Chemokine Binding Research Articles

Abstract 2899: Targeting regulatory T cells with HFB101110, a novel anti-human CCR8 antibody for the treatment of solid tumors

Abstract Regulatory T cells (Tregs) contribute to immunosuppression within the tumor microenvironment and have been associated with poor outcomes in a range of cancers. Targeted depletion of tumor-infiltrating Tregs (TITRs) is an attractive therapeutic strategy with the potential to enhance antitumor immunity in patients who do not respond to current treatments. However, such approaches have been limited to date by a lack of markers that are highly specific for TITRs. CCR8 is a chemokine G protein-coupled receptor (GPCR) that has recently been shown to be specifically expressed on TITRs as compared to Tregs in the periphery or other T cells within tumors. Here, we report the development and characterization of HFB101110, a humanized monoclonal antibody against CCR8 with potent and specific antibody-dependent cellular cytotoxicity (ADCC) activity. HFB101110 specifically recognizes an epitope on the N-terminus extracellular domain of CCR8 and does not recognize the closely related chemokine receptor CCR4. HFB101110 acts through a dual mechanism of action, by both depleting CCR8+ cells via ADCC and blocking binding of the CCL1 chemokine to its receptor CCR8. Blockade by HFB101110 inhibited calcium flux and chemotaxis induced by the interaction between CCL1 and CCR8. Furthermore, HFB101110 showed potent single agent anti-tumor activity associated with depletion of intratumoral Tregs in a human CCR8 knock-in mouse model. HFB101110 mediated specific ex vivo killing of Tregs from primary patient tumor samples both in the presence, and in some cases the absence, of allogeneic NK cells. HFB101110 showed favorable pharmacokinetic properties and a favorable developability profile, and was well-tolerated in both wild-type mice and cynomolgus monkeys. HFB101110 is currently being developed as a novel immunotherapy for the treatment of solid tumors coupled with a patient biomarker strategy derived from HiFiBiO’s Drug Intelligent Science (DIS™) single-cell immune profiling platform. Citation Format: Roshan M. Kumar, Julie Prigent, Hombline Poullain, Ayrin Kok, Carine George, Sami Ellouze, Yun-Yueh Lu, Rebecca Silver, Clarisse Monchecourt, Ross B. Fulton, Qian Zhang, Nicola Beltraminelli, Bernhard Moser, Francisco Adrian, Liang Schweizer. Targeting regulatory T cells with HFB101110, a novel anti-human CCR8 antibody for the treatment of solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2899.

Transcriptomic Evidence of the Immune Response Activation in Individuals With Limb Girdle Muscular Dystrophy Dominant 2 (LGMDD2) Contributes to Resistance to HIV-1 Infection.

LGMDD2 is a rare form of muscular dystrophy characterized by one of the three heterozygous deletions described within the TNPO3 gene that result in the addition of a 15-amino acid tail in the C-terminus.TNPO3 is involved in the nuclear import of splicing factors and acts as a host cofactor for HIV-1 infection by mechanisms not yet deciphered. Further characterization of the crosstalk between HIV-1 infection and LGMDD2 disease may contribute to a better understanding of both the cellular alterations occurring in LGMDD2 patients and the role of TNPO3 in the HIV-1 cycle. To this regard, transcriptome profiling of PBMCs from LGMDD2 patients carrying the deletion c.2771delA in the TNPO3 gene was compared to healthy controls. A total of 545 differentially expressed genes were detected between LGMDD2 patients and healthy controls, with a high representation of G protein-coupled receptor binding chemokines and metallopeptidases among the most upregulated genes in LGMDD2 patients. Plasma levels of IFN-β and IFN-γ were 4.7- and 2.7-fold higher in LGMDD2 patients, respectively. An increase of 2.3-fold in the expression of the interferon-stimulated gene MxA was observed in activated PBMCs from LGMDD2 patients after ex vivo HIV-1 pseudovirus infection. Thus, the analysis suggests a pro-inflammatory state in LGMDD2 patients also described for other muscular dystrophies, that is characterized by the alteration of IL-17 signaling pathway and the consequent increase of metallopeptidases activity and TNF response. In summary, the increase in interferons and inflammatory mediators suggests an antiviral environment and resistance to HIV-1 infection but that could also impair muscular function in LGMDD2 patients, worsening disease evolution. Biomarkers of disease progression and therapeutic strategies based on these genes and mechanisms should be further investigated for this type of muscular dystrophy.

TCR Coexpression Signature Predicts Immunotherapy Resistance in NSCLC.

