Afferent lymph vessels in chronically inflamed skin express CXCL12.

<p>Immunofluorescence staining of frozen sections of chronically inflamed skin (21 days after inflammation elicited by subcutaneous injection of CFA) for CXC12 and CCL21 by LYVE-1⁺ lymph vessels using DAPI counterstaining. One representative image of lymph vessels analyzed in 5 mice is shown. Scale bars, 10 µm.</p

Ovine T cells that exit the inflamed and uninflamed skin efficiently migrate to CXCL12.

<p>Lymphocytes traveling in afferent lymph draining uninflamed or chronically inflamed skin (>d15 post induction of inflammation with CFA) were collected from sheep, and chemotaxis was tested toward mouse CCL21 and human CXCL12 in a Transwell chemotaxis assay. Migration of CD4, CD8 and γδ T cells is expressed as the percentage of each T cell subset that migrated to the lower chamber. Data points represent the mean migration of T cells from individual animals based on triplicate wells for each concentration, and the mean ± SEM for all animals analyzed is shown. N = 5–7 animals per condition and T cell subset.</p

Cxcr4 deficiency does not reduce the tissue exit of Ccr7^−/− T cells from chronically inflamed skin.

<p>(<b>A–C</b>) Adoptive transfer exit experiments with fetal liver chimeric mice as a source of donor splenocytes. As depicted in the experimental scheme (<b>A</b>), fetal liver chimeras were generated by reconstituting lethally irradiated CD45.1⁺ wildtype recipient mice with fetal liver cells from (CD45.2⁺) <i>Cxcr4</i>^−/−<i>Ccr</i>7^−/− or (CD45.2⁺) <i>Ccr7</i>^−/− or (CD45.1⁺) wildtype donors. Splenocytes from the fetal liver chimeras were labeled with eFlour670 and CFSE, so that the combination of congenic maker and fluorescent label uniquely marked cells of each genotype. Labeled splenocytes of all three genotypes were mixed and co-injected into chronically inflamed footpads. (<b>B</b>) Analysis of migrated lymphocyte subsets recovered in the draining popliteal lymph nodes 12 h post cell transfer. Data is presented as the ratio of migrated to input cells for <i>Ccr7</i>^−/− or <i>Cxcr4</i>^−/−<i>Ccr</i>7^−/− relative to wildtype cells of each subset. Connected lines represent individually analyzed recipient mice. Horizontal, non-connecting lines indicate the mean of each group. (<b>C</b>) Flow cytometric analysis of memory (CD45RB^l°) versus naïve (CD45RB^hi) phenotype CD4 and CD8 T cells in injected and migrated populations for each genotype. Data combined from 2 experiments analyzing 5–10 recipient mice per experiment (<b>B</b>) or the cell phenotype of one out two experiment with similar results (<b>C</b>) is shown. WT, wildtype.</p

Pharmacological inhibition of CXCR4 does not impact the tissue exit of wildtype or Ccr7^−/− T cells.

<p>(<b>A–B</b>) Chemotaxis of mouse splenocytes was tested toward mouse CXCL12 (<b>A</b>) and CCL21 (<b>B</b>) in a Transwell chemotaxis assay in the presence or absence of CXCR4 inhibitor AMD3100 at indicated concentrations. Data represent mean ±SD of triplicate wells. One of two similar experiments is shown. (<b>C–E</b>) Mice carrying 3-week-old skin granulomas in the footpads were systemically treated with either PBS or 1000 µg/kg/h AMD3100 through subcutaneously implanted mini osmotic pumps. 12 h after implantation, a mixture of fluorescently labeled <i>Ccr7</i>^−/− and wildtype splenocytes were transferred into the inflamed footpads. The numbers and phenotypes of cells that egressed from the skin and entered the draining lymph node were determined by flow cytometry 12 h after transfer. The numbers of wildtype (<b>C</b>) and <i>Ccr7</i>^−/− (<b>D</b>) lymphocyte subsets that migrated to the draining lymph nodes are shown. Data points represent individually analyzed mice of groups of 7–10 recipient mice per group; horizontal lines indicate the mean of each group. One of two experiments with similar results (<b>C</b> and <b>D</b>) or both experiments combined (<b>E</b>) are shown. WT, wildtype.</p

CD152 enhances chemotaxis of Th1 cells.

