Reduced accumulation of donor T cells in the intestine and increased accumulation of donor T cells in the liver of VAD recipients during acute GvHD.

<p>Mice were analyzed for donor T cell (Thy1.1) occurrence in SPL, mLN, liver and small intestine (SI) at day 21 after transplantation. (A) Each dot represents the percentage of infiltrating Thy1.1⁺CD4⁺ T cells of all Thy1.1⁺CD4⁺ T cells. (B) Each dot represent the percentage of infiltrating Thy1.1⁺CD8⁺ T cells of all Thy1.1⁺CD8⁺ T cells. Bars indicate mean values. N = 4–5/group. **: p<0.01; ***: p<0.001. Data are combined from two independent experiments. (C) Each bar represents absolute numbers of infiltrating Thy1.1⁺CD4⁺ T cells from STD or VAD recipients. Bars indicate mean values. N = 4–5/group. **: p<0.01; ***: p<0.001. Data are combined from two independent experiments. (D) Each bar represents absolute numbers of infiltrating Thy1.1⁺CD4⁺ T cells from STD or VAD recipients. Bars indicate mean values. N = 4–5/group. **: p<0.01; ***: p<0.001. Data are combined from two independent experiments.</p

Vitamin A deficiency of recipient mice leads to increased Th1 cells and decreased FoxP3⁺ Treg cells during GvHD.

<p>STD or VAD recipient mice were analyzed for CD4⁺ T cell polarization status in SPL, mLN, liver and small intestine (SI) at day 21 after transplantation. (<b>A</b>) Each bar represents the percentage of FoxP3⁺ CD4⁺ T cells of CD4⁺ T cells isolated from the indicated organ. (<b>B</b>) Each bar represents the percentage of IFN-γ⁺ CD4⁺ T cells of CD4⁺ T cells isolated from the indicated organ.</p

Donor T cell primed in mesenteric lymph nodes express high levels of gut-homing molecules in recipients under standard (STD) but not under vitamin A deficient (VAD) food.

<p>(<b>A</b>) The expression of CCR9 and integrin-β7 of allogeneic C57BL/6 Thy1.1⁺ CFSE-labeled CD4⁺ and CD8⁺ T cells was determined by flow cytometry three days following adoptive transfer of 2×10⁷ splenocytes into lethally irradiated recipients (BALB/c) fed with STD or VAD diet (N = 3/group). The numbers in the boxes indicate the mean percentage of gated cells +/−SD. Similar results were obtained in at least three repeat experiments. (<b>B</b>) Analysis of donor T cells three days after transfer showed an activated phenotype (CD62L⁻) of CCR9⁺ or β7⁺ T cells. Representative data from one of two experiments are shown. (<b>C</b>) Expression of CCR9 and integrin-β7 of allogeneic C57BL/6 Thy1.1⁺ CD4⁺ and CD8⁺ T cells in mLN and pLN (grey = isotype control, red = STD recipient, blue = VAD recipient). Similar results were obtained in at least three repeat experiments. (<b>D</b>) The proliferation of CFSE-labelled CD4⁺ and CD8⁺ T cells was determined by flow cytometry three days following adoptive transfer of 2×10⁷ splenocytes into lethally irradiated STD or VAD recipients. N = 6/group. Combined data from two experiments (n.s. = not significant).</p

Homing of allo-primed T cells to the intestine is dependent on dietary vitamin A.

<p>2×10⁷ splenocytes (C57BL/6) were transferred into lethally irradiated STD or VAD BALB/c recipients. Three days after transfer donor T cells from mesenteric lymph nodes of STD or VAD BALB/c mice were harvested and then split and differentially labeled with either TAMRA or CFSE. CFSE-labeled cells from STD recipients were mixed with TAMRA-labeled cells from VAD-recipients at a 1∶1 ratio. In cross-labeling experiments, TAMRA-labeled cells from STD-recipients and CFSE-labeled cells from VAD-recipients were used. Mixtures of 5×10⁶ T cells per mouse in total were injected into the tail vein of untreated wt C57BL/6 recipient mice. Eighteen hours after transfer recipient mice were sacrificed and the homing of CD4⁺ and CD8⁺ T cells was analyzed by flow cytometry. The ratio of transferred T cells primed in allogeneic VAD versus STD recipients was analyzed in pLN (pooled per mouse), SPL, mLN, liver, IEL and LPL. Labeling effects were excluded by normalizing the ratio to 1∶1 in each staining group. N = 6/group. Data are combined from two independent experiments.</p

Vitamin A deficiency led to increased hepatic inflammation.

<p>(<b>A</b>) Serum cytokine levels were analyzed using the Cytometric Bead Array, Mouse Inflammation Kit (BD Biosciences). N = 5–6/group. **: p<0.01; ***: p<0.001. Data are combined from two independent experiments. (<b>B</b>) Expression analyses of cytokine levels of GvHD target organs using RT-PCR (N = 6/group) Data are combined from two independent experiments (<b>C</b>) Macroscopic comparison of spleen sizes of VAD and STD recipient mice. N = 3/group. One of two representative experiments is shown. (<b>D</b>) Liver enzymes in the course of GvHD in VAD and STD recipients. N = 5–6/group. **: p<0.01; ***: p<0.001. Data are combined from two independent experiments. (<b>E, F</b>) Representative sections from formalin fixed, paraffin embedded livers and small intestines harvested at 21 days post transplantation. The 4–6 µm slides were stained with hematoxylin and eosin.</p

Vitamin A deficiency in HSCT recipients prolongs survival but does not protect from lethal GvHD.

<p>After lethal irradiation, STD BALB/c or VAD BALB/c recipients received 5×10⁶ C57BL/6 T cell depleted bone marrow cells supplemented with 1×10⁶ T cells (N = 14/group). STD BALB/c or VAD BALB/c recipients that received syngeneic (BALB/c) grafts were used as controls (N = 4/group). Data are combined from two independent experiments.</p