American Society of Hematology

Figure 4

$Figure 4. Immunologic status of cardiac allografts in PPG-treated animals. (A) Characterization of graft-infiltrating cells from NAL-treated (gray bars) or PPG + NAL-treated (black bars) animals. Graft-infiltrating cells were extracted at day 5 after transplantation, counted, and labeled with anti-TCRα/β, anti-CD4, and anti-FoxP3–labeled antibodies, or were stained with anti-CD11b, anti–class II, and anti-CD103 antibodies as described in “Immunostaining and flow cytometry.” Each bar represents the mean ± SEM of 3 animals. (B) “T cell response” panel: graft recipient (LEW.1A) T cells purified from the spleen were stimulated at a 1:1 ratio with irradiated donor-type LEW.1W splenocytes used as APCs. “APC alloreactivity” panel: graft recipient (LEW.1A) DCs from the spleen were used as APCs to stimulate at a 1:10 ratio purified donor-type LEW.1W T cells. White bars represent untreated rejecting recipients; and black bars, recipients treated with PPG + NAL. Cells were harvested on day 6 after transplantation. Proliferation was measured after 5 days of culture. Data are mean ± SD of triplicates from one experiment representative of 3 independent experiments. (C) At day 5 after transplantation, splenocytes from NAL-treated (Control) and PPG + NAL-treated (PPG) were used as APCs to stimulate allogeneic T cells in MLRs. Forty-eight hours later, supernatants were harvested and assessed for IFN-γ secretion. Individual measurements are shown with bars representing the mean. *P < .05. (D) Recipient rats of allogeneic hearts were either treated with NAL alone (gray bars) or with PPG + NAL (black bars). Grafts were harvested on day 5 after transplantation and mRNA levels of indicated factors analyzed by quantitative RT-PCR. Values for NAL alone controls were normalized at an arbitrary value of 1.0. Each bar represents the mean ± SEM of 4 (IFN-γ, IL-12p35, IL-12p40, IL-1b) or 5 (T-Bet) animals. *P < .05. **P < .01. (E-F) At day 5 after transplantation, NAL-treated (Control) and PPG + NAL-treated (PPG) transplants were homogenized, and IL-12 (E) or IFN-γ (F) content was assayed by ELISA in the soluble fraction. Individual measurements are shown with bars representing the mean. *P < .05.$

Immunologic status of cardiac allografts in PPG-treated animals. (A) Characterization of graft-infiltrating cells from NAL-treated (gray bars) or PPG + NAL-treated (black bars) animals. Graft-infiltrating cells were extracted at day 5 after transplantation, counted, and labeled with anti-TCRα/β, anti-CD4, and anti-FoxP3–labeled antibodies, or were stained with anti-CD11b, anti–class II, and anti-CD103 antibodies as described in “Immunostaining and flow cytometry.” Each bar represents the mean ± SEM of 3 animals. (B) “T cell response” panel: graft recipient (LEW.1A) T cells purified from the spleen were stimulated at a 1:1 ratio with irradiated donor-type LEW.1W splenocytes used as APCs. “APC alloreactivity” panel: graft recipient (LEW.1A) DCs from the spleen were used as APCs to stimulate at a 1:10 ratio purified donor-type LEW.1W T cells. White bars represent untreated rejecting recipients; and black bars, recipients treated with PPG + NAL. Cells were harvested on day 6 after transplantation. Proliferation was measured after 5 days of culture. Data are mean ± SD of triplicates from one experiment representative of 3 independent experiments. (C) At day 5 after transplantation, splenocytes from NAL-treated (Control) and PPG + NAL-treated (PPG) were used as APCs to stimulate allogeneic T cells in MLRs. Forty-eight hours later, supernatants were harvested and assessed for IFN-γ secretion. Individual measurements are shown with bars representing the mean. *P < .05. (D) Recipient rats of allogeneic hearts were either treated with NAL alone (gray bars) or with PPG + NAL (black bars). Grafts were harvested on day 5 after transplantation and mRNA levels of indicated factors analyzed by quantitative RT-PCR. Values for NAL alone controls were normalized at an arbitrary value of 1.0. Each bar represents the mean ± SEM of 4 (IFN-γ, IL-12p35, IL-12p40, IL-1b) or 5 (T-Bet) animals. *P < .05. **P < .01. (E-F) At day 5 after transplantation, NAL-treated (Control) and PPG + NAL-treated (PPG) transplants were homogenized, and IL-12 (E) or IFN-γ (F) content was assayed by ELISA in the soluble fraction. Individual measurements are shown with bars representing the mean. *P < .05.

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