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.

NLRP3 regulates CIITA/MHC II axis and interferon-γ-inducible chemokines in Malassezia globosa-infected keratinocytes.

CIITA, a member of NOD-like receptor (NLR) family, is the major MHC II trans-activator and mediator of Th1 immunity, but its function and interaction with NLRP3 have been little studied. We found activation of NLRP3 inflammasome, increased expression of CIITA, CBP, pSTAT1, STAT1, MHC II, IFN-γ and IFN-γ-inducible chemokines (CCL1 and CXCL8), and colocalisation of NLRP3 with CIITA in Malassezia folliculitis lesions, Malassezia globosa-infected HaCaT cells and mouse skin. CoIP with anti-CIITA or anti-NLRP3 antibody pulled down NLRP3 or both CIITA and ASC. NLRP3 silencing or knockout caused CIITA downexpression and their colocalisation disappearance in HaCaT cells and mouse skin of Nlrp3-/- mice, while CIITA knockdown had no effect on NLRP3, ASC, IL-1ß and IL-18 expression. NLRP3 inflammasome inhibitors and knockdown significantly suppressed IFN-γ, CCL1, CXCL8 and CXCL10 levels in M. globosa-infected HaCaT cells. CCL1 and CXCL8 expression was elevated in Malassezia folliculitis lesions and reduced in Nlrp3-/- mice. These results demonstrate that M. globosa can activate NLRP3 inflammasome, CIITA/MHC II signalling and IFN-γ-inducible chemokines in human keratinocytes and mouse skin. NLRP3 may regulate CIITA by their binding and trigger Th1 immunity by secreting CCL1 and CXCL8/IL-8, contributing to the pathogenesis of Malassezia-associated skin diseases.

XCL1, a serum biomarker in neurological diseases; HTLV-1-associated myelopathy and multiple sclerosis.

The XCL1-XCR1 axis has a potential role in the recruitment of immune cells to the site of inflammation. The present study aimed to examine the relation of XCL1 serum levels with Multiple sclerosis (MS) and HTLV-1-associated myelopathy (HAM), as chronic inflammatory diseases of the central nervous system (CNS). DNA was extracted to evaluate HTLV-1 proviral load (PVL) using real-time PCR. Serum levels of XCL1 was determined by using an ELISA assay. The serum level of XCL1 was significantly higher in patients with HAM than that of asymptomatic carriers (ACs) and healthy controls (HCs) (p < 0.001 and p < 0.0001, respectively) and was also higher in MS patients compared to HCs (p < 0.0001). Moreover, the concentration of XCL1 serum level was significantly different between the ACs and HCs group (p < 0.0001). In conclusion, increased expression of XCL1 might contribute to the migration of autoreactive T cells to the central nervous system and play a critical role in the development and pathogenesis of inflammatory neurological diseases including HAM and MS.

Absence of CCR2 Promotes Proliferation of Alveolar Macrophages That Control Lung Inflammation in Acute Respiratory Distress Syndrome in Mice.

Acute respiratory distress syndrome (ARDS) consists of uncontrolled inflammation that causes hypoxemia and reduced lung compliance. Since it is a complex process, not all details have been elucidated yet. In a well-controlled experimental murine model of lipopolysaccharide (LPS)-induced ARDS, the activity and viability of macrophages and neutrophils dictate the beginning and end phases of lung inflammation. C-C chemokine receptor type 2 (CCR2) is a critical chemokine receptor that mediates monocyte/macrophage activation and recruitment to the tissues. Here, we used CCR2-deficient mice to explore mechanisms that control lung inflammation in LPS-induced ARDS. CCR2-/- mice presented higher total numbers of pulmonary leukocytes at the peak of inflammation as compared to CCR2+/+ mice, mainly by enhanced influx of neutrophils, whereas we observed two to six-fold lower monocyte or interstitial macrophage numbers in the CCR2-/-. Nevertheless, the time needed to control the inflammation was comparable between CCR2+/+ and CCR2-/-. Interestingly, CCR2-/- mice presented higher numbers and increased proliferative rates of alveolar macrophages from day 3, with a more pronounced M2 profile, associated with transforming growth factor (TGF)-ß and C-C chemokine ligand (CCL)22 production, decreased inducible nitric oxide synthase (Nos2), interleukin (IL)-1ß and IL-12b mRNA expression and increased mannose receptor type 1 (Mrc1) mRNA and CD206 protein expression. Depletion of alveolar macrophages significantly delayed recovery from the inflammatory insult. Thus, our work shows that the lower number of infiltrating monocytes in CCR2-/- is partially compensated by increased proliferation of resident alveolar macrophages during the inflammation control of experimental ARDS.

