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

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

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

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

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

Infected organs show different levels of vaccination-mediated protection.

<p>C57BL/6 mice were orally vaccinated with attenuated SL1344Δ<i>aro</i>A <i>Salmonella</i> Typhimurium. 40–50 day later vaccinated mice and age-matched controls were orally infected with 1×10⁹ of either SL1344 or SL1344Δ<i>aro</i>A. CFU were determined after two days for single PP, mLN, liver and spleen (spl). Symbols indicate individual organs and bold lines median CFU from 9 or more mice pooled from 2 independent experiments. Numbers indicate organs displaying less than 10 CFU.</p

Vaccination modulates routes of <i>S.</i> Typhimurium dissemination.

<p>Mice were vaccinated with a SL1344Δ<i>aroA</i> strain 40–50 days or left untreated before infection with the WITS library. WITS composition was determined as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004270#ppat-1004270-g003" target="_blank">Fig. 3A</a>. (A) Pie diagrams depict representative examples of WITS composition in pooled PP, mLN, liver and spleen of one non-vaccinated and one vaccinated mouse. (B) Similarity of WITS composition was compared between various compartments. Numbers indicate MHI for the various comparisons and line width/style depicts MHI as indicated in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004270#ppat-1004270-g004" target="_blank">Fig. 4B</a>. 2-way ANOVA reveals that the effect of vaccination on the similarity between compartments (determined by MHI) is significant (p<0.001). Mann-Whitney t test revealed significant effects on distinct compartments. Vaccination significantly affected similarity of the WITS composition between PP and mLN (p = 0.016), mLN and liver (p = 0.026), mLN and spleen (p = 0.016) and liver and spleen (p = 0.004). Vaccination had no significant effect on the similarity in WITS composition observed for the comparison of PP and liver or PP and spleen. (C) Vaccination only moderately reduced invasion of PP. Symbols indicate TSU predicted for individual PP, mLN, liver and spleen of non-vaccinated and vaccinated mice pooled from 3 or more independent experiments. Horizontal bars indicate the mean.</p

Independent Bottlenecks Characterize Colonization of Systemic Compartments and Gut Lymphoid Tissue by <i>Salmonella</i>

<div><p>Vaccination represents an important instrument to control typhoid fever in humans and protects mice from lethal infection with mouse pathogenic serovars of <i>Salmonella</i> species. Mixed infections with tagged <i>Salmonella</i> can be used in combination with probabilistic models to describe the dynamics of the infection process. Here we used mixed oral infections with tagged <i>Salmonella</i> strains to identify bottlenecks in the infection process in naïve and vaccinated mice. We established a next generation sequencing based method to characterize the composition of tagged <i>Salmonella</i> strains which offers a fast and reliable method to characterise the composition of genome-tagged <i>Salmonella</i> strains. We show that initial colonization of <i>Salmonella</i> was distinguished by a non-Darwinian selection of few bacteria setting up the infection independently in gut associated lymphoid tissue and systemic compartments. Colonization of Peyer's patches fuels the sustained spread of bacteria into mesenteric lymph nodes via dendritic cells. In contrast, infection of liver and spleen originated from an independent pool of bacteria. Vaccination only moderately reduced invasion of Peyer's patches but potently uncoupled bacterial populations present in different systemic compartments. Our data indicate that vaccination differentially skews the capacity of <i>Salmonella</i> to colonize systemic and gut immune compartments and provide a framework for the further dissection of infection dynamics.</p></div

WITS similarity reflects routes of <i>S.</i> Typhimurium dissemination.

<p>Mice were treated as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004270#ppat-1004270-g003" target="_blank">Fig. 3A</a>. (A) The frequency of individual WITS in proximal and distal PP (left panel) or middle and distal PP (right panel) was compared. Numbers indicate Pearson correlation coefficients. Symbols represent individual PP pooled from 3 independent experiments. (B) Similarity of WITS representation was calculated as Morisita-Horn index. Lines indicate pairwise comparisons. Numbers indicate the MHI±SD. Line thickness/style depicts average MHI as indicated. Pearson correlation coefficients are higher comparing middle to distal compared to proximal to middle PP. Still, no significance was detected using 2-way ANOVA.</p