2021-09-21, Ghosh, Shirsendu, Feigelson, Sara W., Montresor, Alessio, Shimoni, Eyal, Roncato, Francesco, Legler, Daniel F., Laudanna, Carlo, Haran, Gilad, Alon, Ronen

Leukocyte microvilli are elastic actin-rich projections implicated in rapid sensing and penetration across glycocalyx barriers. Microvilli are critical for the capture and arrest of flowing lymphocytes by high endothelial venules, the main lymph node portal vessels. T lymphocyte arrest involves subsecond activation of the integrin LFA-1 by the G-protein-coupled receptor CCR7 and its endothelial-displayed ligands, the chemokines CCL21 and CCL19. The topographical distribution of CCR7 and of LFA-1 in relation to lymphocyte microvilli has never been elucidated. We applied the recently developed microvillar cartography imaging technique to determine the topographical distribution of CCR7 and LFA-1 with respect to microvilli on peripheral blood T lymphocytes. We found that CCR7 is clustered on the tips of T cell microvilli. The vast majority of LFA-1 molecules were found on the cell body, likely assembled in macroclusters, but a subset of LFA-1, 5% of the total, were found scattered within 20 nm from the CCR7 clusters, implicating these LFA-1 molecules as targets for inside-out activation signals transmitted within a fraction of a second by chemokine-bound CCR7. Indeed, RhoA, the key GTPase involved in rapid LFA-1 affinity triggering by CCR7, was also found to be clustered near CCR7. In addition, we observed that the tyrosine kinase JAK2 controls CCR7-mediated LFA-1 affinity triggering and is also highly enriched on tips of microvilli. We propose that tips of lymphocyte microvilli are novel signalosomes for subsecond CCR7-mediated inside-out signaling to neighboring LFA-1 molecules, a critical checkpoint in LFA-1-mediated lymphocyte arrest on high endothelial venules.

2018-09, Laufer, Julia M., Lyck, Ruth, Legler, Daniel F.

The ζ-associated protein of 70 kDa (ZAP70) is expressed in the aggressive form of B-cell chronic lymphocytic leukemia (CLL). Moreover, the integrin very late antigen (VLA)-1 is highly expressed on subtypes of CLL that are associated with high proliferation rates in the lymph node context. We herein identify a critical role for ZAP70 in chemokine-mediated, inside-out signaling to integrins in trisomy 12 carrying Ohio State University-CLL cell lines derived from a patient with previously treated CLL. We found that ZAP70-positive CLL cells migrated significantly better toward ligands of the lymph node homing chemokine receptors CCR7 and CXCR4 compared with ZAP70-negative cells. In addition, ZAP70-expressing CLL cells adhered more efficiently to integrin ligands under static conditions. We discovered that ZAP70 expression controls chemokine-driven clustering of the integrins VLA-4 and lymphocyte function-associated antigen-1. More precisely, chemokine stimulation resulted in a ZAP70-dependent integrin valency regulation on CLL cells, whereas high-affinity regulation of integrins was independent of ZAP70. Consequently, ZAP70-expressing CLL cells show increased chemokine-driven arrest on immobilized integrin ligands and on chemokine-presenting endothelial cells under physiologic flow conditions. Hence, we describe a novel mechanism showing how ZAP70 controls chemokine-driven valency regulation of integrins and arrest of CLL cells on endothelial cells, a process that might contribute to CLL disease progression.-Laufer, J. M., Lyck, R., Legler, D. F. ZAP70 expression enhances chemokine-driven chronic lymphocytic leukemia cell migration and arrest by valency regulation of integrins.

2011-12-01, Schäuble, Karin, Hauser, Mark A., Singer, Eva, Gröttrup, Marcus, Legler, Daniel F.

Lymphocyte homing to, and motility within, lymph nodes is regulated by the chemokine receptor CCR7 and its two ligands CCL19 and CCL21. There, lymphocytes are exposed to a number of extracellular stimuli that influence cellular functions and determine the cell fate. In this study, we assessed the effect of TCR engagement on CCR7-mediated cell migration. We found that long-term TCR triggering of freshly isolated human T cells through CD3/CD28 attenuated CCR7-driven chemotaxis, whereas short-term activation significantly enhanced CCR7-mediated, but not CXCR4-mediated, migration efficiency. Short-term activation most prominently enhanced the migratory response of naive T cells of both CD4 and CD8 subsets. We identified distinct roles for Src family kinases in modulating CCR7-mediated T cell migration. We provide evidence that Fyn, together with Ca(2+)-independent protein kinase C isoforms, kept the migratory response of naive T cells toward CCL21 at a low level. In nonactivated T cells, CCR7 triggering induced a Fyn-dependent phosphorylation of the inhibitory Tyr505 of Lck. Inhibiting Fyn in these nonactivated T cells prevented the negative regulation of Lck and facilitated high CCR7-driven T cell chemotaxis. Moreover, we found that the enhanced migration of short-term activated T cells was accompanied by a synergistic, Src-dependent activation of the adaptor molecule linker for activation of T cells. Collectively, we characterize a cross-talk between the TCR and CCR7 and provide mechanistic evidence that the activation status of T cells controls lymphocyte motility and sets a threshold for their migratory response.