Cellular Electrical Impedance as a Method to Decipher CCR7 Signalling and Biased Agonism.

The human C-C chemokine receptor type 7 (CCR7) has two endogenous ligands, C-C chemokine ligand 19 (CCL19) and CCL21, displaying biased agonism reflected by a pronounced difference in the level of ß-arrestin recruitment. Detecting this preferential activation generally requires the use of separate, pathway-specific label-based assays. In this study, we evaluated an alternative methodology to study CCR7 signalling. Cellular electrical impedance (CEI) is a label-free technology which yields a readout that reflects an integrated cellular response to ligand stimulation. CCR7-expressing HEK293 cells were stimulated with CCL19 or CCL21, which induced distinct impedance profiles with an apparent bias during the desensitisation phase of the response. This discrepancy was mainly modulated by differential ß-arrestin recruitment, which shaped the impedance profile but did not seem to contribute to it directly. Pathway deconvolution revealed that Gαi-mediated signalling contributed most to the impedance profile, but Gαq- and Gα12/13-mediated pathways were also involved. To corroborate these results, label-based pathway-specific assays were performed. While CCL19 more potently induced ß-arrestin2 recruitment and receptor internalisation than CCL21, both chemokines showed a similar level of Gαi protein activation. Altogether, these findings indicate that CEI is a powerful method to analyse receptor signalling and biased agonism.

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.

Intranasal delivery of a cDC1 targeted influenza vaccine with poly(I:C) enhances T cell responses and protects against influenza infection.

Targeting antigens to dendritic cells represent a promising method for enhancing immune responses against specific antigens. However, many studies have focused on systemic delivery (intravenous or intraperitoneally) of targeted antigen, approaches that are not easily transferable to humans. Here we evaluate the efficacy of an influenza vaccine targeting Xcr1+ cDC1 administered by intranasal immunization. Intranasal delivery of antigen fused to the chemokine Xcl1, the ligand of Xcr1, resulted in specific uptake by lung CD103+ cDC1. Interestingly, intranasal immunization with influenza A/PR/8/34 haemagglutinin (HA) fused to Xcl1, formulated with poly(I:C), resulted in enhanced induction of antigen-specific IFNγ+ CD4+ and IFNγ+ CD8+ T cell responses in lung compared non-targeted anti-NIP-HA (αNIP-HA). Induction of antibody responses was, however, similar in Xcl1-HA and αNIP-HA immunized mice, but significantly higher than in mice immunized with monomeric HA. Both Xcl1-HA and αNIP-HA vaccines induced full protection when mice were challenged with a lethal dose of influenza PR8 virus, reflecting the strong induction of HA-specific antibodies. Our results demonstrate that i.n. delivery of Xcl1-HA is a promising vaccine strategy for enhancing T cell responses in addition to inducing strong antibody responses.

XCL1 Aggravates Diabetic Nephropathy-Mediated Renal Glomerular Endothelial Cell Apoptosis and Inflammatory Response via Regulating p53/Nuclear Factor-Kappa B Pathway.

BACKGROUND: Glomerular endothelial cell damage plays an important role in the occurrence and development of diabetic nephropathy (DN). OBJECTIVES: This study aimed to clarify the role of XCL1 in DN-mediated glomerular endothelial cell apoptosis and whether the function was related to the activation of the p53/nuclear factor-kappa B (NF-κB) signaling pathway. METHODS: Candidate biomarkers were identified by least absolute shrinkage and selection operator (LASSO) regression model analysis. The area under the receiver operating characteristic curve value was calculated and used to evaluate the discriminating ability. Cell viability, apoptosis, and interleukin-1ß and tumor necrosis factor-α expression at messenger RNA and protein levels were detected by using the Cell Counting Kit-8, flow cytometry, ELISA, real-time polymerase chain reaction, and Western blotting assays. In vivo studies were conducted in the DN mice. RESULTS: The LASSO regression model displayed good discriminating performance, with a C-index of 0.803 and good calibration, and high XCL1 expression was identified as the predicting factor for DN in diabetes mellitus patients. XCL1 expression was upregulated in glomeruli of db/db mice, which was closely related to the expression of its receptor (XCR1). XCL1 overexpression played an important role in the apoptosis and inflammatory response of high glucose (HG)-treated human renal glomerular endothelial cells. Meanwhile, the expression of p53 and the levels of inflammatory cytokines were upregulated upon XCL1 overexpression. p53 silencing with its inhibitor blocked the apoptotic response and inflammatory response in XCL1-overexpressed cells exposed to HG. Besides, the XCL1 overexpression-induced downregulation of NF-κB was reversed by pifithrin-α pretreatment. CONCLUSIONS: Our findings in this work provided the mechanistic insights into the effects of XCL1 on the modulation of DN development, illustrating that XCL1 might serve as an essential prognostic indicator and therapeutic target for DN progression.

