Figure 6.
Figure 6. IL-7R–deficient F5 T cells fail to proliferate or survive in replete hosts. Splenocytes from F5 TreIL-7R donors taken off dox food 3 days previously and F5 controls were stimulated with NP68 peptide for 72 hours followed by a further 4-day culture in IL-2. Blasts from cultures of F5 TreIL-7ROFF and control F5 T cells were labeled with CFSE, mixed in equal numbers, and 2 × 107 total cells transferred into either Rag1–/– (n = 3), F5 Rag1–/– Ly5.1 recipients (n = 3), or fully lymphoreplete tolerant bone marrow chimeras (see “Materials and methods”) (C57BL6/J bm chim, n = 3). Recipient mice were bled at different times after transfer and analyzed for CFSE profile, CD8, TCR, and Ly5.1 expression. (A) Dot plots are of Ly5.2 versus Ly5.1 staining by CD8+TCRhi cells, and the square indicates the gate used to identify Ly5.2-positive donor cells. Plots of Ly5.1 versus CFSE are gated on CD8+TCRhi Ly5.2-positive cells for days 1, 11, 19, and 40 after transfer. (B) Mean divisions were calculated from CFSE profiles of F5 control (•) and F5 TreIL-7ROFF cells (○) transferred into Rag1–/– or F5 Rag1–/– Ly5.1 recipients. (C) Graphs show the frequency of F5 control (•) and F5 TreIL-7ROFF cells (○) in PBLs of Rag1–/– or F5 Rag1–/– Ly5.1 and C57BL6/J bone marrow chimera recipients. (D) Graphs show unmanipulated chimerism between F5 control and F5 TreIL-7ROFF cells in Rag1–/– (•), naive F5 Rag1–/– Ly5.1 hosts (○), and C57BL6/J bone marrow chimera recipients (▪) and adjusted chimerisms that exclude the expansive effects of proliferation as predicted by a population CFSE profile (see “Materials and methods”).

IL-7R–deficient F5 T cells fail to proliferate or survive in replete hosts. Splenocytes from F5 TreIL-7R donors taken off dox food 3 days previously and F5 controls were stimulated with NP68 peptide for 72 hours followed by a further 4-day culture in IL-2. Blasts from cultures of F5 TreIL-7ROFF and control F5 T cells were labeled with CFSE, mixed in equal numbers, and 2 × 107 total cells transferred into either Rag1/ (n = 3), F5 Rag1/ Ly5.1 recipients (n = 3), or fully lymphoreplete tolerant bone marrow chimeras (see “Materials and methods”) (C57BL6/J bm chim, n = 3). Recipient mice were bled at different times after transfer and analyzed for CFSE profile, CD8, TCR, and Ly5.1 expression. (A) Dot plots are of Ly5.2 versus Ly5.1 staining by CD8+TCRhi cells, and the square indicates the gate used to identify Ly5.2-positive donor cells. Plots of Ly5.1 versus CFSE are gated on CD8+TCRhi Ly5.2-positive cells for days 1, 11, 19, and 40 after transfer. (B) Mean divisions were calculated from CFSE profiles of F5 control (•) and F5 TreIL-7ROFF cells (○) transferred into Rag1/ or F5 Rag1/ Ly5.1 recipients. (C) Graphs show the frequency of F5 control (•) and F5 TreIL-7ROFF cells (○) in PBLs of Rag1/ or F5 Rag1/ Ly5.1 and C57BL6/J bone marrow chimera recipients. (D) Graphs show unmanipulated chimerism between F5 control and F5 TreIL-7ROFF cells in Rag1/ (•), naive F5 Rag1/ Ly5.1 hosts (○), and C57BL6/J bone marrow chimera recipients (▪) and adjusted chimerisms that exclude the expansive effects of proliferation as predicted by a population CFSE profile (see “Materials and methods”).

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