2006, Legler, Daniel F., Krause, Petra, Scandella, Elke, Singer, Eva, Gröttrup, Marcus

The control of dendritic cell (DC) migration is pivotal for the initiation of cellular immune responses. In this study, we demonstrate that the migration of human monocyte-derived (Mo)DCs as well as of ex vivo peripheral blood DCs toward CCL21, CXCL12, and C5a is stringently dependent on the presence of the proinflammatory mediator PGE2, although DCs expressed CXCR4 and C5aR on their surface and DC maturation was accompanied by CCR7 up-regulation independently of PGE2. The necessity of exogenous PGE2 for DC migration is not due to the suppression of PGE2 synthesis by IL-4, which is used for MoDC differentiation, because maturation-induced endogenous production of PGE2 cannot promote DC migration. Surprisingly, PGE2 was absolutely required at early time points of maturation to enable MoDC chemotaxis, whereas PGE2 addition during terminal maturation events was ineffective. In contrast to mouse DCs, which exclusively rely on EP4 receptor triggering for migration, human MoDCs require a signal mediated by EP2 or EP4 either alone or in combination. Our results provide clear evidence that PGE2 is a general and mandatory factor for the development of a migratory phenotype of human MoDCs as well as for peripheral blood myeloid DCs.

2019-11-01, Ficht, Xenia, Ruef, Nora, Stolp, Bettina, Samson, Guerric P. B., Moalli, Federica, Page, Nicolas, Merkler, Doron, Nichols, Ben J., Diz-Muñoz, Alba, Legler, Daniel F.

Flotillin-1 (Flot1) is an evolutionary conserved, ubiquitously expressed lipid raft-associated scaffolding protein. Migration of Flot1-deficient neutrophils is impaired because of a decrease in myosin II-mediated contractility. Flot1 also accumulates in the uropod of polarized T cells, suggesting an analogous role in T cell migration. In this study, we analyzed morphology and migration parameters of murine wild-type and Flot1-/- CD8+ T cells using in vitro assays and intravital two-photon microscopy of lymphoid and nonlymphoid tissues. Flot1-/- CD8+ T cells displayed significant alterations in cell shape and motility parameters in vivo but showed comparable homing to lymphoid organs and intact in vitro migration to chemokines. Furthermore, their clonal expansion and infiltration into nonlymphoid tissues during primary and secondary antiviral immune responses was comparable to wild-type CD8+ T cells. Taken together, Flot1 plays a detectable but unexpectedly minor role for CD8+ T cell behavior under physiological conditions.

2018-09, Thelen, Marcus, Legler, Daniel F.

Migration of leukocytes is typically mediated by G protein-coupled receptors (GPCRs) upon activation by specific ligands that range from small peptides, chemokines to a variety of lipidic molecules. The heptahelical receptors are highly dynamic structures and the signaling efficiency largely depends on the discrete contact with the ligand. In addition, several allosteric modulators of receptor activity have been reported, which do not induce migration by themselves. Another important mechanism modulating the activity of GPCRs is their local environment. Not only the membrane lipid composition influences the activity, but also direct binding of lipids, in particular cholesterol, was shown to alter receptor signaling properties. Recent findings indicate that also chemokine receptor activity is modulated by membrane lipids. In this short review we discuss this new paradigm and potential consequences for chemokine-induced migration.

2010-09-15, Johannsen, Alexandre, Genolet, Raphael, Legler, Daniel F., Luther, Sanjiv A., Luescher, Immanuel F.

An attractive treatment of cancer consists in inducing tumor-eradicating CD8⁺ CTL specific for tumor-associated Ags, such as NY-ESO-1 (ESO), a strongly immunogenic cancer germ line gene-encoded tumor-associated Ag, widely expressed on diverse tumors. To establish optimal priming of ESO-specific CTL and to define critical vaccine variables and mechanisms, we used HLA-A2/DR1 H-2^-/- transgenic mice and sequential immunization with immunodominant DR1- and A2-restricted ESO peptides. Immunization of mice first with the DR1-restricted ESO_123-137 peptide and subsequently with mature dendritic cells (DCs) presenting this and the A2-restriced ESO_157-165 epitope generated abundant, circulating, high-avidity primary and memory CD8⁺ T cells that efficiently killed A2/ESO_157-165⁺ tumor cells. This prime boost regimen was superior to other vaccine regimes and required strong Th1 cell responses, copresentation of MHC class I and MHC class II peptides by the same DC, and resulted in upregulation of sphingosine 1-phosphate receptor 1, and thus egress of freshly primed CD8⁺ T cells from the draining lymph nodes into circulation. This well-defined system allowed detailed mechanistic analysis, which revealed that 1) the Th1 cytokines IFN-gamma and IL-2 played key roles in CTL priming, namely by upregulating on naive CD8⁺ T cells the chemokine receptor CCR5; 2) the inflammatory chemokines CCL4 (MIP-1beta) and CCL3 (MIP-1alpha) chemoattracted primed CD4⁺ T cells to mature DCs and activated, naive CD8⁺ T cells to DC-CD4 conjugates, respectively; and 3) blockade of these chemokines or their common receptor CCR5 ablated priming of CD8(+) T cells and upregulation of sphingosine 1-phosphate receptor 1. These findings provide new opportunities for improving T cell cancer vaccines.