Interferon-γ-Inducible Chemokines as Prognostic Markers for Lung Cancer.

Interferon (IFN)-γ-inducible chemokines in the CXCR3/ligand axis are involved in cell-mediated immunity and play a significant role in the progression of cancer. We enrolled patients with lung cancer (n = 144) and healthy volunteers as the controls (n = 140). Initial blood samples were collected and concentrations of IFN-γ and IFN-γ-inducible chemokines CXCL9, CXCL10, and CXCL11 were measured using enzyme-linked immunosorbent assay. Of patients with lung cancer, 125 had non-small cell lung cancer (NSCLC) and 19 had small cell lung cancer. The area under the curve (AUC) (95% CI) of CXCL9 was 0.83 (0.80-0.89) for differentiating lung cancer patients from controls. The levels of all the markers were significantly higher in NSCLC patients with stage IV than in those with stages I-III. A Kaplan-Meier survival analysis showed that NSCLC cancer patients with higher levels of all markers showed poorer survival than those with lower levels. In Cox multivariate analysis of patients with NSCLC, independent prognostic factors for overall survival were CXCL9 and CXCL11. CXCL9 was the only independent prognostic factor for cancer-specific survival. Serum IFN-γ-inducible chemokines may be useful as clinical markers of metastasis and prognosis in NSCLC, and CXCL9 levels showed the most significant results.

Resurrected Ancestors Reveal Origins of Metamorphism in XCL1.

In a recent study, Dishman et al. resurrected ancestors of the metamorphic chemokine, XCL1, inferred through phylogenetics, and found that metamorphism arose in the XCL1 lineage ~150 million years ago. A zigzagging evolutionary path suggests that the metamorphic properties are adaptive and reveals three design principles that could be used for technological applications.

Evolution of fold switching in a metamorphic protein.

Metamorphic proteins switch between different folds, defying the protein folding paradigm. It is unclear how fold switching arises during evolution. With ancestral reconstruction and nuclear magnetic resonance, we studied the evolution of the metamorphic human protein XCL1, which has two distinct folds with different functions, making it an unusual member of the chemokine family, whose members generally adopt one conserved fold. XCL1 evolved from an ancestor with the chemokine fold. Evolution of a dimer interface, changes in structural constraints and molecular strain, and alteration of intramolecular protein contacts drove the evolution of metamorphosis. Then, XCL1 likely evolved to preferentially populate the noncanonical fold before reaching its modern-day near-equal population of folds. These discoveries illuminate how one sequence has evolved to encode multiple structures, revealing principles for protein design and engineering.

Specific binding-induced modulation of the XCL1 metamorphic equilibrium.

The metamorphic protein XCL1 switches between two distinct native structures with different functions in the human immune system. This structural interconversion requires complete rearrangement of all hydrogen bonding networks, yet fold-switching occurs spontaneously and reversibly in solution. One structure occupies the canonical α-ß chemokine fold and binds XCL1's cognate G-protein coupled receptor, while the other structure occupies a dimeric, all-ß fold that binds glycosaminoglycans and has antimicrobial activity. Both of these functions are important for the biologic role of XCL1 in the immune system, and each structure is approximately equally populated under near-physiologic conditions. Recent work has begun to illuminate XCL1's role in combatting infection and cancer. However, without a way to control XCL1's dynamic structural interconversion, it is difficult to study the role of XCL1 fold-switching in human health and disease. Thus, a molecular tool that can regulate the fractional population of the two XCL1 structures is needed. Here, we find by heparin affinity chromatography and NMR that an engineered XCL1 variant called CC5 can trigger a dose-dependent shift in XCL1's metamorphic equilibrium such that the receptor binding structure is depleted, and the antimicrobial structure is more heavily populated. This shift likely occurs due to formation of XCL1-CC5 heterodimers in which both protomers occupy the ß-sheet structure. These findings lay the groundwork for future studies seeking to understand the functional role of XCL1 metamorphosis, as well as studies screening for a drug-like molecule that can therapeutically target XCL1 by tuning its metamorphic equilibrium. Moreover, the proof of concept presented here suggests that protein metamorphosis is druggable, opening numerous avenues for controlling biological function of metamorphic proteins by altering the population of their multiple native states.

