Structure-Function Relationship of XCL1 Used for in vivo Targeting of Antigen Into XCR1+ Dendritic Cells.

XCL1 is the ligand for XCR1, a chemokine receptor uniquely expressed on cross-presenting dendritic cells (DC) in mouse and man. We are interested in establishing therapeutic vaccines based on XCL1-mediated targeting of peptides or proteins into these DC. Therefore, we have functionally analyzed various XCL1 domains in highly relevant settings in vitro and in vivo. Murine XCL1 fused to ovalbumin (XCL1-OVA) was compared to an N-terminal deletion variant lacking the first seven N-terminal amino acids and to several C-terminal (deletion) variants. Binding studies with primary XCR1+ DC revealed that the N-terminal region stabilizes the binding of XCL1 to its receptor, as is known for other chemokines. Deviating from the established paradigm for chemokines, the N-terminus does not contain critical elements for inducing chemotaxis. On the contrary, this region appears to limit the chemotactic action of XCL1 at higher concentrations. A participation of the XCL1 C-terminus in receptor binding or chemotaxis could be excluded in a series of experiments. Binding studies with apoptotic and necrotic XCR1-negative cells suggested a second function for XCL1: marking of stressed cells for uptake into cross-presenting DC. In vivo studies using CD8+ T cell proliferation and cytotoxicity as readouts confirmed the critical role of the N-terminus for antigen targeting, and excluded any involvement of the C-terminus in the uptake, processing, and presentation of the fused OVA antigen. Together, these studies provide basic data on the function of the various XCL1 domains as well as relevant information on XCL1 as an antigen carrier in therapeutic vaccines.

Molecular vaccine prepared by fusion of XCL1 to the multi-epitope protein of foot-and-mouth disease virus enhances the specific humoural immune response in cattle.

Targeting antigen to dendritic cells (DCs) is a promising way to manipulate the immune response and to design prophylactic molecular vaccines. In this study, the cattle XCL1, ligand of XCR1, was fused to the type O foot-and-mouth disease virus (FMDV) multi-epitope protein (XCL-OB7) to create a molecular vaccine antigen, and an △XCL-OB7 protein with a mutation in XCL1 was used as the control. XCL-OB7 protein specifically bound to the XCR1 receptor, as detected by flow cytometry. Cattle vaccinated with XCL-OB7 showed a significantly higher antibody response than that to the △XCL-OB7 control (P < 0.05). In contrast, when XCL-OB7 was incorporated with poly (I:C) to prepare the vaccine, the antibody response of the immunized cattle was significantly decreased in this group and was lower than that in the △XCL-OB7 plus poly (I:C) group. The FMDV challenge indicated that cattle immunized with the XCL-OB7 alone or the △XCL-OB7 plus poly (I:C) obtained an 80% (4/5) clinical protective rate. However, cattle vaccinated with △XCL-OB7 plus poly (I:C) showed more effective inhibition of virus replication than that in the XCL-OB7 group after viral challenge, according to the presence of antibodies against FMDV non-structural protein 3B. This is the first test of DC-targeted vaccines in veterinary medicine to use XCL1 fused to FMDV antigens. This primary result showed that an XCL1-based molecular vaccine enhanced the antibody response in cattle. This knowledge should be valuable for the development of antibody-dependent vaccines for some infectious diseases in cattle.

A Requirement for Metamorphic Interconversion in the Antimicrobial Activity of Chemokine XCL1.

Chemokines make up a superfamily of ∼50 small secreted proteins (8-12 kDa) involved in a host of physiological processes and disease states, with several previously shown to have direct antimicrobial activity comparable to that of defensins in efficacy. XCL1 is a unique metamorphic protein that interconverts between the canonical chemokine fold and a novel all-ß-sheet dimer. Phylogenetic analysis suggests that, within the chemokine family, XCL1 is most closely related to CCL20, which exhibits antibacterial activity. The in vitro antimicrobial activity of WT-XCL1 and structural variants was quantified using a radial diffusion assay (RDA) and in solution bactericidal assays against Gram-positive and Gram-negative species of bacteria. Comparisons of WT-XCL1 with variants that limit metamorphic interconversion showed a loss of antimicrobial activity when restricted to the conserved chemokine fold. These results suggest that metamorphic folding of XCL1 is required for potent antimicrobial activity.

Engineering Metamorphic Chemokine Lymphotactin/XCL1 into the GAG-Binding, HIV-Inhibitory Dimer Conformation.

