Chemotaxis of T cell subsets ex vivo

Titelblatt, Inhaltsverzeichnis, Lebenslauf 1\. Einleitung 2\. Material und Methoden 3.1 Ergebnisse Teil 1 3.2 Ergebnisse Teil 2 3.3 Ergebnisse Teil 3 4\. Diskussion 5\. Zusammenfassung / Summary 6\. Tabellenanhang LiteraturverzeichnisChemokine sind wichtig in der Homýostase und Regulation des Immunsystems.Neben ihrer Fýhigkeit Lymphozyten in lymphatische Organe und distinkte Gewebskompartimente zu locken,erfýllen sie wichtige Funktionen in Entzýndung. Von Studien mit in vitro erzeugten Th1-und Th2-Zellen ist bekannt,dass diese Zellen differentiell auf chemotaktische Einflýsse reagieren.Um zu klýren,ob dies auch fýr CD4 + Effektor/Memory T-Zellen gilt,die in vivo entstanden sind,wurden solche Zellen aus Milzen unbeeinflusster und immunisierter Mýuse gewonnen und ex vivo im Chemotaxisassay eingesetzt.Hierbei wurde die Migration zu verschiedenen induzierbaren und homýostatischen Chemokinen getestet. Die Ergebnisse zeigen,dass IL-4 produzierende T-Zellen die hýchste Antwortfýhigkeit gegenýber dem ýTh2-Chemokin"CCL17 zeigten,wýhrend das ýTh1-Chemokin"CXCL9 prýferentiell,aber nicht ausschlieýlich,IFN-? -produzierende T-Zellen anlockte. CXCL13,ein Chemokin,das fýr die Verteilung von T-und B-Zellen in den B-Zell-Follikel lymphatischer Organe verantwortlich ist,hat bevorzugt IL-10-produzierende T-Zellen angelockt,wohingegen andere Zytokinproduzenten weniger reagierten.Nach Immunisierung ýnderte sich dieses Migrationsprofil und IL-4 + T-Zellen waren die am stýrksten reaktive Fraktion zu CXCL13. Der korrespondierende Rezeptor fýr CCL21 und CCL19,CCR7 wurde,wie in der Literatur berichtet,hauptsýchlich auf der in vitro erzeugten Th1-Subpopulation gefunden.Im Gegensatz dazu,konnte ex vivo eine starke Migration von IL-4 und/oder IFN-? - produzierenden T-Zellen zu CCL21 detektiert werden. CCR7 wurde zuvor beschrieben humane Memory T-Zellen in zwei funktionell unterschiedliche Subpopulationen zu trennen,wobei alle Zytokinproduzenten innerhalb der CCR7-negativen Fraktion liegen sollten.In der vorliegenden Studie migrierten dagegen die meisten der murinen oder humanen Effektor/Memory T-Zellen zu CCR7-Liganden.Im Einklang dazu,zeigten die Zellen auch eine positive Oberfýchenfýrbung fýr den Rezeptor. Durch Versuche mit CCR7 -/- -Mýusen konnte gezeigt werden,dass CCR7 der einzige Rezeptor ist,der fýr die Chemotaxis zu CCL21 verantwortlich ist.CCR7-defiziente Th1- Zellen waren in ihrer Fýhigkeit in Lymphknoten und Peyer´sche Plaques zu migrieren stark beeintrýchtigt,aber in der Lage in eine Hautentzýndung einzudringen. Die Ergebnisse weisen daraufhin,dass Effektor/Memory T-Zellen zwar subpopulationsspezifische Prýferenzen fýr induzierbare Chemokine besitzen,diese aber weniger streng ausfallen,als fýr in vitro differenzierte Effektorzellen.Auýerdem behalten in vivo erzeugte Effektor/Memory T-Zellen ihre Fýhigkeit in sekundýre lymphatische Organe durch CCR7 zu rezirkulieren.Chemokines are important in the homeostasis and regulation of the immune system.Besides their capability of targeting lymphocytes into lymphoid organs and respective tissue compartments they fulfill specialized roles in inflammation. It is known from studies with in vitro generated Th1 and Th2 cells that these cells respond differentially to chemotactic agents.To clarify whether this also applies for in vivo primed effector/memory T cells,we isolated them from spleens of untreated and immunized mice and analyzed their migratory properties to various inducible and lymphoid chemokines ex vivo in a chemotaxis assay. The results show,that IL-4 producing T cells exhibited the highest response to the "Th2 chemokine"CCL17,whereas the "Th1 chemokine"CXCL9 preferentially,but not exclusively,attracted IFN-? producing cells. CXCL13 a chemokine that attracts T and B cells into the B cell-follicle of lymphoid tissues was found to preferentially attract IL-10-producing T cells,whereas other cytokine subsets reacted at lower levels.Interestingly,after immunization this migration pattern changed and IL-4-positive T cells exhibited the strongest response toward CXCL13. The corresponding receptor for CCL21 and CCL19 CCR7 was found,as reported,to be dominantly expressed by murine in vitro polarized Th1 cells.Conversely,we detected ex vivo a strong migratory response of IL-4 and/or IFN-? positive T cells toward CCL21. In the human system CCR7 was described to discriminate between two functional subsets of memory T cells,with all effector cytokine-producing T cells within the CCR7-negative subset.In contrast to that,we found the majority of mouse and human cytokine producing T cells responsive toward CCR7 ligands and in addition they also stained on the cell surface for the receptor. By using CCR7 -/- mice we were able to show that CCR7 is the only responsible receptor on CCL21-responsive murine T cells.Th1 cells generated from CCR7 -/- mice were reduced in their capacity to enter lymph nodes and Peyer´s patches,but did enter a site of inflammation. These findings indicate that CD4 + cells producing effector cytokines upon stimulation exhibit subset specific preferences for inducible chemokines,which are less restricted than for in vitro primed effector cells.Additionally,in vivo differentiated effector/memory T cells retain the capacity to recirculate through lymphoid tissues via CCR7