<p>(A) Migration of unpolarized T cells. Recall response of CD4⁺ OVA-specific TCR^tg T cells from CD152^−/− or CD152^+/+ mice was induced by adding 1 µg/ml OVA_323–339 and T cell-depleted APCs. On day 6 of recall response cells were analyzed in chemotaxis assays. Bars indicate migrated CD4⁺ cells as percentage of input cells. (B) Migration of CD152^−/− and CD152^+/+ T cells in primary stimulation: CD4⁺ OVA-specific TCR^tg T cells were stimulated with 1 µg/ml OVA_323–339 and T cell-depleted APCs. On day 6 of primary stimulation cells were analyzed in chemotaxis assays. Bars show the chemotactic index of CD4⁺ cells. (C) Specific migration of antigen-specific stimulated Th1 cells in a recall response: Primary stimulation and recall response of CD4⁺CD62L⁺ OVA-specific TCR^tg T cells were performed under Th1 conditions with 1 µg/ml OVA-peptide in the presence of 200 µg/ml neutralizing anti-CD152 Fab fragments or hamster control Fab fragments. Cells were examined in chemotaxis assay on day 6 of recall response. (D) Chemotactic index of a polyclonally induced recall response of CD152^−/− or CD152^+/+ Th1 cells: Primary stimulation and recall response of splenocytes from CD152^−/− and CD152^+/+ mice were induced polyclonally (as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0005702#s4" target="_blank">Material and Methods</a>) and CD4⁺ cells were used for chemotaxis assays on day 4 of recall response. (E) Migration of CD152^−/− and CD152^+/+ Th1 cells in a recall response is dose dependent: Primary stimulation and recall response of CD4⁺ cells from TCR^tg CD152^−/− and CD152^+/+ mice were induced antigen-specifically using Th1 conditions with indicated amounts of OVA-peptide and cells were analyzed on day 6 of recall response in chemotaxis assays. All data shown represent one out of 3–4 similar experiments.</p

Only CD152^+/+ but not CD152^−/− cells show efficient transendothelial migration.

<p>Primary stimulation and recall response of CD4⁺ OVA-specific TCR^tg T cells from CD152^−/− and CD152^+/+ mice were performed using Th1 conditions with 10 µg/ml OVA-peptide and T cell-depleted APCs. On day 6 of recall response migration of CD4⁺ cells through membranes coated with or without endothelial monolayer was analyzed after 90 min. incubation at 37°C in Transwell chemotaxis assay. CD152 mediated migration of Th1 cells is G-Protein dependent: CD4⁺ OVA-specific TCR^tg CD152^−/− and CD152^+/+ Th1 cells were incubated for 2 hours at 37°C in the presence of 100 ng/ml Pertussis toxin prior to examination in chemotaxis assay. (Inset) Endothelial cells (mlEND) were grown to a confluent monolayer for 48 h on Transwell membranes and confluency was controlled by microscopy. Shown data represent one out of 2 similar experiments.</p

CD152-enhanced migration is IFNγ independent.

<p>CD4⁺ OVA-specific TCR^tg splenocytes from CD152^−/− and CD152^+/+ were stimulated under Th1 conditions (as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0005702#pone-0005702-g002" target="_blank">Fig. 2</a>) in the presence of 20 ng/ml recombinant IFN-γ or 10 µg/ml blocking anti-IFN-γ -antibodies. On day 6 after inducing a recall response cells were analyzed for migration towards CCL4 in chemotaxis assays. The data represent one out of 3 similar experiments.</p

CD152-enhanced migration is primarily mediated by DCs.

