The chemokine XCL1 functions as a pregnancy hormone to program offspring innate anxiety.

Elevated levels of cytokines in maternal circulation increase the offspring's risk for neuropsychiatric disease. Because of their low homeostatic levels, circulating maternal cytokines during normal pregnancies have not been considered to play a role in fetal brain development and offspring behavior. Here we report that the T/NK cell chemotactic cytokine XCL1, a local paracrine immune signal, can function as a pregnancy hormone and is required for the proper development of placenta and male offspring approach-avoidance behavior. We found that circulating XCL1 levels were at a low pregestational level throughout pregnancy except for a midgestational rise and fall. Blunted elevation in maternal plasma XCL1 in dams with a genetic 5HT1A receptor deficit or following neutralization by anti-XCL1 antibodies increased the expression of tissue damage associated factors in WT fetal placenta and led to increased innate anxiety and stress reactivity in the WT male offspring. Therefore, chemokines like XCL1 may act as pregnancy hormones to regulate placenta development and offspring emotional behavior.

Intratumoral delivery of a highly active form of XCL1 enhances antitumor CTL responses through recruitment of CXCL9-expressing conventional type-1 dendritic cells.

Conventional type 1 dendritic cells (cDC1s) play a crucial role in antitumor immunity through the induction and activation of tumor-specific CD8+ cytotoxic T cells (CTLs). The chemokine XCL1 is a major chemotactic factor for cDC1s and its receptor XCR1 is selectively expressed on cDC1s. Here, we investigated the effect of intratumoral delivery of a highly active form of murine XCL1 (mXCL1-V21C/A59C) on cDC1-mediated antitumor immunity using a hydrophilic gel patch. The hydrophilic gel patch containing mXCL1-V21C/A59C increased cDC1 accumulation in the tumor masses and promoted their migration to the regional lymph nodes, resulting in enhanced induction of tumor-specific CTLs. Tumor-infiltrating cDC1s not only expressed XCR1 but also produced CXCL9, a ligand for CXCR3 which is highly expressed on CTLs and NK cells. Consequently, CTLs and NK cells were increased in the tumor masses of mice treated with mXCL1-V21C/A59C, while immunosuppressive cells such as monocyte-derived suppressive cells and regulatory T cells were decreased. We also confirmed that anti-CXCL9 treatment decreased the tumor infiltration of CTLs. The intratumoral delivery of mXCL1-V21C/A59C significantly decreased tumor growth and prolonged survival in E.G7-OVA and B16-F10 tumor-bearing mice. Furthermore, the antitumor effect of mXCL1-V21CA59C was enhanced in combination with anti-programmed cell death protein 1 treatment. Finally, using The Cancer Genome Atlas database, we found that XCL1 expression was positively correlated with tumor-infiltrating cDC1s and a better prognosis in melanoma patients. Collectively, our findings provide a novel therapeutic approach to enhance tumor-specific CTL responses through the selective recruitment of CXCL9-expressing cDC1s into the tumor masses.

New insights into the analgesic properties of the XCL1/XCR1 and XCL1/ITGA9 axes modulation under neuropathic pain conditions - evidence from animal studies.

Recent studies have indicated the involvement of chemokine-C-motif ligand 1 (XCL1) in nociceptive transmission; however, the participation of its two receptors, canonical chemokine-C-motif receptor 1 (XCR1) and integrin alpha-9 (ITGA9), recently recognized as a second receptor, has not been clarified to date. The aim was to explore by which of these receptors XCL1 reveals its pronociceptive properties and how the XCL1-XCR1 and XCL1-ITGA9 axes blockade/neutralization influence on pain-related behavior and opioid analgesia in the model of neuropathic pain. In our studies we used Albino Swiss mice which were exposed to the unilateral sciatic nerve chronic constriction injury (CCI) as a neuropathic pain model. Animals received single intrathecal (i.t.) injection of XCL1, XCL1 neutralizing antibodies, antagonist of XCR1 (vMIP-II) and neutralizing antibodies of ITGA9 (YA4), using lumbar puncture technique. Additionally we performed i.t. co-administration of abovementioned neutralizing antibodies and antagonists with single dose of morphine/buprenorphine. To assess pain-related behavior the von Frey and cold plate tests were used. To measure mRNA and protein level the RT-qPCR and Western Blot/Elisa/immunofluorescence techniques were performed, respectively. Statistical analysis was conducted using ANOVA with a Bonferroni correction. Presented studies have shown time-dependent upregulation of the mRNA and/or protein expression of XCL1 in the spinal cord after nerve injury as measured on day 1, 4, 7, 14, and 35. Our immunofluorescence study showed that XCL1 is released by astroglial cells located in the spinal cord, despite the neural localization of its receptors. Our results also provided the first evidence that the blockade/neutralization of both receptors, XCR1 and ITGA9, reversed hypersensitivity after intrathecal XCL1 administration in naive mice; however, neutralization of ITGA9 was more effective. In addition, the results proved that the XCL1 neutralizing antibody and, similarly, the blockade of XCR1 and neutralization of ITGA9 diminished thermal and mechanical hypersensitivity in nerve injury-exposed mice after 7 days. Additionally, neutralization of XCL1 improves morphine analgesia. Moreover, blockade of XCR1 positively influences buprenorphine effectiveness, and neutralization of ITGA9 enhances not only buprenorphine but also morphine analgesia. Therefore, blockade of the XCL1-ITGA9 interaction may serve as an innovative strategy for the polypharmacotherapy of neuropathic pain in combination with opioids.

