Fig. 6.

Cell-intrinsic defects in lymphocyte development, surface marker expression, survival, and proliferation. (A) Lethally irradiated Rag1 mutant recipients (CD45.2⁺) were transplanted with an equal mixture of wild-type (CD45.1⁺) and heterozygous or homozygous Kdelr1 mutant (CD45.2⁺) bone marrow. Wild-type donor chimerism (CD45.1⁺) was measured 8 wk later in the spleen, bone marrow, and thymus. Bone marrow subsets were gated as follows: preproB (B220⁺IgM⁻), immature (B220^intIgM⁺), mature (B220^hiIgM⁺). Thymic subsets: ETP (Lin⁻CD44⁺CD25⁻CD117⁺), DN2 (Lin⁻CD44⁺CD25⁺), DN3 (Lin⁻CD44⁻CD25⁺), DN4 (Lin⁻CD44⁻CD25⁻), DP (CD4⁺CD8α⁺), CD4SP (CD4⁺CD8α⁻), and CD8SP (CD4⁻CD8α⁺). Lineage markers were CD11b, CD3ε, B220, Ter119, Ly6G, NK1.1, and CD8α. Spleen subsets: T1, T2, Fo, and MZ B cells were gated as in Fig. 5. γδT (CD3ε⁺γδTCR⁺), NKT (NK1.1⁺CD3ε⁺), NK (NK1.1⁺CD3ε⁻), B (CD19⁺), eosinophils (Lin⁻CD11b⁺F4/80⁺SSC^hi), macrophages (Lin⁻CD11b⁺F4/80⁺SSC^lo), and neutrophils (Lin⁻CD11b⁺F4/80⁻Ly6G^hi). Lineage markers included CD19, TCRβ, NK1.1, and the viability dye 7-AAD. (B) Geometric mean surface expression of CD44, CD3ε, and TCRβ on CD4⁺ and CD8⁺ cells in mixed bone marrow chimeras. Surface IgM was measured on CD19⁺ cells. Histogram overlays show relative expression of CD44 on CD4⁺ and CD8⁺ cells. (C) Thymectomized mice were bled at the indicated weeks after surgery, and frequencies of CD4⁺, CD8⁺, and CD19⁺ cells were normalized to the average value in nonthymectomized littermates (set as 1). (D) Mice were injected with the thymidine analog EdU 4 h before organ harvest, and the percentages of EdU⁺ cells within various T-cell subsets was quantified. Asterisks represent P values less than 0.05 as determined by unpaired t tests (only performed at week 8 time point in C). Each symbol in D represents an individual mouse. Data are representative of one (C and D) or two (A and B) experiments, with at least four mice per genotype (error bars represent SEM).