Murine CD146 is widely expressed on endothelial cells and is recognized by the monoclonal antibody ME-9F1

The endothelium plays an important role in the exchange of molecules, but also of immune cells between blood and the underlying tissue. The endothelial molecule S-Endo 1 antigen (CD146) is preferentially located at endothelial junctions and has been claimed to support endothelial integrity. In this study we show that the monoclonal antibody ME-9F1 recognizes the extracellular portion of murine CD146. Making use of ME-9F1 we found CD146 highly expressed and widely spread on endothelial cells in the analyzed murine tissues. In contrast to humans that express CD146 also on T cells or follicular dendritic cells, murine CD146 albeit at low levels was only found on a subset of NK1.1+ cells. The antibody against murine CD146 is useful for immunomagnetic sorting of primary endothelial cells not only from the liver but from various other organs. In vitro, no evidence was seen that the formation and integrity of endothelial monolayers or the transendothelial migration of T cells was affected by antibody binding to CD146 or by crosslinking of the antigen. This makes the antibody ME-9F1 an excellent tool especially for the ex vivo isolation of murine endothelial cells intended to be used in functional studies

CD152 (CTLA-4) Determines CD4 T Cell Migration In Vitro and In Vivo

BACKGROUND:Migration of antigen-experienced T cells to secondary lymphoid organs and the site of antigenic-challenge is a mandatory prerequisite for the precise functioning of adaptive immune responses. The surface molecule CD152 (CTLA-4) is mostly considered as a negative regulator of T cell activation during immune responses. It is currently unknown whether CD152 can also influence chemokine-driven T cell migration. METHODOLOGY/PRINCIPAL FINDINGS:We analyzed the consequences of CD152 signaling on Th cell migration using chemotaxis assays in vitro and radioactive cell tracking in vivo. We show here that the genetic and serological inactivation of CD152 in Th1 cells reduced migration towards CCL4, CXCL12 and CCL19, but not CXCL9, in a G-protein dependent manner. In addition, retroviral transduction of CD152 cDNA into CD152 negative cells restored Th1 cell migration. Crosslinking of CD152 together with CD3 and CD28 stimulation on activated Th1 cells increased expression of the chemokine receptors CCR5 and CCR7, which in turn enhanced cell migration. Using sensitive liposome technology, we show that mature dendritic cells but not activated B cells were potent at inducing surface CD152 expression and the CD152-mediated migration-enhancing signals. Importantly, migration of CD152 positive Th1 lymphocytes in in vivo experiments increased more than 200% as compared to CD152 negative counterparts showing that indeed CD152 orchestrates specific migration of selected Th1 cells to sites of inflammation and antigenic challenge in vivo. CONCLUSIONS/SIGNIFICANCE:We show here, that CD152 signaling does not just silence cells, but selects individual ones for migration. This novel activity of CD152 adds to the already significant role of CD152 in controlling peripheral immune responses by allowing T cells to localize correctly during infection. It also suggests that interference with CD152 signaling provides a tool for altering the cellular composition at sites of inflammation and antigenic challenge