Traumatic brain injury in mice induces changes in the expression of the XCL1/XCR1 and XCL1/ITGA9 axes.

BACKGROUND: Every year, millions of people suffer from various forms of traumatic brain injury (TBI), and new approaches with therapeutic potential are required. Although chemokines are known to be involved in brain injury, the importance of X-C motif chemokine ligand 1 (XCL1) and its receptors, X-C motif chemokine receptor 1 (XCR1) and alpha-9 integrin (ITGA9), in the progression of TBI remain unknown. METHODS: Using RT-qPCR/Western blot/ELISA techniques, changes in the mRNA/protein levels of XCL1 and its two receptors, in brain areas at different time points were measured in a mouse model of TBI. Moreover, their cellular origin and possible changes in expression were evaluated in primary glial cell cultures. RESULTS: Studies revealed the spatiotemporal upregulation of the mRNA expression of XCL1, XCR1 and ITGA9 in all the examined brain areas (cortex, thalamus, and hippocampus) and at most of the evaluated stages after brain injury (24 h; 4, 7 days; 2, 5 weeks), except for ITGA9 in the thalamus. Moreover, changes in XCL1 protein levels occurred in all the studied brain structures; the strongest upregulation was observed 24 h after trauma. Our in vitro experiments proved that primary murine microglial and astroglial cells expressed XCR1 and ITGA9, however they seemed not to be a main source of XCL1. CONCLUSIONS: These findings indicate that the XCL1/XCR1 and XCL1/ITGA9 axes may participate in the development of TBI. The XCL1 can be considered as one of the triggers of secondary injury, therefore XCR1 and ITGA9 may be important targets for pharmacological intervention after traumatic brain injury.

The chemokine lymphotactin and its recombinant variants in oral cancer cell regulation.

BACKGROUND: The expression of XCR1 receptor and its metamorphic ligand lymphotactin (hLtn) has been shown in cancers but their precise role in tumorigenesis is poorly understood including the significance of the physiologically existing hLtn monomeric (CC3) and dimeric (W55D) confirmations where the latter thought to function as the receptor antagonist. The aim of this study was to explore the functional role of bioengineered hLtn variants and the role of fibroblasts in XCR1/hLtn expression regulation in oral cancer cells (OCCL). MATERIAL AND METHODS: qRT-PCR and flow cytometry were performed to evaluate mRNA and protein expression of XCR1 and hLtn. Recombinant hLtn variants (wild-type, CC3 and W55D mutant) were designed, expressed, purified and evaluated using proliferation, adhesion and chemotaxis assays. XCR1 and hLtn expression regulation by fibroblasts was determined using indirect co-culture. XCR1 and hLtn expression in primary and metastatic OSCC tissue was assessed using immunohistochemistry. RESULTS: hLtn caused a significant decrease in OCCL XCR1 surface protein expression. hLtn CC3 mutant was highly functional facilitating proliferation and migration. Conditioned media from primary cancer-associated and senescent fibroblasts significantly upregulated XCR1 and hLtn mRNA expression in OCCL. Immunohistochemistry revealed higher XCR1 and hLtn expression in metastatic tumour deposits and surrounding stroma compared to primary OSCC tissue. CONCLUSIONS: The development of hLtn biological mutants, regulation of XCR1 expression by its ligand hLtn and crosstalk with fibroblasts are novel findings suggesting an important role for the XCR1/hLtn axis within the OSCC tumour microenvironment. These discoveries build upon previous studies and suggest that the hLtn/XCR1 axis has a significant role in stromal crosstalk and OSCC progression.