Approaches to overcome flow cytometry limitations in the analysis of cells from veterinary relevant species

BACKGROUND: Flow cytometry is a powerful tool for the multiparameter analysis of leukocyte subsets on the single cell level. Recent advances have greatly increased the number of fluorochrome-labeled antibodies in flow cytometry. In particular, an increase in available fluorochromes with distinct excitation and emission spectra combined with novel multicolor flow cytometers with several lasers have enhanced the generation of multidimensional expression data for leukocytes and other cell types. However, these advances have mainly benefited the analysis of human or mouse cell samples given the lack of reagents for most animal species. The flow cytometric analysis of important veterinary, agricultural, wildlife, and other animal species is still hampered by several technical limitations, even though animal species other than the mouse can serve as more accurate models of specific human physiology and diseases. RESULTS: Here we present time-tested approaches that our laboratory regularly uses in the multiparameter flow cytometric analysis of ovine leukocytes. The discussed approaches will be applicable to the analysis of cells from most animal species and include direct modification of antibodies by covalent conjugation or Fc-directed labeling (Zenon™ technology), labeled secondary antibodies and other second step reagents, labeled receptor ligands, and antibodies with species cross-reactivity. CONCLUSIONS: Using refined technical approaches, the number of parameters analyzed by flow cytometry per cell sample can be greatly increased, enabling multidimensional analysis of rare samples and giving critical insight into veterinary and other less commonly analyzed species. By maximizing information from each cell sample, multicolor flow cytometry can reduce the required number of animals used in a study

T Cell Migration from Inflamed Skin to Draining Lymph Nodes Requires Intralymphatic Crawling Supported by ICAM-1/LFA-1 Interactions.

T cells are the most abundant cell type found in afferent lymph, but their migration through lymphatic vessels (LVs) remains poorly understood. Performing intravital microscopy in the murine skin, we imaged T cell migration through afferent LVs in vivo. T cells entered into and actively migrated within lymphatic capillaries but were passively transported in contractile collecting vessels. Intralymphatic T cell number and motility were increased during contact-hypersensitivity-induced inflammation and dependent on ICAM-1/LFA-1 interactions. In vitro, blockade of endothelial cell-expressed ICAM-1 reduced T cell adhesion, crawling, and transmigration across lymphatic endothelium and decreased T cell advancement from capillaries into lymphatic collectors in skin explants. In vivo, T cell migration to draining lymph nodes was significantly reduced upon ICAM-1 or LFA-1 blockade. Our findings indicate that T cell migration through LVs occurs in distinct steps and reveal a key role for ICAM-1/LFA-1 interactions in this process

Secreted IgM Modulates IL-10 Expression in B Cells

IL-10+ B cells are critical for immune homeostasis and restraining immune responses in infection, cancer, and inflammation; however, the signals that govern IL-10+ B cell differentiation are ill-defined. Here we find that IL-10+ B cells expand in mice lacking secreted IgM ((s)IgM–/–) up to 10-fold relative to wildtype (WT) among all major B cell and regulatory B cell subsets. The IL-10+ B cell increase is polyclonal and presents within 24 hours of birth. In WT mice, sIgM is produced prenatally and limits the expansion of IL-10+ B cells. Lack of the high affinity receptor for sIgM, FcμR, in B cells translates into an intermediate IL-10+ B cell phenotype relative to WT or sIgM–/– mice. Our study thus shows that sIgM regulates IL-10 programming in B cells in part via B cell-expressed FcμR, thereby revealing a function of sIgM in regulating immune homeostasis

