Fig 1.
Human T cells are the predominant cell population within the peripheral blood in humanized mice.
NRG mice were injected i.v. with 6.5x107-1x108 hPBMCs. (A) All flow cytometric analyses throughout this study were performed by gating on living cells in the FSC/SSC plot, excluding cell doublets in an FCS/FSC-W plot, and gating on hCD45+ cells. (B) Percentages of hCD45+ cells in the peripheral blood of NRG mice were analyzed by flow cytometric analysis before reconstitution (n = 3) or on day 8–16 after reconstitution (n = 73). <; not detectable. (C) Cell composition within hCD45+ cells of freshly isolated hPBMCs (input; n = 6) or peripheral blood (n = 54), spleen (n = 14), PEC (n = 6), LN (n = 9), and thymus (n = 9) of humanized mice on day 8–16 after repopulation was analyzed by flow cytometric analysis. Data shown in (A) are representative for all flow cytometric analyses performed throughout this study. Data shown in (B) are taken from 1–13 independent experiments. Data shown in (C) are taken from 2–7 independent experiments.
Fig 2.
Loss of hCD45+ or hCD3+ cells from the peripheral blood upon TGN1412 or OKT3 treatment.
Humanized mice were injected i.v. with 20 μg OKT3 or 20 μg TGN1412 per 10 grams body weight. (A) Before (black bars) and 2–6 hours post OKT3 (n = 11–12) or TGN1412 (n = 18–20) application (time point of sacrifice; gray bars), percentage and composition of hCD45+ cells in peripheral blood of reconstituted mice were analyzed by flow cytometric analysis. PBS-treated mice (n = 12) were used as control. Each line represents an individual mouse. *** p < 0.001; n.s., not significant (paired t-test for difference after-before). (B) Before and 4 hours post OKT3 application (n = 3) CD3 expression on hCD4+ or hCD8+ T cells in peripheral blood of humanized mice was investigated by flow cytometric analysis. Data from one individual animal, being representative for the indicated groups, are shown. The relative number of hCD4+ or hCD8+ T cells 4 hours after OKT3 application (gray bars) is given as percentages of total hCD4+ or hCD8+ T cells, normalized to “before”. *** p < 0.001 (paired t-test for difference after-before). (C) CD3 expression on human CD4+/CD8+ T cells in peripheral blood of humanized mice was investigated by flow cytometric analysis after OKT3 in vivo application (n = 3; upper panel). CD3 expression on CD4+/CD8+ T cells was investigated by flow cytometry before and after in vitro stimulation of purified human T cells with coated OKT3 (n = 3; lower panel). As control, primary human T cells were left unstained (light gray-shaded curve). Data from one individual animal or independent T cell donor, being representative for the indicated groups, are shown. Data shown in (A) are taken from 5–8 independent experiments. Data shown in (B) are representative for 2, in (C) for 3 independent experiments.
Fig 3.
OKT3 application selectively downmodulates CD3 on T cells in humanized mice.
Humanized mice were injected i.v. with 20 μg OKT3 or 20 μg TGN1412 per 10 grams body weight. (A) 2–6 hours (time point of sacrifice) post OKT3 (gray bars; n = 6–16) or TGN1412 (white bars; n = 6–16) application, percentage and composition of hCD45+ cells in spleen, PEC, LN, and thymus of humanized mice were analyzed by flow cytometry. PBS-treated mice (black bars; n = 6–16) were used as control. *** p < 0.001; n.s., not significant (t-test, for comparison of hCD45+ cells adjusted according Dunnett for multiple comparisons). (B) 4 hours post OKT3 (n = 3) application, CD3 expression on hCD4+ or hCD8+ T cells in spleen, PEC, LN, and thymus of humanized mice was analyzed by flow cytometry. PBS-treated mice (n = 3) were used as control to adjust quadrants for definition of positive and negative cells. Data from one individual animal, being representative for the indicated groups, are shown. (C) 4 hours post OKT3 (n = 3) or TGN1412 (n = 3) application, mAbs bound by their target CD3 or CD28 on human CD4+/CD8+ T cells in spleen, PEC, LN, and thymus of humanized mice were analyzed by flow cytometry (black curves). In vivo receptor occupancy of CD3 or CD28 was analyzed by exogenously adding OKT3 or TGN1412 to cells isolated from spleen, PEC, LN, and thymus of in vivo mAb-treated humanized mice (dotted curve). PBS-treated mice (n = 3) were used as control (gray-shaded curves). Data from one individual animal, being representative for the indicated groups, are shown. Data shown in (A) are taken from 2–6 independent experiments. Data shown in (B) and (C) are representative for 2 independent experiments.
Fig 4.
Release of human cytokines in humanized mice upon OKT3 and TGN1412 treatment.
Humanized mice were injected i.v. with 20 μg OKT3 or 20 μg TGN1412 per 10 gram body weight. As control, humanized mice were injected with PBS. Before and 2–6 hours (time point of sacrifice) post OKT3 (n = 16) or TGN1412 (n = 16) application, blood was collected and investigated for human IFN-γ, TNF-α, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12p70, and IL-1β by human FlowCytomix Th1/Th2 11plex analysis. PBS-treated mice (n = 12) were used as control. *** p < 0.001; ** p < 0.01; * p < 0.05 (paired t-test for differences after-before). Data shown are taken from 4–6 independent experiments.
Fig 5.
Humanized mice succumb to antibody application.
Humanized mice were injected i.v. with 20 μg OKT3 or 20 μg TGN1412 per 10 grams body weight. 1 hour before, indicated humanized mice were treated with 10 μg anti-human TNF-α mAb per mouse (C-E). As control, humanized mice were injected with PBS (A-B). (A) Before and 2–6 hours (time point of sacrifice) post OKT3 (n = 6) or TGN1412 (n = 6) application, body temperature of mice was monitored. PBS-treated mice (n = 5) were used as control. ** p < 0.01; *** p < 0.001 (Dunnett-adjusted t-test). (B) 2–6 hours post OKT3 (n = 22) or TGN1412 (n = 22) application mice were sacrificed if severe signs of illness were detectable. PBS-treated mice (n = 18) were sacrificed 6 hours post treatment, the latest time point survived by mAb-treated mice. *** p < 0.001 (Dunnett-HSU-adjusted Logrank test). (C) Before (be.) and after (af.) OKT3 (n = 11) or TGN1412 (n = 6) application and 1 hour post anti-human TNF-α mAb (α-hTNF-α) application (before OKT3 or TGN1412 treatment) and 0.33–7 hours (time point of sacrifice) post OKT3 (n = 14) or TGN1412 (n = 11) application, body temperature of mice was monitored. * p < 0.05; *** p < 0.001 (paired t-test for difference after-before). (D) 0.33–7 hours post OKT3 (n = 11), anti-human TNF- α mAb + OKT3 (n = 14), TGN1412 (n = 6), or anti-human TNF-α mAb + TGN1412 (n = 11) application mice were sacrificed if severe signs of illness were detectable. (E) Before (be.) and after (af.) OKT3 (n = 11) or TGN1412 (n = 6) application and 1 hour post anti-human TNF-α mAb application (before OKT3 or TGN1412 treatment; black bars) and 0.33–7 hours post OKT3 (n = 14) or TGN1412 (n = 11) application (time point of sacrifice; gray bars), percentages of hCD45+ cells in peripheral blood of reconstituted mice were analyzed by flow cytometric analysis. * p < 0.05; ** p < 0.01; *** p < 0.001 (Wilcoxon signed rank test for difference after-before). Data shown in (A) are taken from 2, in (B) from 6–9, and in (C), (D), and (E) from 3 independent experiments.