Heterodimeric IL-15 delays tumor growth and promotes intratumoral CTL and dendritic cell accumulation by a cytokine network involving XCL1, IFN-γ, CXCL9 and CXCL10.

BACKGROUND: Interleukin-15 (IL-15) promotes growth and activation of cytotoxic CD8+ T and natural killer (NK) cells. Bioactive IL-15 is produced in the body as a heterodimeric cytokine, comprising the IL-15 and IL-15 receptor alpha chains (hetIL-15). Several preclinical models support the antitumor activity of hetIL-15 promoting its application in clinical trials. METHODS: The antitumor activity of hetIL-15 produced from mammalian cells was tested in mouse tumor models (MC38 colon carcinoma and TC-1 epithelial carcinoma). The functional diversity of the immune infiltrate and the cytokine/chemokine network within the tumor was evaluated by flow cytometry, multicolor immunohistochemistry (IHC), gene expression profiling by Nanostring Technologies, and protein analysis by electrochemiluminescence and ELISA assays. RESULTS: hetIL-15 treatment resulted in delayed primary tumor growth. Increased NK and CD8+ T cell tumoral infiltration with an increased CD8+/Treg ratio were found by flow cytometry and IHC in hetIL-15 treated animals. Intratumoral NK and CD8+ T cells showed activation features with enhanced interferon-γ (IFN-γ) production, proliferation (Ki67+), cytotoxic potential (Granzyme B+) and expression of the survival factor Bcl-2. Transcriptomics and proteomics analyses revealed complex effects on the tumor microenvironment triggered by hetIL-15 therapy, including increased levels of IFN-γ and XCL1 with intratumoral accumulation of XCR1+IRF8+CD103+ conventional type 1 dendritic cells (cDC1). Concomitantly, the production of the chemokines CXCL9 and CXCL10 by tumor-localized myeloid cells, including cDC1, was boosted by hetIL-15 in an IFN-γ-dependent manner. An increased frequency of circulating CXCR3+ NK and CD8+ T cells was found, suggesting their ability to migrate toward the tumors following the CXCL9 and CXCL10 chemokine gradient. CONCLUSIONS: Our results show that hetIL-15 administration enhances T cell entry into tumors, increasing the success rate of immunotherapy interventions. Our study further supports the incorporation of hetIL-15 in tumor immunotherapy approaches to promote the development of antitumor responses by favoring effector over regulatory cells and by promoting lymphocyte and DC localization into tumors through the modification of the tumor chemokine and cytokine milieu.

XCL1 expression correlates with CD8-positive T cells infiltration and PD-L1 expression in squamous cell carcinoma arising from mature cystic teratoma of the ovary.

Molecular characteristics of carcinoma arising from mature cystic teratoma of the ovary (MCT) remain unclear due to its rarity. We analyzed RNA-sequencing data of 2322 pan-cancer [1378 squamous cell carcinomas (SCC), 6 adenosquamous carcinomas (ASC), and 938 adenocarcinomas (AC)] including six carcinomas arising from MCT (four SCCs, one ASC, and one AC). Hierarchical clustering and principal component analysis showed that gene expression profiles of carcinomas arising from MCT were different between each histological type and that gene expression profiles of SCCs arising MCT (MCT-SCCs) was apparently similar to those of lung SCCs. By epidermis-associated pathways activity based on gene set enrichment analysis, 1030 SCCs were divided into two groups: epidermis-signature high (head and neck, esophagus, and skin) and low (cervix, lung, and MCT). In addition to pan-SCC transcriptome analysis, cytokeratin profiling based on immunohistochemistry in the independent samples of 21 MCT-SCCs clarified that MCT-SCC dominantly expressed CK18, suggesting the origin of MCT-SCC was columnar epithelium. Subsequently, we investigated differentially expressed genes in MCT-SCCs compared with different SCCs and identified XCL1 was specifically overexpressed in MCT-SCCs. Through immunohistochemistry analysis, we identified XCL1 expression on tumor cells in 13/24 (54%) of MCT-SCCs but not in MCTs. XCL1 expression was also significantly associated with the number of tumor-infiltrating CD8-positive T cells and PD-L1 expression on tumor cells. XCL1 produced by tumor cells may induce PD1/PD-L1 interaction and dysfunction of CD8-positive T cells in tumor microenvironment. XCL1 expression may be a novel biomarker for malignant transformation of MCT into SCC and a biomarker candidate for therapeutic response to an anti-PD1/PD-L1 therapy.