Involvement of the ERK/HIF-1α/EMT Pathway in XCL1-Induced Migration of MDA-MB-231 and SK-BR-3 Breast Cancer Cells.

Chemokine-receptor interactions play multiple roles in cancer progression. It was reported that the overexpression of X-C motif chemokine receptor 1 (XCR1), a specific receptor for chemokine X-C motif chemokine ligand 1 (XCL1), stimulates the migration of MDA-MB-231 triple-negative breast cancer cells. However, the exact mechanisms of this process remain to be elucidated. Our study found that XCL1 treatment markedly enhanced MDA-MB-231 cell migration. Additionally, XCL1 treatment enhanced epithelial-mesenchymal transition (EMT) of MDA-MB-231 cells via E-cadherin downregulation and upregulation of N-cadherin and vimentin as well as increases in ß-catenin nucleus translocation. Furthermore, XCL1 enhanced the expression of hypoxia-inducible factor-1α (HIF-1α) and phosphorylation of extracellular signal-regulated kinase (ERK) 1/2. Notably, the effects of XCL1 on cell migration and intracellular signaling were negated by knockdown of XCR1 using siRNA, confirming XCR1-mediated actions. Treating MDA-MB-231 cells with U0126, a specific mitogen-activated protein kinase kinase (MEK) 1/2 inhibitor, blocked XCL1-induced HIF-1α accumulation and cell migration. The effect of XCL1 on cell migration was also evaluated in ER-/HER2+ SK-BR-3 cells. XCL1 also promoted cell migration, EMT induction, HIF-1α accumulation, and ERK phosphorylation in SK-BR-3 cells. While XCL1 did not exhibit any significant impact on the matrix metalloproteinase (MMP)-2 and -9 expressions in MDA-MB-231 cells, it increased the expression of these enzymes in SK-BR-3 cells. Collectively, our results demonstrate that activation of the ERK/HIF-1α/EMT pathway is involved in the XCL1-induced migration of both MDA-MB-231 and SK-BR-3 breast cancer cells. Based on our findings, the XCL1-XCR1 interaction and its associated signaling molecules may serve as specific targets for the prevention of breast cancer cell migration and metastasis.

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.

Blockade of XCL1/Lymphotactin Ameliorates Severity of Periprosthetic Osteolysis Triggered by Polyethylene-Particles.

Periprosthetic osteolysis induced by orthopedic implant-wear particles continues to be the leading cause of arthroplasty failure in majority of patients. Release of the wear debris results in a chronic local inflammatory response typified by the recruitment of immune cells, including macrophages. The cellular mediators derived from activated macrophages favor the osteoclast-bone resorbing activity resulting in bone loss at the site of implant and loosening of the prosthetic components. Emerging evidence suggests that chemokines and their receptors are involved in the progression of periprosthetic osteolysis associated with aseptic implant loosening. In the current study, we investigated the potential role of chemokine C-motif-ligand-1 (XCL1) in the pathogenesis of inflammatory osteolysis induced by wear particles. Expressions of XCL1 and its receptor XCR1 were evident in synovial fluids and tissues surrounding hip-implants of patients undergoing revision total hip arthroplasty. Furthermore, murine calvarial osteolysis model induced by ultra-high molecular weight polyethylene (UHMWPE) particles was used to study the role of XCL1 in the development of inflammatory osteolysis. Mice received single injection of recombinant XCL1 onto the calvariae after implantation of particles exhibited significantly greater osteolytic lesions than the control mice. In contrast, blockade of XCL1 by neutralizing antibody significantly reduced bone erosion and the number of bone-resorbing mature osteoclasts induced by UHMWPE particles. In consistence with the results, transplantation of XCL1-soaked sponge onto calvariae caused osteolytic lesions coincident with excessive infiltration of inflammatory cells and osteoclasts. These results suggested that XCL1 might be involved in the development of periprosthetic osteolysis through promoting infiltration of inflammatory cells and bone resorbing-osteoclasts. Our further results demonstrated that supplementing recombinant XCL1 to cultured human monocytes stimulated with the receptor activator of nuclear factor kappa-B ligand (RANKL) promoted osteoclastogenesis and the osteoclast-bone resorbing activity. Moreover, recombinant XCL1 promoted the expression of inflammatory and osteoclastogenic factors, including IL-6, IL-8, and RANKL in human differentiated osteoblasts. Together, these results suggested the potential role of XCL1 in the pathogenesis of periprosthetic osteolysis and aseptic loosening. Our data broaden knowledge of the pathogenesis of aseptic prosthesis loosening and highlight a novel molecular target for therapeutic intervention.