Unlike other chemokines, XCL1 undergoes a distinct metamorphic interconversion between a canonical monomeric chemokine fold and a unique ß-sandwich dimer. The monomeric conformation binds and activates the receptor XCR1, whereas the dimer binds extracellular matrix glycosaminoglycans and has been associated with anti-human immunodeficiency virus (HIV) activity. Functional studies of WT-XCL1 are complex, as both conformations are populated in solution. To overcome this limitation, we engineered a stabilized dimeric variant of XCL1 designated CC5. This variant features a new disulfide bond (A36C-A49C) that prevents structural interconversion by locking the chemokine into the ß-sandwich dimeric conformation, as demonstrated by NMR structural analysis and hydrogen/deuterium exchange experiments. Functional studies analyzing glycosaminoglycan binding demonstrate that CC5 binds with high affinity to heparin. In addition, CC5 exhibits potent inhibition of HIV-1 activity in primary peripheral blood mononuclear cells (PBMCs), demonstrating the importance of the dimer in blocking viral infection. Conformational variants like CC5 are valuable tools for elucidating the biological relevance of the XCL1 native-state interconversion and will assist in future antiviral and functional studies.

The CD8-derived chemokine XCL1/lymphotactin is a conformation-dependent, broad-spectrum inhibitor of HIV-1.

CD8+ T cells play a key role in the in vivo control of HIV-1 replication via their cytolytic activity as well as their ability to secrete non-lytic soluble suppressive factors. Although the chemokines that naturally bind CCR5 (CCL3/MIP-1α, CCL4/MIP- 1ß, CCL5/RANTES) are major components of the CD8-derived anti-HIV activity, evidence indicates the existence of additional, still undefined, CD8-derived HIV-suppressive factors. Here, we report the characterization of a novel anti-HIV chemokine, XCL1/lymphotactin, a member of the C-chemokine family that is produced primarily by activated CD8+ T cells and behaves as a metamorphic protein, interconverting between two structurally distinct conformations (classic and alternative). We found that XCL1 inhibits a broad spectrum of HIV-1 isolates, irrespective of their coreceptor-usage phenotype. Experiments with stabilized variants of XCL1 demonstrated that HIV-1 inhibition requires access to the alternative, all-ß conformation, which interacts with proteoglycans but does not bind/activate the specific XCR1 receptor, while the classic XCL1 conformation is inactive. HIV-1 inhibition by XCL1 was shown to occur at an early stage of infection, via blockade of viral attachment and entry into host cells. Analogous to the recently described anti-HIV effect of the CXC chemokine CXCL4/PF4, XCL1-mediated inhibition is associated with direct interaction of the chemokine with the HIV-1 envelope. These results may open new perspectives for understanding the mechanisms of HIV-1 control and reveal new molecular targets for the design of effective therapeutic and preventive strategies against HIV-1.

The chemokines CXCL10 and XCL1 recruit human annulus fibrosus cells.

STUDY DESIGN: Human annulus fibrosus tissue and cells were analyzed for the presence of chemokine receptors and the migratory effect of selected chemokines. OBJECTIVE: To investigate spontaneous repair mechanisms and underlying cell recruitment in response to annular tears and degenerative defects. SUMMARY OF BACKGROUND DATA: Resorption of herniated disc tissue and the attempt to close annulus tears with repair tissue occur spontaneously. Although chemokines are suggested to play a role in resorption of herniated disc tissue, the role of chemokines in annulus fibrosus homeostasis and repair remains unclear. METHODS: Cells were isolated from annulus fibrosus tissue and expanded in the presence of human serum. Multiwell chemotaxis assays were used to analyze the migratory effect of human serum and 0 to 1000 nM concentrations of the chemokines CXCL7, CXCL10, CXCL12, CCL25, and XCL1 on annulus fibrosus cells (AFCs) (n = 9 per chemokine and dose). Presence of corresponding chemokine receptors in AFCs was determined by real-time polymerase chain reaction analysis and immunohistochemistry. RESULTS: Serum (0.1%-10%) significantly (P < 0.01) stimulates the migration of AFCs. Compared with untreated cells, the migration of cells was significantly (P < 0.01) enhanced upon stimulation with 100 to 1000 nM CXCL10 and 1000 nM XCL1. Chemokine receptors showed low expression levels in expanded AFCs as assessed by polymerase chain reaction. Immunohistochemical staining of the CXCL10 receptor CXCR3 and the XCL1 receptor XCR1 showed that the presence of the particular receptors in AFCs expanded under conventional cell culture conditions. In native annulus fibrosus tissue, CXCR3 was evident, whereas XCR1 could not be detected. CONCLUSION: The findings suggest that chemokines, in particular CXCL10, effectively recruit isolated AFCs. This suggests that chemokines are involved in annulus fibrosus homeostasis and potentially in spontaneous annulus repair attempts. This might have important implications for biological annulus-sealing strategies.