A functional variant near XCL1 gene improves breast cancer survival via promoting cancer immunity.

Most genome-wide association studies (GWASs) identify genetic variants for breast cancer occurrence. In contrast, few are for recurrence and mortality. We conducted a GWAS on breast cancer survival after diagnosis in estrogen receptor-positive patients, including 953 Taiwanese patients with 159 events. Through Cox proportional hazard models estimation, we identified 24 risk SNPs with p < 1 × 10-5 . Based on imputation and integrated analysis, one SNP, rs1024176 (located in 1q24.2, p = 2.43 × 10-5 ) was found to be a functional variant associated with breast cancer survival and XCL1 gene expression. A series of experimental approaches, including cell-based analyses and CRISPR/Cas9 genome-editing system, were then used and identified the transcription factor MYBL2 was able to discriminately bind to the A allele of rs1024176, the protective variant for breast cancer survival, which promoted XCL1 expression, but not to the G allele of rs1024176. The chemokine XCL1 attracts type 1 dendritic cells (DC1s) to the tumor microenvironment. In breast cancer tissues, we applied a two-step Mendelian randomization analysis, using expression quantitative trait loci as instrumental variables, to confirm higher XCL1 expression was correlated with higher DC1 signatures and favorable disease progression, through the causal effect of rs1024176-A allele. Our study supports the genetic effect on preventing breast cancer survival through XCL1-induced DC1 recruitment in tumor microenvironment.

Structure-function guided modeling of chemokine-GPCR specificity for the chemokine XCL1 and its receptor XCR1.

Chemokines interact with their G protein-coupled receptors (GPCRs) through a two-step, two-site mechanism and, through this interaction, mediate various homeostatic and immune response mechanisms. Upon initial recognition of the chemokine by the receptor, the amino terminus of the chemokine inserts into the orthosteric pocket of the GPCR, causing conformational changes that trigger intracellular signaling. There is considerable structural and functional evidence to suggest that the amino acid composition and length of the chemokine amino terminus is critical for GPCR activation, complementing the size and amino acid composition of the orthosteric pocket. However, very few structures of a native chemokine-receptor complex have been solved. Here, we used a hybrid approach that combines structure-function data with Rosetta modeling to describe key contacts within a chemokine-GPCR interface. We found that the extreme amino-terminal residues of the chemokine XCL1 (Val1, Gly2, Ser3, and Glu4) contribute a large fraction of the binding energy to its receptor XCR1, whereas residues near the disulfide bond-forming residue Cys11 modulate XCR1 activation. Alterations in the XCL1 amino terminus changed XCR1 activation, as determined by assessing inositol triphosphate accumulation, intracellular calcium release, and directed cell migration. Computational analysis of XCL1-XCR1 interactions revealed functional contacts involving Glu4 of XCL1 and Tyr117 and Arg273 of XCR1. Subsequent mutation of Tyr117 and Arg273 led to diminished binding and activation of XCR1 by XCL1. These findings demonstrate the utility of a hybrid approach, using biological data and homology modeling, to study chemokine-GPCR interactions.

Endocytosis Deficient Murine Xcl1-Fusion Vaccine Enhances Protective Antibody Responses in Mice.

Targeting antigen to surface receptors on dendritic cells (DCs) can improve antibody response against subunit vaccines. We have previously observed that human XCL1-fusion vaccines target murine Xcr1+ DCs without actively inducing endocytosis of the antigen, resulting in enhanced antibody responses in mice. However, the use of foreign chemokines for targeting is undesirable when translating this observation to human or veterinary medicine due to potential cross-reactive responses against the endogenous chemokine. Here we have identified a mutant version of murine Xcl1, labeled Xcl1(Δ1) owing to removal of a conserved valine in position 1 of the mature chemokine, that retains specific binding to Xcr1+ DCs without inducing endocytosis of the receptor. DNA immunization with Xcl1(Δ1) conjugated to influenza hemagglutinin (HA) induced improved antibody responses, with higher end point titers of IgG compared to WT Xcl1-HA. The Xcl1(Δ1) fusion vaccine also resulted in an increased number of HA reactive germinal center B cells with higher avidity toward the antigen, and serum transfer experiments show that Xcl1(Δ1)-HA induced antibody responses provided better protection against influenza infection as compared to WT Xcl1-HA. In summary, our observations indicate that targeting antigen to Xcr1+ DCs in an endocytosis deficient manner enhances antibody responses. This effect was obtained by introducing a single mutation to Xcl1, suggesting our strategy may easily be translated to human or veterinary vaccine settings.

