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

Human bone marrow samples show highly individual Ig-Repertoires.

<p>(a) Schematic overview of human VDJ-amplicons. Amplicon libraries for each Ig class contain framework (FR2, 3) and complementary determining regions (CDR1, 2, 3). (b) In a set of 4000 IgG-sequences per bone marrow sample, clones with 95% CDR3 sequence identity and identical VJ-usage were clustered as clonotypes. Frequencies of clonotypes are indicated by color code; clonotypes comprising more than 5% of the repertoire are highlighted in blue and numbers indicate their frequencies; grey color indicates absence of a given clonotype. Clontypes were sorted according to their frequency in healthy donor (HD) 1 and the 100 most frequent clonotypes of HD1 and their abundance in HD2-4 were displayed as heatmap. Similarity of repertoires was expressed as Morisita-Horn index (MHI), comparing 4000 clustered CDR3 sequences of HD1-4. Symbols represent pairwise comparisons. (c) The IgG-repertoire of HD4 (I) was re-investigated as independent V_H1DJ-amplificate (II) or de-novo cDNA plus V_H1DJ-amplificate (III). Repertoire-similarity of I to its technical replicates II and III is illustrated by heatmap and MHI, evaluating 2500 clustered sequences of each sample.</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

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

Switch Ig repertoires after HSCT comprise a spectrum of low and highly mutated clones.

<p>Numbers of somatic hypermutations in bone marrow switch IgA and IgG repertoires. Lines represent individual samples. Evaluation was based on all available sequences. Diagrams in (a) show results of four healthy donor repertoires (HD1-4), compared to (b) patient 1 and 6 evaluated separately for IgG (left column) and IgA (right column).</p

Clinical characteristics of AML-patients investigated for repertoire analysis before and after allogeneic HSCT.

<p>Clinical characteristics of AML-patients investigated for repertoire analysis before and after allogeneic HSCT.</p

Ig repertoire analysis reveals clonal sharing between isotypes and persistence of clonotypes after HSCT.

<p>(a) IgA, IgG and IgE repertoires sequences were clustered (4000 sequences; 95% CDR3 sequence identity; same VJ-usage), and sorted according to the 100 most abundant clonotypes present in the IgA (left panel), IgG (middle panel), or IgE (right panel) repertoire and plotted as heatmap (see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0168096#pone.0168096.g001" target="_blank">Fig 1</a>). Comparison of repertoire similarity of IgA, IgG and IgE repertoires of HD1 based on the analysis of 4000 sequences each. (b, c) IgG sequences of eleven patients pre- and post-HSCT (patient 1: 2000 sequences; other patients: 4000 sequences) were assigned to clonotypes. (b) Heatmaps show CDR3-overlap of the 100 most abundant IgG clonotypes of patients 1 and 5 pre- and post-HSCT. Repertoire similarity is described as Morisita-Horn index (MHI). (c) IgG-repertoire and (d) IgA-repertoire similarity quantified as MHI. Symbols represent comparisons of patient 1-11(X) pre- versus post-HSCT (X_pre vs. X_post) or versus itself as control (X_pre vs. X_pre). In addition, repertoires of patients 1 and 5 pre-HSCT were compared to all other patients pre HSCT (X₁ or X₅ vs.Y_pre). Pairwise match of all pre-HSCT or post-HSCT IgG repertoires investigated results in a mean MHI of 0.0001±0.0005 for all pre- and 0.0001±0.0004 and for all post-HSCT samples (mean±SD). Pairwise match of all pre-HSCT or post-HSCT IgA repertoires investigated results in MHI of 0.00007±0.00033 for all pre- and 0.00002±0.00011 for all post-HSCT samples (mean±SD). Statistical analysis was performed using Kruskal-Wallis test with Dunn’s multiple comparison post hoc test, * p<0.05.</p

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

Repertoire diversity is reduced after allogeneic HSCT.

<p>(a) The Exponent Shannon (e^Shannon) indices were calculated based on 4000 clustered IgA and IgG repertoires of four healthy donors (HD1-4). Symbols indicate individual samples, horizontal lines indicate the mean. (b) Diversity (e^Shannon) of IgG and IgA repertoires pre- and post-HSCT was compared for eleven AML-patients. Exponent Shannon indices were calculated for each sample based on 2000 (patient 9) or 4000 (all other patients) clustered IgG and IgA sequences. Matched samples (pre- and post-transplantation) are connected by lines, numbers indicate the different patients. Statistical analysis was performed using paired T-test with Wilcoxon signed rank test, ** p< 0.01. Patient 11 (grey lines and symbols) was not considered for statistical analysis.</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