Figure 2.

nfκb1^−/− CD8SP thymocytes acquire memory markers independently of the IL-4 producing PLZF⁺ population. (A) Expression of PLZF in wt and nfκb1^−/− thymocyte subsets. Data represent four mice per genotype. (B) CD4 and CD8 expression by wt and nfκb1^−/− TCRγδ⁺ thymocytes. Mean (±s.e.m.) numbers of wt and nfκb1^−/− TCRγδ⁺CD4SP thymocytes, determined by total numbers of TCRγδ⁺ thymocytes and proportion of TCRγδ⁺CD4SP cells (n=3–4 mice per genotype). (C) Expression of IL-4 and IFN-γ in wt, nfκb1^+/− and nfκb1^−/− thymocytes 2 h after stimulation with PMA plus ionomycin. NKT cells (CD1d tetramer⁺ TCRβ⁺) stained with CD1d tetramer loaded with PBS-44 were analysed as an internal positive control for IL-4 and IFN-γ (left panel). CD4SP thymocytes gated on TCRβ⁺ cells were examined for IL-4 and IFN-γ (right panel). (D) Percentages (mean±s.e.m.) of IL-4-producing CD4 T cells and NKT cells from thymuses of wt (n=4), nfκb1^+/− (n=2) and nfκb1^−/− (n=7) mice. Results are representative of three (B) and four (A, C, D) experiments. (E) Expression of Eomes, CD122, CD44 and CD24 by wt (Ly5.1⁺) CD8SP thymocytes (bold) isolated from wt+ (wt Ly5.1⁺) (shaded histograms) or nfκb1^−/− + (wt Ly5.1⁺) (black lines) chimera mice. Memory phenotype of nfκb1^−/− CD8SP cells from intact nfκb1^−/− mice (hatched lines) served as concurrent positive control. Data are representative of three different chimera cohorts (n>6 mice per group). BM chimera mice were established by engrafting wt (Ly5.2⁺) hosts with a mix (50:50) of nfκb1^−/− and wt (Ly5.1⁺) haematopoietic cells. (F) NKT cells (CD1d tetramer⁺ TCRβ⁺) and CD4 T cells (TCRβ⁺CD4⁺CD8⁻) were assessed for IL-4 and IFNγ expression. Thymocytes were isolated from chimeras and stimulated in vitro with PMA and ionomycin for 2 h and then analysed for intracellular levels of IL-4 and IFN-γ by flow cytometry. Wt and nfκb1^−/− thymocytes were defined as Ly5.1⁺ and Ly5.1⁻, respectively (left panel).