Genetic diversity of chemokine XCL1 and its receptor XCR1 in murine rodents.

The chemokine ligand XCL1 plays critical roles in immune responses with diverse physiological and pathological implications through interactions with a cognate G protein-coupled receptor XCR1. To shed insight into their versatile nature, we analyzed genetic variations of XCL1 and XCR1 in murine rodents, including commonly-used model organisms Mus musculus (house mouse) and Rattus norvegicus (Norway rat). Our results showed that adaptive selection has contributed to the genetic diversification of these proteins in murine lineage. Moreover, in both M. musculus and R. norvegicus, the chemokine and its receptor exhibit similar signs of selective sweeps resulting from positive selection. In light of currently available structural and interaction information for chemokines and their receptors, the similarity of XCL1/XCR1 evolutionary patterns among murine species and the parallels of their evolutionary footprints within individual species suggest that interplay could exist between the adaptively selected changes, or between the domains on which the identified changes are located, and consequently preserve the physiological interaction of XCL1 and XCR1.

Combination of Synthetic Long Peptides and XCL1 Fusion Proteins Results in Superior Tumor Control.

Cross-presenting Xcr1+CD8α DCs are attractive APCs to target for therapeutic cancer vaccines, as they are able to take up and process antigen from dying tumor cells for their MHCI-restricted presentation to CD8 T cells. To this aim, we developed fusion proteins made of the Xcr1 ligand Xcl1 fused to an OVA synthetic long peptide (SLP) and IgG1 Fc fragment. We demonstrated the specific binding and uptake of the Xcl1 fusion proteins by Xcr1+ DCs. Most importantly, their potent adjuvant effect on the H-2Kb/OVA specific T cell response was associated with a sustained tumor control even against the poorly immunogenic B16-OVA melanoma tumor. The increased tumor protection correlated with higher tumor infiltration of antigen-specific CD8+ T cells, increased IFNγ production and degranulation potential. Altogether, these results demonstrate that therapeutic cancer vaccines may be greatly improved by the combination of SLP antigen and Xcl1 fusion proteins.

Intratumoral Immunotherapy with XCL1 and sFlt3L Encoded in Recombinant Semliki Forest Virus-Derived Vectors Fosters Dendritic Cell-Mediated T-cell Cross-Priming.

: Multiple lines of evidence indicate a critical role of antigen cross-presentation by conventional BATF3-dependent type 1 classical dendritic cells (cDC1) in CD8-mediated antitumor immunity. Flt3L and XCL1, respectively, constitute a key growth/differentiation factor and a potent and specific chemoattractant for cDC1. To exploit their antitumor functions in local immunotherapy, we prepared Semliki Forest Virus (SFV)-based vectors encoding XCL1 and soluble Flt3L (sFlt3L). These vectors readily conferred transgene expression to the tumor cells in culture and when engrafted as subcutaneous mouse tumor models. In syngeneic mice, intratumoral injection of SFV-XCL1-sFlt3L (SFV-XF) delayed progression of MC38- and B16-derived tumors. Therapeutic activity was observed and exerted additive effects in combination with anti-PD-1, anti-CD137, or CTLA-4 immunostimulatory mAbs. Therapeutic effects were abolished by CD8ß T-cell depletion and were enhanced by CD4 T-cell depletion, but not by T regulatory cell predepletion with anti-CD25 mAb. Antitumor effects were also abolished in BATF3- and IFNAR-deficient mice. In B16-OVA tumors, SFV-XF increased the number of infiltrating CD8 T cells, including those recognizing OVA. Consistently, following the intratumoral SFV-XF treatment courses, we observed increased BATF3-dependent cDC1 among B16-OVA tumor-infiltrating leukocytes. Such an intratumoral increase was not seen in MC38-derived tumors, but both resident and migratory cDC1 were boosted in SFV-XF-treated MC38 tumor-draining lymph nodes. In conclusion, viral gene transfer of sFlt3L and XCL1 is feasible, safe, and biologically active in mice, exerting antitumor effects that can be potentiated by CD4 T-cell depletion. SIGNIFICANCE: These findings demonstrate that transgenic expression of sFLT3L and XCL1 in tumor cells mediates cross-priming of, and elicits potent antitumor activity from, CD8 T lymphocytes, particularly in combination with CD4 T-cell depletion.

