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Interleukin-7 Optimizes FOXP3+CD4+ Regulatory T Cells Reactivity to Interleukin-2 by Modulating CD25 Expression

<div><p>The vast majority of Foxp3 regulatory T cells (Treg) exhibits constitutive expression of CD25 (IL-2Rα), which allows the constitution of the high affinity IL-2Rαβγ receptor, ensuring efficient IL-2 binding by Treg. Maintenance of CD25 expression at Treg surface depends on both cell intrinsic factors and environmental stimuli such as IL-2 itself. Whether other factors can participate to maintenance of CD25 expression <i>in vivo</i> is at present unknown. In the present work we demonstrated that IL-7, a gamma-chain cytokine exerting a crucial role in T cell development and homeostasis, is able and necessary to sustain the expression of high levels of CD25 at Treg surface. We demonstrated that, during <i>in vitro</i> cultures performed in the absence of IL-2, IL-7 is able to sustain CD25 expression at Treg surface through a transcriptional mechanism. By studying mice in which IL-7 signaling is either genetically impaired or increased and by employing adoptive transfer murine models, we demonstrated that IL-7 is necessary for sustained expression of CD25 at Treg surface <i>in vivo</i>. To ascertain the biological impact of IL-7 mediated modulation of CD25 expression, we demonstrated that IL-7 modulation of CD25 expression at Treg surface affected their ability to efficiently bind IL-2 and transduce IL-2 signaling. Finally, we demonstrated that IL-7 dependent modulation of CD25 associated with potentiated IL-2 induced expansion of Treg <i>in vivo</i>. Collectively, our results identify IL-7 as a necessary factor contributing to sustained CD25 expression at Treg surface <i>in vivo</i> thereby affecting their ability to efficiently react to IL-2.</p></div

IL-7 induced expansion of both conventional and regulatory T cells is thymic independent.

<p>CFSE labeled total CD4 T cells from CD45.1 mice were transferred into CD45.2 C57Bl/6 hosts. Mice were then treated with IL-7/anti-IL-7 complexes as in Fig. 2. (A) Representative dot plots of CD45.1 vs FOXP3 expression on CD4⁺ TCRβ cells recovered from lymph nodes and spleens of PBS or IL-7 complexes treated mice. (B) Total numbers of adoptively transferred CD45.1 FOXP3⁻ conventional T cells (left panels) and CD45.1 FOXP3⁺ regulatory T cells (right panels) from lymph nodes and spleen.</p

IL-7 complexes treatment induced both conventional and regulatory T cell expansion.

<p>C57Bl/6 mice were i.p. injected with PBS or IL-7/anti-IL-7 complexes (1 µg rmIL-7 plus 5 µg M25) three times at 2 day intervals. LN and SP cells were analyzed 7 days after the first injection. (A) Representative dot plots of CD25 vs FOXP3 expression on CD4⁺TCRβ⁺ cells recovered from PBS or IL-7 complexes treated mice. (B) Total number of CD4⁺TCRβFOXP3⁻ conventional T cells (left panels) and CD4⁺ TCRβ FOXP3⁺ regulatory T cells (right panels) from lymph nodes and spleen.</p

Confirmation of differential expression of IFNs in tissues by RT-qPCR and at the protein level in plasma.

<p>(<b>A</b>) IFN-α mRNA expression in PLNs by RT-qPCR. (<b>B</b>) IFN-β mRNA expression in PLNs by RTqPCR. (<b>C</b>) IFN-α mRNA expression in RB by RT-qPCR. (<b>D</b>) IFN-γmRNA expression in PLNs by RT-qPCR. (<b>E</b>) MXA mRNA expression in PLNs by RT-qPCR. (<b>F</b>) IRF9 mRNA expression in PLNs by RT-qPCR. (<b>G</b>) Type-I IFN antiviral activity measured in plasma (MDBK/VSV biological assay) and (<b>H</b>) plasma IFN-γ concentration (measured by Luminex). ULD = Under the Limit of Detection. The Lower Limit of Quantification (LLOQ) is represented as a red dotted line in figure 3H. P values are given for non-parametric Wilcoxon rank sum test and was considered significant if p < 0.05.</p

SIV infection induces ISG expression during both phases of infection in PLNs.

