8 research outputs found
Priming of CD4 T-cells by endogenous antigen in the inflamed liver.
<p>(A) Eight million OT-I T-cells were transferred intravenously into TF-OVA mice (+ Hepatitis), or mice were left untreated (- Hepatitis). ALT levels were determined at day 6. Values from individual mice and mean ± SEM are depicted (*** p<0.0001 by Mann-Whitney test). (B) Four million CFSE-labeled OT-II T-cells were transferred intravenously into TF-OVA mice at day 6 after transfer of OT-I T-cells (+ Hepatitis) or into untreated TF-OVA mice (- Hepatitis). Non-parenchymal cells were isolated from liver and spleen analyzed for the presence of CFSE<sup>+</sup> cells. The absolute number of CFSE<sup>+</sup> cells in liver or spleen was determined 20 hours after transfer of OT-II T-cells. Cumulative results are depicted from n = 6 mice per group (mean ± SEM, ** p<0.005 by Student's t-test). (C) Mice were treated as in (B). Proliferation was analyzed 20 and 44 hours after transfer of OT-II T-cells. Events to the right of the vertical line represent the undivided population. Events at the far left of the plot represent unlabeled endogenous cells. Representative results are depicted from n = 6 mice in each group. All plots display data gated on CD4<sup>+</sup>Vα2<sup>+</sup> cells.</p
Priming of naive CD4 T-cells by liver-derived antigen <i>in vivo.</i>
<p>OT-II T-cells were purified from the lymph nodes and spleen of OT-II mice and labeled with CFSE. Four million cells were transferred intravenously into splenectomized TF-OVA mice (A), MHC-II<sup>−/−</sup> → TF-OVA chimeras (B), or TF-OVA mice (control). Cells from the indicated organs were isolated 44 (A) or 68 hours (B) after cell transfer and analyzed for the presence of proliferating OT-II T-cells by detection of CFSE-dilution. All plots depict data gated on CD4<sup>+</sup> cells. Events to the right of the vertical line represent the undivided population. Events at the far left of the plot represent unlabeled endogenous cells. Representative results from n = 4 bone-marrow chimeras and n = 4 control mice (A), and n = 6 splenectomized and n = 4 control mice (B) are shown.</p
CD4 T-cells primed by liver-derived antigen display deficient Th1-effector function.
<p>(A) Four million CFSE-labeled OT-II T-cells were transferred intravenously into TF-OVA mice or into B6 control mice. After 68 hours, non-parenchymal cells were purified from liver and spleen and incubated <i>in vitro</i> with PMA/ionomycin. Production of Interferon-γ and IL-2 was analyzed after 4 hours. Representative results are depicted (n = 6). (B) Mice were treated as in (A), but cells were analyzed for CD25 and FoxP3 expression immediately after purification of cells from the indicated organs. Representative results are depicted from n = 6 mice in each group. All plots depict data gated on CD4<sup>+</sup>CFSE<sup>+</sup> cells. CD25/FoxP3 plots on the right depict the frequency of CD25<sup>+</sup>FoxP3<sup>+</sup> double positive cells.</p
Hepatitis is amplified by effector but not naive CD4 T-cells.
<p>(A) Four million naïve (open squares) or effector (open circles) OT-II T-cells were transferred alone or together with 4 million naïve OT-I T-cells (filled squares and circles) into TF-OVA mice. Naïve OT-II T-cells were also transferred three days prior to transfer of OT-I T-cells to allow timely redistribution to the liver (half-filled squares). As a control, 4 million naïve OT-I T-cells (filled triangles) were transferred alone. Alanine aminotransferase levels were determined at day 5. Individual values and mean ± SEM are depicted (**p<0.005 by Mann-Whitney test). (B) One million naïve OT-I T-cells were transferred alone (filled triangles) or together with 4 million effector OT-II T-cells (half-filled circles) into TF-OVA mice. Alanine aminotransferase levels were determined at day 5. Individual values and mean ± SEM are depicted (**p<0.005 by Mann-Whitney test). (C) Four million effector OT-II T-cells were transferred alone (CD4) or together with 1 million naïve OT-I T-cells (CD8+CD4) into TF-OVA mice. As controls, 1 million naïve OT-I CD8 T-cells (CD8) were transferred alone or no cells were transferred (control). At day 6, equal numbers of CFSE<sup>high</sup> SIINFEKL-pulsed and CFSE<sup>low</sup> unpulsed B6 splenocytes were injected. After 5 hours cells from the indicated organs were analyzed for CFSE staining. Histogram blots depict data gated on CFSE-positive cells. (D) Antigen-specific cytolysis was calculated as described in methods. Cumulative results are depicted from n = 6 mice per group (mean ± SEM; ** p<0.005 by Mann-Whitney test).</p
Priming of naive CD4 T-cells by liver-derived antigen <i>in vitro.</i>
<p>Dendritic cells (DC), Kupffer cells (KC) and liver sinusoidal cells (LSEC) were isolated from livers of TF-OVA mice, DCs were also isolated from spleens of TF-OVA mice. APCs (2×10<sup>5</sup> cells) were allowed to settle for 24 h before addition of 1×10<sup>5</sup> CFSE labeled T-cells. After three days of co-culture, cells were harvested, stained for CD8 or CD4, and proliferation of OT-I or OT-II T-cells was analyzed (black line) by CFSE dilution. OT-II and OT-I T-cells incubated alone were used as negative control (filled gray histogram). Representative results from n = 3–6 experiments are shown. Plots depict data gated on CD8<sup>+</sup>CFSE<sup>+</sup> or CD4<sup>+</sup>CFSE<sup>+</sup> cells.</p
Effector CD4 T-cells accumulate in the liver of TF-OVA mice.
<p>(A) Effector OT-II T-cells with a Th1-phenotype were generated <i>in vitro</i> with the cognate peptide antigen in the presence of IL-12, Interferon-γ, and anti-IL-4 antibody. After 6 days in culture, cells were restimulated with PMA/ionomycin and stained for Interferon-γ and IL-4. (B) Four million CFSE-labeled naïve or effector OT-II T-cells were transferred into TF-OVA or B6 control mice. Non-parenchymal cells from the indicated organs were isolated after 20 or 68 hours, and analyzed for the presence of proliferating OT-II T-cells by CFSE dilution. All plots depict data gated on CD4<sup>+</sup>Vα2<sup>+</sup> cells. Events to the right of the vertical line represent the undivided population. Events at the far left of the plot represent unlabeled endogenous cells. Representative results from n = 4–6 mice in each group are shown. (C) Cells from the indicated organs were isolated at the indicated days after transfer of 4 million naïve or effector OT-II T-cells, and numbers of CD4<sup>+</sup>Vα2<sup>+</sup> cells were enumerated. Data shown are derived from n = 3–6 mice per group at each time point. Note the different scales for spleen and liver/liver lymph node (mean ± SEM; * p<0.05, *** p<0.001 by Student's t-test). (D) Liver sections from TF-OVA and B6 mice were stained with H&E 6 days after transfer of 4 million effector OT-II T-cells. Representative images from n = 6 mice per group (magnification 100x) are depicted.</p