Interaktion von T-Zellen mit sinusoidalen Endothelzellen der Leber

Auch unter physiologischen Bedingungen finden sich T-Zellen und andere Leukozyten nicht nur in den Sinusoiden, sondern auch im Parenchym der Leber. Da die Leber u. a. verschiedene Aufgaben für das Immunsystem übernimmt (z. B. Deletion aktivierter T Zellen, Induktion peripherer Toleranz), könnte die Akkumulation der T-Zellen in der Leber - neben der immunologischen Überwachung der Leber - Voraussetzung für ihre Modulation sein. In der vorliegenden Arbeit wurde der Einfluss von Leber-sinusoidalen Endothelzellen (LSEC), der Barriere zwischen Blut und Leber-Parenchym, auf CD4+ T-Zellen untersucht. Zum einen zeigte sich, dass die LSEC sowohl die spontane Transmigration der T-Zellen, als auch ihre Chemotaxis zu CXCL9 und CXCL12 effizienter unterstützen als andere Endothelien. Eine endotheliale Aktivierung durch die Chemokine wurde als Mechanismus ausgeschlossen. Dagegen schien eine effiziente Präsentation der Chemokine auf der luminalen LSEC-Oberfläche nach Aufnahme von abluminal für die gesteigerte Transmigration der T Zellen verantwortlich zu sein. Die LSEC könnten somit in vivo an der Rekrutierung von T-Zellen in die Leber beteiligt sein, indem sie eine rasche Wanderung der T-Zellen aus dem Blut ins Parenchym und möglicherweise auch zurück in die Zirkulation zulassen. Des Weiteren konnte gezeigt werden, dass die LSEC fähig sind, naive CD4+ T-Zellen in vitro Antigen-spezifisch zu aktivieren. Im Vergleich zu professionellen APZ war hierfür eine höhere Antigen-Dosis notwendig, die Expansion schwächer und es waren kaum Effektorzytokin-Produzenten detektierbar. Diese konnten jedoch durch Restimulierung mit professionellen APZ induziert werden (reversibler Phänotyp), was auf einen unreifen Differenzierungsstatus der T-Zellen schließen ließ. Es bleibt zu prüfen, in welchem Maße die Aktivierung naiver CD4+ T-Zellen durch LSEC in vivo stattfindet und diese durch LSEC aktivierten CD4+ T-Zellen funktionelle Bedeutung, z. B. regulatorische Kapazität, für das Immunsystem besitzen.The liver plays a major role for the metabolism, but it is also of general importance for the immune system, e.g. for the deletion of activated T cells or the induction of peripheral tolerance. Under physiological conditions T cells and other leukocytes can be found in the liver, in the sinusoids as well as in the parenchyma. This hepatic accumulation of T cells might be due to immunosurveillance, but it would also be a prerequisite for modulation of T cells by hepatic cells. The present study investigated two different aspects of the interaction of liver sinusoidal endothelial cells (LSEC), the barrier between the sinusoidal lumen and the hepatic parenchyma, and CD4+ T cells. In the first part of the study it could be demonstrated that LSEC support the spontaneous transmigration of CD4+ T cells as well as their chemotaxis to CXCL12 and CXCL9 more efficiently than other endothelial cells. Whereas a direct endothelial activation by chemokines could be excluded the efficient chemokine presentation at the luminal LSEC surface (after abluminal uptake) might be responsible for the enhanced T cell transmigration. The findings suggest that LSEC might be involved in the recruitment of T cells by supporting a rapid transendothelial migration. The second part of the study focused on the characteristics of LSEC in the context of antigen presentation. LSEC were able to prime and expand naïve CD4+ T cells in vitro but less effective than professional APC as proven by weaker expansion of cells, a requirement for higher antigen concentration and the lack of cytokine producing T cells. The “immature effector” phenotype of the CD4+ T cells primed on LSEC was reversible since it could be overcome by restimulation on professional APC. In conclusion these data suggest that antigen presentation by LSEC results in activation but incomplete differentiation of CD4+ T cells

Enhanced T cell transmigration across the murine liver sinusoidal endothelium is mediated by transcytosis and surface presentation of chemokines

Transmigration through the liver endothelium is a prerequisite for the homeostatic balance of intrahepatic T cells and a key regulator of inflammatory processes within the liver. Extravasation into the liver parenchyma is regulated by the distinct expression patterns of adhesion molecules and chemokines and their receptors on the lymphocyte and endothelial cell surface. In the present study, we investigated whether liver sinusoidal endothelial cells (LSEC) inhibit or support the chemokine-driven transmigration and differentially influence the transmigration of pro-inflammatory or anti-inflammatory CD4(+) T cells, indicating a mechanism of hepatic immunoregulation. Finally, the results shed light on the molecular mechanisms by which LSEC modulate chemokine-dependent transmigration. LSEC significantly enhanced the chemotactic effect of CXC-motif chemokine ligand 12 (CXCL12) and CXCL9, but not of CXCL16 or CCL20, on naive and memory CD4(+) T cells of a T helper 1, T helper 2, or interleukin-10-producing phenotype. In contrast, brain and lymphatic endothelioma cells and ex vivo isolated lung endothelia inhibited chemokine-driven transmigration. As for the molecular mechanisms, chemokine-induced activation of LSEC was excluded by blockage of G(i)-protein-coupled signaling and the use of knockout mice. After preincubation of CXCL12 to the basal side, LSEC took up CXCL12 and enhanced transmigration as efficiently as in the presence of the soluble chemokine. Blockage of transcytosis in LSEC significantly inhibited this effect, and this suggested that chemokines taken up from the basolateral side and presented on the luminal side of endothelial cells trigger T cell transmigration. CONCLUSION: Our findings demonstrate a unique capacity of LSEC to present chemokines to circulating lymphocytes and highlight the importance of endothelial cells for the in vivo effects of chemokines. Chemokine presentation by LSEC could provide a future therapeutic target for inhibiting lymphocyte immigration and suppressing hepatic inflammation

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