. As previously documented (15), MHC-II-restricted CD8 lineage T cells develop in 8DP4 experimental mice despite their expression of mismatching MHC-II TCR and CD8 coreceptors. Thymocytes were stained for CD4, CD8, CD24, and CD5. Mature CD8 lineage cells (i.e., CD8+CD24low) from 8DP4 mice are shown in green, while mature CD8 lineage cells from E8I-CD4 + 8DP4 mice are shown in red. Numbers above the two-color histograms indicate total numbers of cells, while numbers in boxes indicate the frequency (percent) of cells in that box (left panels). Numbers in the CD5 histogram (right panel) indicate CD5 mean fluorescent intensity (MFI). Data are displayed on a 4-decade log scale and are representative of three independent experiments. C, Absolute numbers of TCRβhighCD8+ thymocytes and TCRβ+CD8+ LN T cells in WT (black bar), 8DP4 experimental mice (green bar), and 8DP4 experimental mice expressing the E8I-CD4 transgene (red bar). Data are average ±SEM) of three mice per group and were compared with a two-tailed Student’s t test. N.S. indicates not significant (p > 0.05).