XCL1-XCR1 pathway promotes trophoblast invasion at maternal-fetal interface by inducing MMP-2/MMP-9 activity.

PROBLEM: Certain chemokines with their receptors can promote or inhibit trophoblast cell migration and invasion in human first-trimester placenta. Whether the lymphotactin (Lptn; XCL1)-XC chemokine receptor 1 (XCR1) chemokine pathway affects trophoblast cell migration and invasion in human first-trimester placenta remains unclear. METHOD OF STUDY: The expression pattern of chemokine XCL1 and its receptor XCR1 was detected in human first-trimester by qRT-PCR, and the effect of recombinant human XCL1 (rhXCL1) on trophoblast cell function was tested by wound healing and Transwell assays. Matrix metalloproteinase (MMP) activity in trophoblast cells treated with rhXCL1 was assessed via qRT-PCR and gelatin zymography. RESULTS: Abundant XCR1 mRNA was expressed in the first-trimester decidua and villi. XCL1 and XCR1 mRNA were expressed at a higher level in the first-trimester than in the term placenta. RhXCL1 promoted trophoblast cell migration and invasion by increasing MMP-9 and MMP-2 activity and that of the MMP-2/tissue inhibitor of metalloproteinases 2 (TIMP-2) complex via the phosphatidylinositol 3-kinase (PI3K)/AKT kinase (AKT), mitogen-activated protein kinase (MEK), and JUN N-terminal kinase (JNK) signaling pathways. CONCLUSION: XCL1-XCR1 chemokine pathway promotes trophoblast invasion by increasing matrix metalloproteinase activity in human first-trimester placenta.

A Novel Role for Lymphotactin (XCL1) Signaling in the Nervous System: XCL1 Acts via its Receptor XCR1 to Increase Trigeminal Neuronal Excitability.

Chemokines are known to have a role in the nervous system, influencing a range of processes including the development of chronic pain. To date there are very few studies describing the functions of the chemokine lymphotactin (XCL1) or its receptor (XCR1) in the nervous system. We investigated the role of the XCL1-XCR1 axis in nociceptive processing, using a combination of immunohistochemical, pharmacological and electrophysiological techniques. Expression of XCR1 in the rat mental nerve was elevated 3 days following chronic constriction injury (CCI), compared with 11 days post-CCI and sham controls. XCR1 co-existed with neuronal marker PGP9.5, leukocyte common antigen CD45 and Schwann cell marker S-100. In the trigeminal root and white matter of the brainstem, XCR1-positive cells co-expressed the oligodendrocyte marker Olig2. In trigeminal subnucleus caudalis (Vc), XCR1 immunoreactivity was present in the outer laminae and was colocalized with vesicular glutamate transporter 2 (VGlut2), but not calcitonin gene-related peptide (CGRP) or isolectin B4 (IB4). Incubation of brainstem slices with XCL1 induced activation of c-Fos, ERK and p38 in the superficial layers of Vc, and enhanced levels of intrinsic excitability. These effects were blocked by the XCR1 antagonist viral CC chemokine macrophage inhibitory protein-II (vMIP-II). This study has identified for the first time a role for XCL1-XCR1 in nociceptive processing, demonstrating upregulation of XCR1 at nerve injury sites and identifying XCL1 as a modulator of central excitability and signaling via XCR1 in Vc, a key area for modulation of orofacial pain, thus indicating XCR1 as a potential target for novel analgesics.

The invariant natural killer T cell-mediated chemokine X-C motif chemokine ligand 1-X-C motif chemokine receptor 1 axis promotes allergic airway hyperresponsiveness by recruiting CD103+ dendritic cells.