Selective expression of the chemokine receptor XCR1 on cross-presenting dendritic cells determines cooperation with CD8+ T cells.

The expression of the chemokine receptor XCR1 and the function of its ligand XCL1 (otherwise referred to as ATAC, lymphotactin, or SCM-1) remained elusive to date. In the present report we demonstrated that XCR1 is exclusively expressed on murine CD8(+) dendritic cells (DCs) and showed that XCL1 is a potent and highly specific chemoattractant for this DC subset. CD8(+) T cells abundantly secreted XCL1 8-36 hr after antigen recognition on CD8(+) DCs in vivo, in a period in which stable T cell-DC interactions are known to occur. Functionally, XCL1 increased the pool of antigen-specific CD8(+) T cells and their capacity to secrete IFN-gamma. Absence of XCL1 impaired the development of cytotoxicity to antigens cross-presented by CD8(+) DCs. The XCL1-XCR1 axis thus emerges as an integral component in the development of efficient cytotoxic immunity in vivo.

Production of biologically active human lymphotactin (XCL1) by Lactococcus lactis.

Lymphotactin-XCL1 is a chemokine produced mainly by activated CD8+ T-cells and directs migration of CD4+ and CD8+ lymphocytes and natural killer (NK) cells. We expressed human lymphotactin (LTN) by the lactic-acid bacterium Lactococcus lactis. Biological activity of LTN was confirmed by chemo-attraction of human T-cells by chemotaxis demonstrating, for the first time, how this chemokine secreted by a food-grade prokaryote retains biological activity and chemoattracts T lymphocytes. This strain thus represents a feasible well-tolerated vector to deliver active LTN at a mucosal level.

Signals emanating from leukocyte-endothelium interactions during inflammation

Leukocyte trafficking throughout the vasculature is a crucial step for the development of innate and adaptive immunity (1). The coordinate function of adhesion receptors, cytoskeleton and signaling molecules in both cellular types is fundamental during leukocyte extravasation. Hence, the correct integration of “outside-in” and “inside-out” signals in leukocytes and endothelium during each stage of the process is critical to allow the completion of the so-called “multi-step paradigm” (leukocyte tethering and rolling involving selectins and their ligands, followed by leukocyte firm adhesion and crawling mediated by integrins and their endothelial counter-receptors and the subsequent diapedesis also mediated by junctional proteins) (2, 3). This review focuses on the signaling pathways triggered during the extravasation that allow leukocytes to efficiently migrate towards the inflammatory foci to exert their effector functions (AU)

El tráfico leucocitario a través de la vasculatura es un paso crucial para el desarrollo de la inmunidad innata y adaptativa. El funcionamiento coordinado de los receptores de adhesión, el citoesqueleto y las moléculas señalizadoras tanto en el leucocito como en el endotelio es fundamental durante el proceso de extravasación. Así, la correcta integración de las señales del exterior hacia el interior y del interior hacia el exterior en ambos tipos celulares durante cada etapa del proceso es crítica para la consecución del llamado “paradigma multi-secuencial” (el rodamiento de los leucocitos en el que están implicadas las selectinas y sus ligandos, seguido de la adhesión firme mediada por las integrinas leucocitarias y sus contra-receptores endoteliales, así como el subsiguiente paso de diapedesis en el que también están implicadas moléculas típicas de uniones intercelulares). Esta revisión se centra en las vías de señalización que se desencadenan durante la extravasación que permiten a los leucocitos migrar de manera eficiente hacia los focos inflamatorios donde ejercen sus funciones efectoras (AU)

Immunomodulation by herpesvirus U51A chemokine receptor via CCL5 and FOG-2 down-regulation plus XCR1 and CCR7 mimicry in human leukocytes.