Developmental Stage, Phenotype, and Migration Distinguish Naive- and Effector/Memory-like CD4+ Regulatory T Cells

Regulatory T cells (Tregs) fulfill a central role in immune regulation. We reported previously that the integrin αEβ7 discriminates distinct subsets of murine CD4+ regulatory T cells. Use of this marker has now helped to unravel a fundamental dichotomy among regulatory T cells. αE−CD25+ cells expressed L-selectin and CCR7, enabling recirculation through lymphoid tissues. In contrast, αE-positive subsets (CD25+ and CD25−) displayed an effector/memory phenotype expressing high levels of E/P-selectin–binding ligands, multiple adhesion molecules as well as receptors for inflammatory chemokines, allowing efficient migration into inflamed sites. Accordingly, αE-expressing cells were found to be the most potent suppressors of inflammatory processes in disease models such as antigen-induced arthritis

The Lack of Natural IgM Increases Susceptibility and Impairs Anti-Vi Polysaccharide IgG Responses in a Mouse Model of Typhoid

Circulating IgM present in the body prior to any apparent Ag exposure is referred to as natural IgM. Natural IgM provides protective immunity against a variety of pathogens. Salmonella enterica serovar Typhi (S. Typhi) is the causative agent of typhoid fever in humans. Because mice are not permissive to S. Typhi infection, we employed a murine model of typhoid using S. enterica serovar Typhimurium expressing the Vi polysaccharide (ViPS) of S. Typhi (S. Typhimurium strain RC60) to evaluate the role of natural IgM in pathogenesis. We found that natural mouse IgM binds to S. Typhi and S. Typhimurium. The severity of S. Typhimurium infection in mice is dependent on presence of the natural resistance-associated macrophage protein 1 (Nramp1) allele; therefore, we infected mice deficient in secreted form of IgM (sIgM) on either a Nramp1-resistant (129S) or -susceptible (C57BL/6J) background. We found that the lack of natural IgM results in a significantly increased susceptibility and an exaggerated liver pathology regardless of the route of infection or the Nramp1 allele. Reconstitution of sIgM-/- mice with normal mouse serum or purified polyclonal IgM restored the resistance to that of sIgM+/+ mice. Furthermore, immunization of sIgM-/- mice with heat-killed S. Typhi induced a significantly reduced anti-ViPS IgG and complement-dependent bactericidal activity against S. Typhi in vitro, compared with that of sIgM+/+ mice. These findings indicate that natural IgM is an important factor in reducing the typhoid severity and inducing an optimal anti-ViPS IgG response to vaccination

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

CXCR4 is dispensable for T cell egress from chronically inflamed skin via the afferent lymph.

T cell recirculation through extralymphoid tissues is essential to immune surveillance, host defense and inflammation. In this process, T cells enter the tissue from the blood and subsequently leave via the afferent lymph. In the absence of inflammation, T cells require CCR7 expression to egress from the skin or lung, which is consistent with the constitutive expression of the CCR7 ligand CCL21 on lymphatic endothelium. However, during chronic inflammation alternative chemoattractants come into play, allowing Ccr7-deficient (Ccr7-/-) T cells to egress efficiently from affected skin. As T cell egress from inflamed sites is a potential control point of the inflammatory response, we aimed to determine alternative T cell exit receptors using a mouse and a sheep model. We show that CCR7+ and CCR7- T cells exiting from the chronically inflamed skin were highly responsive to the CXCR4 ligand CXCL12, which was induced in the lymphatics in the inflamed site. Based on these findings, we hypothesized that CXCR4 mediates T cell egress from inflamed skin. However, pharmacological inhibition of CXCR4 did not affect the tissue egress of wildtype or Ccr7-/- CD4 and CD8 T cells after adoptive transfer into chronically inflamed skin. Similarly, adoptively transferred Cxcr4-/- Ccr7-/- and Ccr7-/- T cells egressed from the inflamed skin equally well. Based on these data, we conclude that, while CXCR4 might play an essential role for other cell types that enter the afferent lymphatics, it is dispensable for T cell egress from the chronically inflamed skin