BACKGROUND: The chemokine X-C motif chemokine ligand 1 (XCL1)-X-C motif chemokine receptor 1 (XCR1) axis has been reported to play a role in immune homeostasis and inflammation. However, it is not known whether this axis has a critical function in patients with allergic asthma. OBJECTIVE: In the present study we explored whether the invariant natural killer T (iNKT) cell-mediated XCL1-XCR1 axis regulated allergic asthma. METHODS: Ovalbumin (OVA)- or house dust mite-induced asthma was developed in XCL1 or XCR1 knockout (KO) mice. RESULTS: XCL1 or XCR1 KO mice showed attenuation in airway hyperresponsiveness (AHR), numbers of CD103+ dendritic cells (DCs), and TH2 responses in the lungs compared with wild-type (WT) mice during OVA- or house dust mite-induced asthma. These effects were reversed by intratracheal administration of recombinant XCL1 or adoptive transfer of CD103+ DCs but not CD11b+ DCs into XCL1 KO mice. Moreover, iNKT cells highly expressed XCL1 both in vitro and in vivo. On intranasal α-galactosyl ceramide challenge, CD103+ DC numbers in the lungs were increased in WT but not XCL1 KO mice. Furthermore, adoptive transfer of WT iNKT cells increased AHR, CD103+ DC recruitment, and TH2 responses in the lungs of CD1d KO mice during OVA-induced asthma, whereas adoptive transfer of XCL1-deficient iNKT cells did not. In human patients, percentages and XCL1 production capacity of iNKT cells from PBMCs were greater in patients with asthma than in healthy control subjects. CONCLUSION: These data demonstrate that the iNKT cell-mediated XCL1-XCR1 axis promotes AHR by recruiting CD103+ DCs into the lung in patients with allergic asthma.

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.

CD14+CD16+ monocytes are the main target of Zika virus infection in peripheral blood mononuclear cells in a paediatric study in Nicaragua.

The recent Zika pandemic in the Americas is linked to congenital birth defects and Guillain-Barré syndrome. White blood cells (WBCs) play an important role in host immune responses early in arboviral infection. Infected WBCs can also function as 'Trojan horses' and carry viruses into immune-sheltered spaces, including the placenta, testes and brain. Therefore, defining which WBCs are permissive to Zika virus (ZIKV) is critical. Here, we analyse ZIKV infectivity of peripheral blood mononuclear cells (PBMCs) in vitro and from Nicaraguan Zika patients and show CD14+CD16+ monocytes are the main target of infection, with ZIKV replication detected in some dendritic cells. The frequency of CD14+ monocytes was significantly decreased, while the CD14+CD16+ monocyte population was significantly expanded during ZIKV infection compared to uninfected controls. Viral RNA was detected in PBMCs from all patients, but in serum from only a subset, suggesting PBMCs may be a reservoir for ZIKV. In Zika patients, the frequency of infected cells was lower but the percentage of infected CD14+CD16+ monocytes was significantly higher compared to dengue cases. The gene expression profile in monocytes isolated from ZIKV- and dengue virus-infected patients was comparable, except for significant differences in interferon-γ, CXCL12, XCL1, interleukin-6 and interleukin-10 levels. Thus, our study provides a detailed picture of the innate immune profile of ZIKV infection and highlights the important role of monocytes, and CD14+CD16+ monocytes in particular.

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.

The anti-influenza M2e antibody response is promoted by XCR1 targeting in pig skin.

XCR1 is selectively expressed on a conventional dendritic cell subset, the cDC1 subset, through phylogenetically distant species. The outcome of antigen-targeting to XCR1 may therefore be similar across species, permitting the translation of results from experimental models to human and veterinary applications. Here we evaluated in pigs the immunogenicity of bivalent protein structures made of XCL1 fused to the external portion of the influenza virus M2 proton pump, which is conserved through strains and a candidate for universal influenza vaccines. Pigs represent a relevant target of such universal vaccines as pigs can be infected by swine, human and avian strains. We found that cDC1 were the only cell type labeled by XCR1-targeted mCherry upon intradermal injection in pig skin. XCR1-targeted M2e induced higher IgG responses in seronegative and seropositive pigs as compared to non-targeted M2e. The IgG response was less significantly enhanced by CpG than by XCR1 targeting, and CpG did not further increase the response elicited by XCR1 targeting. Monophosphoryl lipid A with neutral liposomes did not have significant effect. Thus altogether M2e-targeting to XCR1 shows promises for a trans-species universal influenza vaccine strategy, possibly avoiding the use of classical adjuvants.