<p>(<b>A</b>) Venn diagram showing the number of ISGs expressed at D9, M3, or both time points. (<b>B</b>) Comparison of mean ISG expression FC at D9 and M3 p.i. for the 51 ISGs expressed at both time points. (<b>C</b>) Venn diagram of ISGs expressed at D9 p.i., splitting them into those only inducible by type I IFN (Type I), only inducible by type II IFN (Type II), and those inducible by both IFN types (left) and comparison of their mean induction levels (right). (<b>D</b>) Venn diagram of ISGs expressed at M3 p.i., splitting them into those only inducible by type I IFN, those only inducible by type II IFN, and those inducible by both IFN types (left) and comparison of their induction levels (right). (<b>E</b>) Venn diagram of ISGs expressed at D9 p.i., splitting them into those displaying at least one ISRE, one GAS, or both sequence motifs in their promoters (left) and comparison of their induction levels (right). (<b>F</b>) Venn diagram of ISGs expressed at M3 p.i., splitting them into those displaying at least one ISRE, one GAS, or both in their promoters (left) and comparison of their induction levels (right). (<b>G</b>) Functional enrichment of type I-, type II-, and type I&II ISG subsets. The p value was calculated by the Fisher test with the Benjamini-Hochberg correction for multiple comparisons. The -log(p-value) is given for the most significant processes. In C, D, E, and F, the p value is given for Welch's unequal variances <i>t</i>-test, considering p < 0.05 to be significant.</p

Transcriptome profiling reveals inter-tissue differences and the evolution of differential gene expression post-infection.

<p>Two critical time points, day 9 (D9) and month 3 (M3) p.i., were compared to baseline (before viral infection) (<b>A</b>) Number of genes with significant differential expression relative to baseline in RBs, PLNs, and PBMCs. A two-sample t-tests (p < 0.05) was used to identify differentially expressed genes (<b>B</b>) Multidimensional scaling (MDS) representation showing the segregation of biological samples based on the altered expression of the 1,638 genes with FC > 2. (<b>C</b>) Venn diagram showing the distribution of differentially expressed genes over time in RBs, PLNs, and PBMCs. (<b>D</b>) Evolution of RNA expression of interferons and interferon receptors in RBs, PLNs, and PBMCs at D9 and M3 p.i. Heatmap representing the FC from baseline for the six SIVmac251 infected macaques.</p

Infection of six cynomolgus macaques with SIVmac251 and establishment of chronic infection.

<p>(<b>A</b>) vRNA load in plasma. (<b>B</b>) Evolution of total CD4 T cell numbers in the blood over time (percentage of baseline). (<b>C</b>) vRNA in RB. (<b>D</b>) vRNA in PLNs. ULD = under the limit of detection. P values are given for non-parametric Wilcoxon rank sum test and was considered significant if p < 0.05.</p

Evolution of the ISG-associated functional signature during infection.

<p>Functional enrichment of ISGs differentially expressed at either (<b>A</b>) D9 p.i., (<b>B</b>) M3 p.i., or (<b>C</b>) both time points (D9&M3), was performed using IPA. The right-tailed Fisher Exact Test with the Benjamini-Hochberg correction for multiple tests was used to identify processes showing statistically significant over-representation of focus ISGs. Over-represented functional or pathway processes have more focus genes than expected by chance and the–log(p-values) of the most significant functions are plotted in the radar plot.</p

Heat-map signatures of differentially expressed ISGs.

<p>The differentially expressed genes of the ISG cluster with the most significant functions are represented as heat-maps. The color intensity shown at each time point is related to the FC in RNA expression: grey for not differentially expressed (not DEG), yellow for FC values between 1 and 2, orange for FC values between 2 and 5, and red for FC values above 5.</p