Fig. S9.

Characterization of transduced thymocytes in stromal cell culture systems. (A) β-Transduced thymocytes were cultured with OP9-DL4 stromal cells and analyzed for preTCR expression using anti-Cβ mAb H-57 compared with vector-only transduced controls. Levels are quantitated using mean fluorescent intensities (MFIs). Levels in DN and DP stages after 6–7 d in culture are shown. Vector data are not available for DP, because vector-transduced thymocytes do not develop beyond DN3. (A) N15β WT, M2₂, or M2₃ or vector-transduced thymocytes. (B) N30β WT vs. N30β mutants, N15β WT, or vector. (C) N15β WT vs. β-patch mutants or vector. (D and E) AnnexinV levels are inversely correlated with proliferation in OP9-DL4–cultured, β-transduced thymocytes without regard for identity of transductant. (F) Scheme for transduction of B6 Rag2^−/− thymocytes with WT or mutant β within two-color FTOC. Flow cytometry panel shows gating of DP thymocytes before quantitating CFP⁺ (N15β WT; control) or GFP⁺ cells (N15β WT, M2₂, or M2₃; test), allowing for automatic correction for variability because of size or other thymic lobe differences in FTOC. (G) Proportion of GFP⁺ test β-transductants (WT or mutant; green symbols) and CFP⁺ control transductants (WT only; gray symbols) within the DP population of doubly transduced thymocytes (n = 11). Actual percentages are given. Note that fewer M2₂-transduced thymocytes (P = 0.001) after a 7-d FTOC become DP thymocytes relative to WT. In contrast, M2₃ was not found to be significantly different from WT (P > 0.1), and there were no CFP⁺ control samples found to differ between groups (P > 0.1). CFP-containing vector was less effective than GFP-containing vector at supporting β WT development in this system, so that a normalized ratio to WT of 0.702 ± 0.082 for M2₂ and 1.117 ± 0.073 for M2₃ was also calculated from the mutant GFP/WT CFP ratio divided by WT GFP/WT CFP, supporting the diminished developmental progression capacity of M2₂.