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Article Figures & Data

Figures

  • Fig. 1.

    Human thymus repopulation in NOD/SCID mice.

    Conditioning refers to radiotherapy only. Typical phenotypic profile of a poorly reconstituted thymus of NOD/SCID mice injected with CD3-depleted UCB 12 to 15 weeks earlier. Gates were set on viable human CD45+ cells.26

  • Fig. 2.

    Influence of TM-β1 on human cell engraftment in NOD/SCID mice.

    Percentages (A) and absolute numbers (B) of human CD45+cells within murine organs—bone marrow (BM), spleen (SPL), mesenteric lymph nodes (MLN), peripheral blood (PB), and thymus (THY)—of NOD/SCID mice injected with CD3-depleted human UCB 12 to 15 weeks earlier. Mouse conditioning consisted of radiotherapy alone (░) or radiotherapy and injection with TM-β1 (▪). Each bar represents the mean ± SD of 28 mice (RT) or 39 mice (RT + TM-β1), respectively. Calculation of absolute numbers of cells was performed as described before.26 Differences in engraftment between 2 groups were significant for all organs (P < .0005 for bone marrow, spleen, mesenteric lymph nodes, and peripheral blood,P = .002 for thymus).

  • Fig. 3.

    Phenotypical analysis of human thymocytes recovered from NOD/SCID mice.

    Conditioning indicates radiotherapy + TM-β1. Unless otherwise indicated, gates were set on viable human CD45+ cells (A, B, C, E, H, I). Within this population subsequent maturational stages were identified: CD4+CD8+ (68%), CD3+CD69+ (13%), CD3+CD27+ (7%), and CD3+CD1 (2%). Virtually all CD3+cells were TCR-αβ+. (D) Gated on CD45+CD3+ cells. (F, G) Gated on CD3+CD1 cells, as indicated.

  • Fig. 4.

    Vβ-Cβ RT-PCR heteroduplex analysis of thymus and bone marrow.

    RNA was isolated from thymus (sorted CD45+CD3+CD27+CD69+fraction) (A) and bone marrow (total, unsorted) (B), and after reverse transcription cDNA was amplified with PCR. On heteroduplex analysis of the thymus, polyclonal smears can be observed for most Vβ families. Bone marrow shows a pauciclonal pattern superimposed on polyclonal background smears.

  • Fig. 5.

    Peripheral T cells after injection of CD34+CD3 UCB cells.

    (A, B) Expression of CD34 and CD3 before (CD34+-enriched UCB cells) and after cell sorting, respectively, is shown. Presence of human CD3+ T cells in murine thymus (C) and spleen (D) 12 weeks after injection of CD34+CD3 cells is shown. Plots are gated on human CD45+ cells.

  • Fig. 6.

    Phenotypical analysis of peripheral human cells.

    Phenotypical profile of CD45RO and CD45RA surface markers on CD3+ cells harvested from spleen from NOD/SCID mice pretreated with radiotherapy and TM-β1. Plots representative for 6 of 13 mice (A) and 7 of 13 mice (B, C) are shown. (A-C) Gated on CD3+ cells. (D, E) Gated on CD45+cells. Monocytes (CD14+), B cells (CD19+), and T cells (CD3+) were present in the periphery of all 10 mice.

  • Fig. 7.

    TREC analysis on human thymocytes and peripheral T cells harvested from mice injected with purified CD34+ cells.

    Real-time PCR quantification of TRECs was performed on human CD3+ cells harvested from several organs of mice injected with purified CD34+ UCB cells 3 months earlier. CD3+CD45RAlow and CD45+CD3+CD45RA+ cells sorted from spleen and mesenteric lymph nodes (MesLN) and CD45+CD3+CD27/69+ cells sorted from the thymus.

Tables

  • Table 1.

    Success rate of human thymopoiesis according to mouse strain and conditioning

    N (mice)NOD/SCID RTNOD/SCID RT + TM-β1Rag2−/−γc−/− mice RT
    Total283925
    DP in thymus (no.)5224
    Success rate (%)18566
    • Comparison of thymus repopulation frequency between irradiated NOD/SCID mice (RT), irradiated NOD/SCID mice pretreated with injection of TM-β1 (RT + TM-β1), and irradiated Rag2−/−γc−/− mice (RT). Of each group, the total number of mice and number/percentage of mice having DP (≥ 5%) in their thymi are shown.

  • Table 2.

    Kinetics of appearance of peripheral T cells

    Time after injection (w)
    4-78-1112-15
    n (total)305140
    n (T cells +)0817
    % (T cells +/total)01643
    • Mice injected with CD3-depleted UCB were bled at several time points (4-7 weeks, 8-11 weeks, and 12-15 weeks). Percentage of mice with human T cells in the peripheral blood increased up to 15 weeks.

  • Table 3.

    Cytokine production after stimulation of human T cells from spleen and thymus

    ExperimentIL-2 (pg/mL)IL-4 (pg/mL)IFN-γ (pg/mL)
    144.53.0316.5
    213.321.9289.0
    3157.513.5424.0
    • Human cells were cultured from murine spleen by stimulation with PHA. After washing, these cells were stimulated with anti-CD3 + TPA or ConA. Supernatants were harvested 24 hours later, and cytokine content was measured. Data shown are from 3 mice injected from the same UCB donor, and numbers shown are averages from 2 stimulation methods.