2019-07, Impellizzieri, Daniela, Ridder, Frederike, Raeber, Miro E., Egholm, Cecilie, Woytschak, Janine, Kolios, Antonios G.A., Legler, Daniel F., Boyman, Onur

Background
Type 2 immunity serves to resist parasitic helminths, venoms, and toxins, but the role and regulation of neutrophils during type 2 immune responses are controversial. Helminth models suggested a contribution of neutrophils to type 2 immunity, whereas neutrophils are associated with increased disease severity during type 2 inflammatory disorders, such as asthma.

Objective
We sought to evaluate the effect of the prototypic type 2 cytokines IL-4 and IL-13 on human neutrophils.

Methods
Human neutrophils from peripheral blood were assessed without or with IL-4 or IL-13 for (1) expression of IL-4 receptor subunits, (2) neutrophil extracellular trap (NET) formation, (3) migration toward CXCL8 in vitro and in humanized mice, and (4) CXCR1, CXCR2, and CXCR4 expression, as well as (5) in nonallergic versus allergic subjects.

Results
Human neutrophils expressed both types of IL-4 receptors, and their stimulation through IL-4 or IL-13 diminished their ability to form NETs and migrate toward CXCL8 in vitro. Likewise, in vivo chemotaxis in NOD-scid-Il2rg−/− mice was reduced in IL-4–stimulated human neutrophils compared with control values. These effects were accompanied by downregulation of the CXCL8-binding chemokine receptors CXCR1 and CXCR2 on human neutrophils on IL-4 or IL-13 stimulation in vitro. Ex vivo analysis of neutrophils from allergic patients or exposure of neutrophils from nonallergic subjects to allergic donor serum in vitro impaired their NET formation and migration toward CXCL8, thereby mirroring IL-4/IL-13–stimulated neutrophils.

Conclusion
Signaling in human neutrophils affects several neutrophil effector functions, which bears important implications for immunity in type 2 inflammatory disorders.

2017-04, Legler, Daniel F., Matti, Christoph, Laufer, Julia M., Jakobs, Barbara, Purvanov, Vladimir, Uetz-von Allmen, Edith, Thelen, Marcus

Chemokine receptors are seven transmembrane-domain receptors belonging to class A of G protein-coupled receptors (GPCRs). The receptors together with their chemokine ligands constitute the chemokine system, which is essential for directing cell migration and plays a crucial role in a variety of physiological and pathological processes. Given the importance of orchestrating cell migration, it is vital that chemokine receptor signaling is tightly regulated to ensure appropriate responses. Recent studies highlight a key role for cholesterol in modulating chemokine receptor activities. The steroid influences the spatial organization of GPCRs within the membrane bilayer, and consequently can tune chemokine receptor signaling. The effects of cholesterol on organization and function of chemokine receptors and GPCRs in general include direct and indirect effects. Here, we review how cholesterol and some key metabolites modulate the chemokine system functions by multiple ways. We emphasize on the role of cholesterol in chemokine receptor oligomerization, thereby promoting the formation of a signaling hub enabling integration of distinct signaling pathways at the receptor-membrane interface. Moreover, we discuss the role of cholesterol in stabilizing particular receptor conformations and its consequence for chemokine binding. Finally, we highlight how cholesterol accumulation, its deprivation or cholesterol metabolites contribute to modulate cell orchestration during inflammation, upon induction of an adaptive immune response, as well as in dampening the anti-tumor immune response.

2010, Metzner, Christoph, Legler, Daniel F., Dangerfield, John A.

This chapter covers the use of glycosylphosphatidylinositol (GPI)-anchored proteins for surface modification of diverse types of biomembrane covered entities ranging from viruses and virus-related particles (section 1), to cells (section 2) and other natural and engineered micro- and nano-scaled particles (section 3). The aim is to present and review state-of-the-art research in this area and to discuss the future direction of GPI painting technology relating to applications in research, biotechnology and biomedicine.