(A) Schematic workflow of the BM-niche age influence assay. (B) A representative flow cytometry analysis shows donor congenic marker gates (left panels), and profiles of T-lineage (Thy1.2⁺) vs. myeloid-lineage (Mac1⁺) cells derived from old (18 months old) and young (2 months old) IL7R^−/− host BM niches after culture on OP9-DL1 stromal cell monolayer (right panels). These cells were originally from the same pool of young WT BM progenitors. (C) A summary of panel B in % myeloid-lineage cells (left) and % T-lineage cells (right) derived from old (striped bar) and young (grey bar) IL7R^−/− host BM niches. Data show Mean ± SEM in all bar graphs, n = IL7R^−/− host mouse number. (D) A representative culture result of Panel A shows absolute cell numbers (Mean ± SD) of myeloid cells vs. T-lineage cells derived from old or young IL7R^−/− host BM niche-modulated young WT donor BM progenitors (~2000 sorted LSK cells loaded per well) after 14 days in culture on OP9-DL1 stromal cell monolayer (n = IL7R^−/− host animal #).

(A) Results of competitive repopulation of grafted fetal thymus by “young and old” or “old and young” natural thymus-seeding cells from the first and second hosts. Left panel shows % T-lineage thymocytes (gated on DP, CD4⁺ and CD8⁺ SPs) in the grafted thymus naturally seeded by young (~2 month old, circles) and old (22 months old, triangles) BM progenitors in the different seeding orders (initial seeding: filled circles or triangles; subsequent/second seeding: open circles or triangles). Each triangle or circle represents one animal. An unpaired Student's t-test shows p > 0.05 (no significant). The table in the A panel shows absolute cell numbers per grafted lobe (each host mouse was grafted with 2-3 fetal thymic lobes, n = animal number). (B) A representative result of differentiated CD4⁺ and CD8⁺ T cells from the grafted fetal thymic lobes (bottom panels). The thymocytes are derived from first and second seeded young- and old-BM progenitors, identified by CD45.1 and CD45.2 congenic markers (top panels).

(A) A schematic diagram of workflow. (B) Left panel shows the gross appearance of young IL7R^−/− mouse thymus size from a representative experiment, 5 weeks after infusion with PBS, or equal numbers of 2-, 8-, 12-, 18-, or 22-month-old WT BM cells. Right panel shows a summary of total thymocyte number in young IL7R^−/− host mouse thymus, derived from donor WT mouse BM cells of different ages. (C) Left panel shows the gross appearance of 1- (left column) and 12-month-old (right column) IL7R^−/− mouse thymus size from a representative experiment, 5 weeks after transplantation with equal numbers of ~2-month-old WT BM cells (top row) or PBS (bottom row). Right panel shows a summary of total thymocyte number derived from young donor WT mouse BM cells in IL7R^−/− host niches of different ages. (D) Left panel shows the linear regression of thymocyte number derived from donor WT BM cells of different ages in young IL7R^−/− host niches (blue line, Exp-A) and from young donor WT BM cells in IL7R^−/− host niches of different ages (red line, Exp-B). Test for equal slopes for the blue (slope −3.72 ~ −1.28) and red (slope −7.23 ~ −3.97) gives a (2-sided) p-value of 0.016 (significantly different). Right panel shows donor-derived thymocyte numbers from 11-to-13-month-old donor WT BM cells in young IL7R^−/− host niches (left bar) and young donor WT BM cells in 11-to-13-month-old IL7R^−/- host niches (right bar). Data show mean ± SEM in all bar graphs, n = IL7R^−/− host animal number, each triangle and square in C represents one animal. Experiments were repeated over 5 times.

(A) Thymocyte number in dGUO-treated (top panel) or intact (bottom panel) grafted fetal thymic lobes, at various weeks after transplanted under the kidney capsules of young (~2 months) and old (20 - 22 months) WT mice. The images shown are representative results of grafted thymic lobe size in the hosts? kidney capsules. Each data point (triangle or square) represents 2-3 host mice. (B) A representative result shows differentiation profiles (CD44 vs. CD25 at one-week time point, and CD4 vs. CD8 at all other longer time points) of thymocytes from grafted fetal thymic lobes under the young and old kidney capsules.

(A) Schematic workflow of the comprehensive competitive culture assay showing the recruitment of old and young natural thymus-seeding cells in vivo to the in vitro competitive co-culture on OP9-DL1 monolayer stromal cells. (B) A representative result of % T-lineage cells derived from old (CD45.2⁺) and young (CD45.1⁺) thymus-seeding LPCs after competitive co-culture on OP9-DL1 stromal cell monolayer. (C) A summary of % T-lineage cells derived from old (CD45.2⁺) and young (CD45.1⁺) thymus-seeding LPCs after competitive co-culture on OP9-DL1 stromal cells. (D) A representative flow cytometry dot-plot shows CD4 vs. CD8 profile of T-lineage cells from the grafted fetal thymic lobes 7 days after KCT (left panels); purification of DN cells after negative-selection with beads (middle panels); and CD4 vs. CD8 profile of T-lineage cells after competitive co-culture on OP9-DL1 stromal cells (right panels). (E) A summary of % CD4⁺CD8⁺ (DP) cells derived from old (CD45.2⁺) and young (CD45.1⁺) thymus-seeding LPCs after competitive co-culture on OP9-DL1 stromal cells. Data in C and E panels show mean ± SEM, n = competitive co-culture wells; total host animal number is 5 young and 5 old WT mice. The experiment was conducted 5 times (i.e. 5 FTOC, 5 sorts, and 5 cultures).