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.

Transcutaneous immunization with a highly active form of XCL1 as a vaccine adjuvant using a hydrophilic gel patch elicits long-term CD8+ T cell responses.

Memory CD8+ cytotoxic T-lymphocytes (CTLs) play a key role in protective immunity against infection and cancer. However, the induction of memory CTLs with currently available vaccines remains difficult. The chemokine receptor XCR1 is predominantly expressed on CD103+ cross-presenting dendritic cells (DCs). Recently, we have demonstrated that a high activity form of murine lymphotactin/XCL1 (mXCL1-V21C/A59C), a ligand of XCR1, can induce antigen-specific memory CTLs by increasing the accumulation of CD103+ DCs in the vaccination site and the regional lymph nodes. Here, we combined a hydrophilic gel patch as a transcutaneous delivery device and mXCL1-V21C/A59C as an adjuvant to further enhance memory CTL responses. The transcutaneous delivery of ovalbumin (OVA) and mXCL1-V21C/A59C by the hydrophilic gel patch increased CD103+ DCs in the vaccination site and the regional lymph nodes for a prolonged period of time compared with the intradermal injection of OVA and mXCL1-V21C/A59C. Furthermore, the hydrophilic gel patch containing OVA and mXCL1-V21C/A59C strongly induced OVA-specific memory CTLs and efficiently inhibited the growth of OVA-expressing tumors more than the intradermal injection of OVA and mXCL1-V21C/A59C. Collectively, this type of hydrophilic gel patch and a high activity form of XCL1 may provide a useful tool for the induction of memory CTL responses.

Switchable Membrane Remodeling and Antifungal Defense by Metamorphic Chemokine XCL1.

Antimicrobial peptides (AMPs) are a class of molecules which generally kill pathogens via preferential cell membrane disruption. Chemokines are a family of signaling proteins that direct immune cell migration and share a conserved α-ß tertiary structure. Recently, it was found that a subset of chemokines can also function as AMPs, including CCL20, CXCL4, and XCL1. It is therefore surprising that machine learning based analysis predicts that CCL20 and CXCL4's α-helices are membrane disruptive, while XCL1's helix is not. XCL1, however, is the only chemokine known to be a metamorphic protein which can interconvert reversibly between two distinct native structures (a ß-sheet dimer and the α-ß chemokine structure). Here, we investigate XCL1's antimicrobial mechanism of action with a focus on the role of metamorphic folding. We demonstrate that XCL1 is a molecular "Swiss army knife" that can refold into different structures for distinct context-dependent functions: whereas the α-ß chemokine structure controls cell migration by binding to G-Protein Coupled Receptors (GPCRs), we find using small angle X-ray scattering (SAXS) that only the ß-sheet and unfolded XCL1 structures can induce negative Gaussian curvature (NGC) in membranes, the type of curvature topologically required for membrane permeation. Moreover, the membrane remodeling activity of XCL1's ß-sheet structure is strongly dependent on membrane composition: XCL1 selectively remodels bacterial model membranes but not mammalian model membranes. Interestingly, XCL1 also permeates fungal model membranes and exhibits anti-Candida activity in vitro, in contrast to the usual mode of antifungal defense which requires Th17 mediated cell-based responses. These observations suggest that metamorphic XCL1 is capable of a versatile multimodal form of antimicrobial defense.