Journal: PLoS ONE

Article Title: TGN1412 Induces Lymphopenia and Human Cytokine Release in a Humanized Mouse Model

doi: 10.1371/journal.pone.0149093

Figure Lengend Snippet: 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.

Article Snippet: 24-well flat-bottom tissue culture plates (Sarstedt, Nümbrecht, Germany) were coated before with 5 μg/ml OKT3 (Janssen-Cilag, Neuss, Germany) in 200 μl PBS (Biochrom, Berlin, Germany) per well at 4°C for 24 h. Plates were washed twice with 400 μl PBS to remove unbound antibody prior to addition of human T cells in 1 ml X-VIVO 15 medium.

Techniques: Injection, Expressing, In Vivo, Flow Cytometry, In Vitro, Purification

Journal: PLoS ONE

Article Title: TGN1412 Induces Lymphopenia and Human Cytokine Release in a Humanized Mouse Model

doi: 10.1371/journal.pone.0149093

Figure Lengend Snippet: 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.

Article Snippet: 24-well flat-bottom tissue culture plates (Sarstedt, Nümbrecht, Germany) were coated before with 5 μg/ml OKT3 (Janssen-Cilag, Neuss, Germany) in 200 μl PBS (Biochrom, Berlin, Germany) per well at 4°C for 24 h. Plates were washed twice with 400 μl PBS to remove unbound antibody prior to addition of human T cells in 1 ml X-VIVO 15 medium.

Techniques: Injection, Flow Cytometry, Expressing, In Vivo, Isolation

Journal: PLoS ONE

Article Title: TGN1412 Induces Lymphopenia and Human Cytokine Release in a Humanized Mouse Model

doi: 10.1371/journal.pone.0149093

Figure Lengend Snippet: 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.

Article Snippet: 24-well flat-bottom tissue culture plates (Sarstedt, Nümbrecht, Germany) were coated before with 5 μg/ml OKT3 (Janssen-Cilag, Neuss, Germany) in 200 μl PBS (Biochrom, Berlin, Germany) per well at 4°C for 24 h. Plates were washed twice with 400 μl PBS to remove unbound antibody prior to addition of human T cells in 1 ml X-VIVO 15 medium.

Techniques: Injection

Journal: PLoS ONE

Article Title: TGN1412 Induces Lymphopenia and Human Cytokine Release in a Humanized Mouse Model

doi: 10.1371/journal.pone.0149093

Figure Lengend Snippet: 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.

Article Snippet: 24-well flat-bottom tissue culture plates (Sarstedt, Nümbrecht, Germany) were coated before with 5 μg/ml OKT3 (Janssen-Cilag, Neuss, Germany) in 200 μl PBS (Biochrom, Berlin, Germany) per well at 4°C for 24 h. Plates were washed twice with 400 μl PBS to remove unbound antibody prior to addition of human T cells in 1 ml X-VIVO 15 medium.

Techniques: Injection