<p>(A) Surface expression of CD152 on CD4⁺ cells is up-regulated by stimulation with DCs. Naϊve CD4⁺CD62L⁺ TCR^tg T cells were stimulated with 1 µg/ml OVA-peptide presented by matured DCs or by LPS-activated B cells. After 48 hours surface expression of CD152 was detected by liposome staining technique and subsequent FACS analysis. Left panels show CD152 staining, right panels show blocking controls. Numbers indicate the percentage of CD152⁺ CD4⁺ based on total CD4⁺ cells. (B) Equal proliferation of T cells stimulated with activated B cells or matured DCs. Naϊve CD4⁺CD62L⁺ TCR^tg T cells were labeled with CFSE and antigen-specifically stimulated with activated B cells or matured DCs. Proliferation of T cells was determined by flow cytometry 48 hours (filled curve) and 72 hours later (black line). T cells cultured with different APCs but without antigenic stimulation are shown as heavy grey line. Numbers indicate the percentage of divided T cells 72 hours after stimulation relating to unstimulated controls (after 48 h 48% of Dc-stimulated T cells and 46% of Bc-stimulated T cells proliferated). (C) Kinetics of CD152 surface expression on CD4⁺ T cells. Indicated time after onset of stimulation cells cultured as described in 5A were stained with liposome staining technique for CD152. Percentages of surface expressing CD152 cells of total CD4⁺ cells are shown. (D) Chemotactic index of Th1 cells stimulated with different APCs. CD4⁺CD62L⁺ TCR^tg T cells were stimulated under Th1 conditions with 1 µg/ml OVA peptide presented by activated B-cells or bone marrow derived DCs. On day 6 of primary stimulation, CD4⁺ cells were analyzed for migration capacity in chemotaxis assay. All data show representative data from at least two experiments.</p

Th1 cell homing to the site of inflammation and lymph nodes is enhanced by CD152.

<p>(A) Distribution of TCR^tg CD152^−/− and CD152^+/+ Th1 cells 48 hours after transfer. <i>In vitro</i> induced Th1 cells from a recall response were radioactively labeled and injected i.v. (5×10⁶ cells per mouse). After 24 hours OVA-peptide (250 ng) in IFA was administered s.c. into one footpad (DTH); PBS/IFA-injection in the other footpad served as control. Different organs were analyzed after 48 hours for radioactivity. Recovered radioactivity in organs was calculated as percentage of total amount of radioactivity per mouse (7 mice per group). (LN: mesenteric and axillary lymph nodes; drLN: draining (popliteal and inguinal) lymph nodes; PBL: peripheral blood lymphocytes). Results from one out of two similar experiments are shown. (B) Specific migration of transferred CD152^−/− and CD152^+/+ Th1 cells to the site of inflammation. Radioactively labeled CD152^−/− and CD152^+/+ Th1 cells were transferred into mice and DTH was induced in footpads as described in 7A. Shown is the antigen-dependent accumulation of Th1 cells to the site of inflammation indicating the ratio of recovered radioactivity in the inflamed footpad versus the PBS injected footpad. (C) Similar expression of activation induced markers and similar amount of IFN-γ-producers. TCR^tg CD152^−/− and CD152^+/+ CD4⁺ T cells were either left unstimulated or were <i>in vitro</i> stimulated under Th1 conditions and a recall response was performed. Unstimulated cells were stained on day 3 (upper panel) and stimulated cells on day 5 of recall response for activation induced molecules (lower panel). Intracellular staining for IFN-γ of fixed cells was performed on day 3 of recall response after restimulation with PMA/Ionomycin for 6 hours; Brefeldin A was added for the last 2 hours of incubation. One out of two similar experiments is shown.</p

rIL-27 treatment at an early stage of infection aggravates disease severity and impairs viral clearance.

<p>C57BL/6 mice were challenged with a sublethal dose influenza virus then treated daily with rIL-27 from 1–7 d.p.i. Non-treated control mice (NT) were injected with PBS. (<b>A</b>) Weight loss of rIL-27 treated (<i>n</i> = 5) or NT (<i>n</i> = 5) mice. Arrows (↓) indicate time of treatment. At 7 d.p.i., (<b>B</b>) histological scores of H&E-stained lungs from treated or NT groups and (<b>C</b>) viral <i>pa</i> mRNA expression were determined. (<b>D</b>) Numbers of influenza virus peptide-specific IL-17 or IFN-γ-producing T cells, (<b>E</b>) influx of neutrophils, (<b>F</b>) monocytes and NK cells in the lungs at 9 d.p.i. were determined by FACS. Gated cells in FACS plots in <b>e</b> indicate the percentage of neutrophils from total lung cells. Data from <b>A</b> are representative of two independent treatment experiments. Data from <b>B</b> to <b>F</b> are pooled from at least two independent experiments. <i>P</i> values were determined by unpaired two-tailed Student's <i>t</i> test. Values are means ± s.d. except for A, s.e.m.; ns, not significant.</p