Human herpesvirus-6A (HHV-6A) betachemokine-receptor U51A binds inflammatory modulators CCL2, CCL5, CCL11, CCL7, and CCL13. This unique specificity overlaps that of human chemokine receptors CCR1, CCR2, CCR3, and CCR5. In model cell lines, expression leads to CCL5 down-regulation with both constitutive and inducible signaling. Here, immunomodulation pathways are investigated in human leukocytes permissive for infection. Constitutive signaling was shown using inositol phosphate assays and inducible calcium signaling by response to CCL2, CCL5 and CCL11. Constitutive signaling targets were examined using an immune response-related microarray and RT-PCR, showing down-regulation of CCL5 and FOG-2, a hematopoietic transcriptional repressor. By RT-PCR and siRNA reversion, CCL5 and FOG-2 were shown down-regulated, during peak U51A expression post infection. Two further active ligands, XCL1 and CCL19, were identified, making U51A competitor to their human receptors, XCR1 and CCR7, on T lymphocytes, NK and dendritic cells. Finally, U51A-expressing cell lines and infected ex vivo leukocytes, showed migration towards chemokine-gradients, and chemokine internalization. Consequently, U51A may affect virus dissemination or host transmission by chemotaxis of infected cells to sites of chemokine secretion specific for U51A (for example the lymph node or lung, by CCL19 or CCL11, respectively) and evade immune-effector cells by chemokine diversion and down-regulation, affecting virus spread and inflammatory pathology.

An engineered second disulfide bond restricts lymphotactin/XCL1 to a chemokine-like conformation with XCR1 agonist activity.

Chemokines adopt a conserved tertiary structure stabilized by two disulfide bridges and direct the migration of leukocytes. Lymphotactin (Ltn) is a unique chemokine in that it contains only one disulfide and exhibits large-scale structural heterogeneity. Under physiological solution conditions (37 degrees C and 150 mM NaCl), Ltn is in equilibrium between the canonical chemokine fold (Ltn10) and a distinct four-stranded beta-sheet (Ltn40). Consequently, it has not been possible to address the biological significance of each structural species independently. To stabilize the Ltn10 structure in a manner independent of specific solution conditions, Ltn variants containing a second disulfide bridge were designed. Placement of the new cysteines was based on a sequence alignment of Ltn with either the first (Ltn-CC1) or third disulfide (Ltn-CC3) in the CC chemokine, HCC-2. NMR data demonstrate that both CC1 and CC3 retain the Ltn10 chemokine structure and no longer exhibit structural rearrangement. The ability of each mutant to activate the Ltn receptor, XCR1, has been tested using an intracellular Ca2+ flux assay. These data support the conclusion that the chemokine fold of Ltn10 is responsible for receptor activation. We also examined the role of amino- and carboxyl-terminal residues in Ltn-mediated receptor activation. In contrast to previous reports, we find that the 25 residues comprising the novel C-terminal extension do not participate in receptor activation, while the native N-terminus is absolutely required for Ltn function.

Effects of CC, CXC, C, and CX3C chemokines on proliferation of myeloid progenitor cells, and insights into SDF-1-induced chemotaxis of progenitors.

Chemokines have been implicated in the regulation of stem/progenitor cell proliferation and movement. The purpose of the present study was to assess a number of new chemokines for suppressive activity and to delve further into SDF-1-mediated chemotaxis of progenitor cells. This report extends the list of chemokines that have suppressive activity against immature subsets of myeloid progenitors stimulated to proliferate by multiple growth factors to include: MCP-4/CK beta-10, MIP-4/CK beta-7, I-309, TECK, GCP-2, MIG and lymphotactin. The suppressive activity of a number of other chemokines was confirmed. Additionally, pretreatment of the active chemokines with an acetylnitrile solution enhanced specific activity of a number of these chemokines. The new chemokines found to be lacking suppressive activity include: MCP-2, MCP-3, eotaxin-1, MCIF/HCC-1/CK beta-1, TARC, MDC, MPIF-2/eotaxin-2/CK beta-6, SDF-1 and fractalkine/neurotactin. Overall, 19 chemokines, crossing the CC, CXC, and C subgroups, have now been found to be myelosuppressive, and 14 chemokines crossing the CC, CXC and CX3C subgroups have been found to lack myelosuppressive activity under the culture conditions of our assays. Because of the redundancy in chemokine/chemokine receptor interactions, it is not yet clear through which chemokine receptors many of these chemokines signal to elicit suppressive activities. It was also found that SDF-1-induced chemotaxis of progenitors can occur in the presence of fibronectin (FN) and extracellular matrix components and that FN effects involve activation of beta 1-, and possibly alpha 4-, integrins.