Supplementary Figure 4: The redirected CD8⁺ T cells in B2m^–/– chimeras reconstituted with Tcf7^–/–Lef1^–/– BM exhibit true CD8⁺ T cell characteristics.

BM cells from Tcf7^-/-, Tcf7^-/-Lef1^-/-, or littermate controls were transplanted into lethally irradiated CD45.1⁺ congenic β2m^-/- mice. Six weeks later, splenocytes were isolated from the BM chimeras and used for downstream analysis. (a) and (b) The redirected CD8⁺ T cells in the absence of TCF-1 or both TCF-1 and LEF-1 persist in the periphery. Donor-derived CD45.2⁺TCRβ⁺ splenocytes were analyzed for CD4⁺ and CD8⁺ lineage distribution. Representative contour plots (a) are from 4 independent experiments with ≥ 4 recipients analyzed in each experiment. Numbers of mature CD4⁺ and CD8⁺ splenocytes in the BM chimeras are shown in b as means ± s.d. (n ≥ 14). *, p<0.05; **, p<0.01***; p<0.001. (c) The redirected Tcf7^-/-Lef1^-/- CD8⁺ T cells express CD8⁺ T cell-characteristic genes. CD4⁺ and CD8⁺ splenic T cells were sorted from WT C57BL/6 mice, and CD8⁺CD4^– and CD8*4 CD45.2⁺TCRβ⁺ splenocytes were sorted from the Tcf7^-/-Lef1^-/--reconstituted β2m^-/- BM chimeras (Tcf7^-/-Lef1^-/- BM chimeras), followed by gene expression analysis. (d) The redirected Tcf7^-/-Lef1^-/- CD8⁺ T cells proficiently produce granzyme B and interferon-γ upon stimulation. Splenic T cells were isolated from WT B6 mice or Tcf7^-/-Lef1^-/- BM chimeras, and then activated by plate-bound anti-CD3 antibody and soluble anti-CD28 antibody in the presence of IL-2. Three days later, the cells were stimulated with PMA/Ionomycin in the presence of Golgi plug, and then surface-stained for CD40L, intracellularly stained for granzyme B, interferon-γ, and IL-2. For c and d, similar results were obtained for redirected Tcf7^-/- CD8⁺ T cells (not shown).