(A) Representative macroscopic appearance of thymic implant in humanized mouse. NOD/SCID mice that received Thy/Liv/CD34⁺ FLCs were sacrificed to evaluate the development of human thymocytes in human thymic grafts 18 weeks after implantation. The humanized mouse graft shown in the photomicrograph contained 210×10⁶ thymocytes. (B) Phenotype of thymocytes in thymic grafts of humanized mouse (right) and human fetal thymus (left) are shown. (C) Comparison of phenotypic distribution of CD4 or CD8 single positive T cells of thymus graft and PBMC of the same animals. Humanized mice (n = 4) were sacrificed 21 weeks post-transplantation. Mononuclear cells of thymic graft and blood were isolated from each individual mouse by ficoll separation. Single-cell suspensions were stained for markers of T cells (CD4 and CD8) and phenotypic markers of T cells (CD45RA, CD45RO and CCR7), and analyzed by FCM. Open bars and closed bars represent distribution of indicated phenotypic subpopulations (CD45 RO−RA+, CD45 RO+RA−, CD45 RO+RA+ and CCR7+ cells) among CD4 and CD8 single positive T cells in thymus grafts and PBMC, respectively. All data are expressed as mean ± SEM. Experiments were repeated twice with similar results. *** p < 0.001, comparing thymus grafts and PBMC (two-way ANOVA, Bonferroni test). (D) Phenotypes and Tregs of CD4 single positive thymocytes in thymic grafts of humanized mouse (lower panels) and human fetal thymus (upper panels). The thymic graft specimen shown in (A) and human fetal donor thymus were analyzed. The bold line represents staining of the indicated gated subpopulations with fluorochrome-labelled mAb for the indicated molecule. The filled area represents staining with isotype control mAb. Representative profiles are shown. (E) Helios expression in human CD45+CD4+CD8−CD25+CD127lo thymocytes was assessed by flow cytometry in the thymic graft of humanized mice 18 weeks post-transplant. Representative FCM profile is shown (left) and total FoxP3+Helios+ and FoxP3+Helios− cells were calculated from n=2 (right).

Humanized mice (closed circles in B and C, n = 26) or healthy human volunteers (open circles in B and C; n = 6) were bled to measure distribution of regulatory T cells and their phenotype in PBMCs. Blood samples of humanized mice were obtained 16–18wk post-transplantation and isolated PBMC were stained for CD4, CD8, CD25, CD127, CD45RA, CD45RO and HLA-DR. Samples were fixed and further stained intracellulary for FoxP3. (A) Phenotypic characteristics of regulatory T cells in PBMC from representative healthy adult human (upper row) and humanized mouse (lower row), respectively. CD4⁺CD25⁺FoxP3⁺ human T cell populations were gated and their surface expression of CD45RA, CD45RO and HLA-DR is shown in the histograms. The bold line represents staining with fluorochrome-labelled mAb for the indicated molecule, and the filled area represents staining with isotype control mAb. (B and C) Comparison of proportion of CD25⁺CD127⁻ cells among total CD4⁺ T cells, FoxP3⁺ cells among CD4⁺CD25⁺CD127⁻ T cells, and of CD25⁺FoxP3⁺ regulatory T cells among total CD4⁺ T cells (B) and proportion of CD45RO⁺CD25⁺FoxP3⁺ activated regulatory T cells among total CD4⁺ T cells (C) in PBMC of humanized mice (closed circles) or healthy adult human volunteers (open circles). Each circle represents results from an individual animal or donor and each line represents mean value. Results are shown from the combination of three similar experiments. *** p < 0.001, comparing humanized mice and human volunteers (Mann-Whitney test).

Humanized mice were sacrificed 21 weeks post-transplantation and mononuclear cells of spleen, LNs, thymic grafts and blood isolated from each individual mouse were analyzed by FCM. Mononuclear cells from each organ were isolated by ficoll separation and analyzed by FCM as in Figure 3. (A) Comparison of proportion of CD25⁺FoxP3⁺ regulatory T cells among CD4⁺ T cells in indicated organs. Each circle represents cells from an individual animal or donor. (B) Phenotypic characteristics of CD4⁺CD25⁺FoxP3⁺ regulatory T cells in indicated organs from representative humanized mouse. Histograms show expression of each indicated molecule. The bold line represents staining with fluorochrome-labelled mAb for the indicated molecule, and the filled area represents staining with isotype control mAb. (C) Comparison of phenotypic distribution of regulatory T cells among CD4⁺CD25⁺FoxP3⁺ cells. Each circle represents a result from an individual animal. * p < 0.05, ** p < 0.01, *** p < 0.001, comparing indicated combination (Kruskal Wallis test, Dunn’s multiple comparison test). (D) Natural Tregs were assessed in splenic (SPL) and peripheral lymph node (PLN) populations by flow cytometry, gating on human CD45+CD4+CD8−CD25+CD127lo and analyzing FoxP3+ cells for expression of Helios as shown (right panels). Total cells are calculated in FoxP3+Helios+ and FoxP3+Helios− cells from n=4 humanized mice.

Panel (A) shows a representative phenotypic profile and purity of CD4⁺CD25⁻ (responders, left) and CD4⁺CD25⁺CD127⁻ (suppressors, middle) T cells of a humanized mouse after FACS sorting. The frequency of CD25⁺CD127⁻ cells among gated CD4 single positive T cells in each cell fraction is shown. Right panel shows intracellular FoxP3 expression of the indicated fraction. The shaded line represents the staining with isotype control mAb and the thick line and dashed line represent the test staining of suppressor and responder cells, respectively. (B and C) In vitro suppression of proliferation of CD4⁺CD25⁻ T cells (responders) by FACS-sorted CD4⁺CD25⁺CD127⁻T cells (suppressors) isolated from normal human PBMCs or pooled spleen and lymph nodes of humanized mice 20 wks post-transplantation. CD4⁺CD25⁻ T cells (2×10⁴) isolated from human PBMC or pooled spleen and LN cells of 3 humanized mice were incubated with syngeneic CD4⁺CD25⁺CD127⁻ T cells in indicated ratios in the presence of plate-bound anti-CD3 mAb (1 μg/ml) and soluble anti-CD28 mAb (2.5 μg/ml) for 4 days (B) or in the presence of 30Gy-irradiated allogeneic PBMC as stimulators for 5 days (C). Cultures were pulsed with [³H]thymidine at day 3 (B) or 4 (C) and harvested 16 h later. Data are expressed as mean of triplicates.