Skin and blood T cell subsets demonstrate differential gene expression by quantitative PCR.

<p>(a) qPCR was performed for selected genes differentially expressed between skin- and blood-derived T cells (combined CD8⁺, CD4⁺ and regulatory T cells) as identified on microarray analysis (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0148351#pone.0148351.t001" target="_blank">Table 1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0148351#pone.0148351.s005" target="_blank">S3 Table</a>). Expression values were normalized to the geometric mean of two housekeeping genes. (b) Expression of lineage-related genes in the CD8⁺, CD4⁺ and regulatory T cells (combined blood and skin samples). *P≤0.05; **P≤0.01 using two-tailed Mann-Whitney U-test.</p

T cells in human skin are transcriptionally distinct from skin-tropic T cells in the blood.

<p>Principal component analysis of gene expression of sorted skin and blood CD4⁺, CD8⁺ and regulatory T cells. Graphs plotted showing (a) principal components 1,2 and 3 and (b) principal components 18 and 21. Each symbol represents one array; 5 arrays/cell type. (c) Venn diagram showing overlap of the significantly differentially expressed genes (DEGs, listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0148351#pone.0148351.s005" target="_blank">S3 Table</a>; P≤0.05) in pairwise comparison between skin and blood CD4⁺, CD8⁺ and regulatory T cells using RUVinv analysis. (d) Venn diagram of DEGs between T cell lineages in blood and skin (listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0148351#pone.0148351.s006" target="_blank">S4 Table</a>; P≤0.05) showing overlaps between categories. Numbers in brackets indicate total number of DEGs from each pairwise comparison. Treg, regulatory T cells.</p

Human skin T cell transcription profiles are enriched for signature resident memory T cell genes defined in the mouse.

<p>(a) The fold change of various genes in the murine skin T_RM core signature in human blood versus skin CD4⁺, CD8⁺ and T_reg cells on microarray analysis. Green (upregulated in T_RM) and red arrows (downregulated in T_RM) below indicate the expected direction of expression in T_RM. Asterisks indicate significantly differentially expressed genes (P≤0.05; n = 5 arrays per cell type). (b) Enrichment scores for the various skin T cell types following Gene Set Enrichment Analysis using gene set lists containing lung, gut and skin T_RM gene signatures. All gene sets shown are significantly enriched at false discovery rate <25%.</p

CD4⁺ T cells in the skin are transcriptionally active for cytokine and inflammatory response genes.

<p>Graphic representation of relative gene expression between skin CD4⁺ T cells and skin-tropic T cells derived from blood. Cytokines and inflammatory response pathway (WP530) depicted using PathVisio v3.2.1 and WikiPathways. Red-green colour bar denotes magnitude of log₂fold-change (green = upregulated in skin, red = upregulated in blood). Similar results were found in CD8⁺ T cells and for regulatory T cells (data not shown).</p

Transcriptional signature of T cells resident in human skin.

<p>Transcriptional signature of T cells resident in human skin.</p

Shared genes of human skin T cells and murine T_RM signature with greatest contribution to the gene set enrichment.

<p>Shared genes of human skin T cells and murine T_RM signature with greatest contribution to the gene set enrichment.</p

CD8⁺ T cells in the skin overexpress genes in the oxidative stress pathway.

<p>Graphic representation of relative gene expression between skin and blood CLA⁺ CD8⁺ T cells in the Oxidative Stress pathway (WP408) using PathVisio 3.2.1 and WikiPathways. Similar results were found for CD4⁺ T cells and for regulatory T cells (data not shown). Red-green colour bar denotes magnitude of log₂fold-change (green = upregulated in skin, red = upregulated in blood). TF = transcription factor.</p

Multiple subsets of skin-tropic T cells are present in human skin and blood.

<p>Representative images are shown to demonstrate gating strategy for fluorescence activated cell sorting of T cells from blood and skin. In each case, gates were used to exclude debris, doublets and non-viable cells, and a dump channel was used to exclude irrelevant cell types. (a) For blood samples, T cells were identified based on CD3 expression, then divided into CD4⁺ and CD8⁺ populations. Skin-tropic CD8⁺ and CD4⁺ memory T cells were isolated based on their expression of memory marker CD45RO and skin addressin CLA. Skin-tropic regulatory T cells (T_reg) were identified based on their CD25_hiCD127_lo surface profile. (b) For skin samples, CD45 was used to eliminate cells of non-haematopoietic origin. CLA⁺ T cells were identified based on CD3 expression. CD8⁺CD103^- T cells were captured; CD4⁺ T cells (CD127_hi) and T_reg (CD25_hiCD127_lo) were identified on the basis of CD25 and CD127 expression. FSC = forward scatter; PI = propidium iodide; SSC = side scatter.</p

A Practical, Protecting-Group-Free Synthesis of a PI3K/mTOR Inhibitor

We report a practical and protecting-group-free synthesis amenable to produce multikilogram amounts of PI3K/mTOR inhibitor <b>GDC-0980</b>. The route employed metalation/formylation and reductive amination followed by a metal catalyzed Suzuki–Miyaura cross-coupling. The metalation was performed via triarylmagnesiate intermediates allowing formylation under noncryogenic conditions. 2-Picoline·BH₃ was employed to replace Na­(OAc)₃BH in the reductive amination and to eliminate the use of molecular sieves. A concise one-step synthesis was developed for the selective monoamidation of piperazine with (<i>S</i>)-lactate to produce the piperazine lactamide starting material. The boronic acid was produced from 2-amino-5-bromopyrimidine in a one-step and protecting-group-free approach. The final crystallization in 1-propanol and water afforded the API in 59% overall yield in four steps and >99% purity by HPLC

Two subsets of CD8⁺ T cells are retained within human lymphoid tissues.

<p>The co-expression of CD69 and CD103 by CD8⁺ T cells isolated from spleen and tonsils were determined by flow cytometry. (A and B) Representative flow cytometry plots (left) and graphs (right; mean ± SEM; n = 10) show the proportion of CD69⁺CD8⁺ T cells expressing CD103 in human spleen (A) and tonsils (B). (C—E) Representative flow cytometry plots showing the expression levels of CCR7, CD45RA and CD11a between CD69^—CD103^—(blue), CD69⁺CD103^—(red) and CD69⁺CD103⁺ (green) CD8⁺ T cell subsets from the spleen (C) and tonsils (D) and the expression of PD-1, TIM3 and BTLA in tonsils (E). Data is representative of 3–5 independent experiments. (F) Relative expression of <i>KLF2</i> and <i>S1PR1</i> in purified CD8⁺ T subsets from the spleen (left panels) and tonsils (right panels). Individual dots represent different donor samples (n = 8 for spleen and n = 3 for tonsils). Statistical analysis was performed using one-way ANOVA and Tukey test. P<0.05 is noted with * and P<0.0005 is noted with ***.</p