Background: Lung cancer has the highest morbidity and mortality rate among types of malignant tumors, and as such, research into prolonging the survival time of patients is vital. The emergence of immune checkpoint inhibitors (ICIs) has greatly improved the survival of patients with non-small cell lung cancer (NSCLC), however, the lack of effective biomarkers to predict the prognosis of immunotherapy has made it difficult to maximize the benefits. T cell receptor (TCR) is one of the most important components for recognizing tumor cells, and with this study we aim to clarify the relationship between TCR coexpression and the prognosis of NSCLC patients receiving immunotherapy. Methods: Univariate COX regression, logistics regression, and KM survival analysis were used to evaluate the relationship between TCR coexpression and the prognosis of immunotherapy. Additionally, CIBERSORT, Gene Set Enrichment Analysis (GSEA), and single-sample GSEA (ssGSEA) algorithms were used to evaluate the tumor immune microenvironment (TIME) of NSCLC patients. Results: Univariate Cox regression analysis showed that the TCR coexpression signature can be used as a clinical prognostic indicator for NSCLC patients receiving immunotherapy (p = 0.0205). In addition, those in the NSCLC group with a high TCR coexpression signature had significantly improved progression-free survival (PFS) (p = 0.014). In the ICI treatment cohort (GSE35640). In addition, there was a high infiltration of CD8+T cells, activated memory CD4+T cells, and M1 macrophages in the TIME of those with a high TCR coexpression signature. The results of pathway enrichment analysis showed that patients with a high TCR coexpression signature had significantly activated signal pathways such as lymphocyte proliferation and activation, chemokine binding, and inflammatory cytokine production. Also, we found that patients with a high TCR coexpression signature had an elevated T cell inflammation gene expression profile (GEP). Conclusion: We show that the TCR coexpression signature may be useful as a new biomarker for the prognosis of NSCLC patients undergoing immunotherapy, with high signatures indicating better treatment response. Additionally, we found that patients with a high TCR coexpression signature had tumor immune microenvironments with beneficial anti-tumor characteristics.

MMP9 Expression Correlates With Cisplatin Resistance in Small Cell Lung Cancer Patients.

Background: Cisplatin is the basis of the primary treatment for SCLC chemotherapy. However, the limited objective response rate and definite drug resistance greatly restrict the clinical potential and therapeutic benefits of cisplatin use. Therefore, it is essential to identify biomarkers that can discern the sensitivity of SCLC patients to cisplatin treatment. Methods: We collected two SCLC cohorts treated with cisplatin that included mutation data, prognosis data and expression data. The sensitivity of cisplatin was evaluated by the pRRophetic algorithm. MCPcounter, quanTIseq, and xCell algorithms were used to evaluate immune cell score. GSEA and ssGSEA algorithms were used to calculate immune-related pathway scores. Univariate and multivariate Cox regression models were employed, and survival analysis was used to evaluate the prognostic value of the candidate genes. Results: MMP9-High is related to improved clinical prognoses of patients with SCLC (HR = 0.425, p = 0.0085; HR = 0.365, p = 0.0219). Multivariate results showed that MMP-High could be used as an independent predictor of the prognosis of SCLC after cisplatin treatment (HR = 0.216, p = 0.00153; HR = 0.352; p = 0.0199). In addition, MMP9-High displayed a significantly lower IC50 value of cisplatin and higher immunogenicity than MMP9-Low SCLC. Compared with MMP9-Low SCLC, MMP9-High included significantly increased levels of T-cells, cytoxic lymphocytes, B-cells, NK-cells, and dense cells (DCS). Similarly, the activity of cytokine binding, B-cell, NK-cell mediated immune response chemokine binding, and antigen presentation pathways in MMP9-High was significantly higher than that in MMP9-Low. Conclusion: In this study, we identified that MMP9-High could be potentially considered a novel biomarker used to ascertain the improved prognosis of SCLC patients after cisplatin treatment. Furthermore, we indicated that the tumor immune microenvironment of MMP9-High SCLC is mainly characterized by a large number of infiltrated activated immune cells as well as activated immune-related pathways.

Modulating tenascin-C functions by targeting the MAtrix REgulating MOtif, “MAREMO”

The extracellular matrix molecule Tenascin-C (TNC) promotes cancer and chronic inflammation by multiple mechanisms. Recently, TNC was shown to promote an immune suppressive tumor microenvironment (TME) through binding soluble chemoattracting factors, thus retaining leukocytes in the stroma. TNC also binds to fibronectin (FN) and other molecules, raising the question of a potential common TNC binding mechanism. By sequence comparison of two TNC-interacting domains in FN, the fifth (FN5) and thirteenth (FN13) fibronectin type III domains we identified a MAtrix REgulating MOtif “MAREMO” or M-motif that is highly conserved amongst vertebrates. By sequence analysis, structural modeling and functional analysis we found also putative M-motifs in TNC itself. We showed by negative staining electron microscopic imaging that the M-motif in FN mediates interactions with FN as well as with TNC. We generated two M-motif mimetic peptides P5 and P13 resembling the M-motif in FN5 and FN13, respectively. By using structural information we modelled binding of these M-motif mimetics revealing a putative MAREMO binding site MBS in FN5 and TN3, respectively overlapping with the M-motif. We further demonstrated that the M-motif mimetic peptides blocked several functions of TNC, such as binding of TNC to FN, cell rounding on a mixed FN/TNC substratum, FN matrix expression and subsequent assembly, TNC-induced signaling and gene expression, TNC chemokine binding and dendritic cell retention, thus providing novel opportunities to inhibit TNC actions. Our results suggest that targeting the MAREMO/MBS interaction could be exploited for reducing inflammation and matrix functions in cancer and fibrosis.

Identification of Inflammation-Related Biomarker Pro-ADM for Male Patients With Gout by Comprehensive Analysis.

ObjectiveGout is a local inflammatory disease caused by the deposition of monosodium urate (MSU) crystals in joints or adjacent tissues. When some gout occurs without hyperuricemia, or its clinical symptoms and signs are not typical, the diagnosis of gout will be delayed, so there is an urgent need to find a new biomarker to predict and diagnose of gout flare. Our research attempts to find the key genes and potential molecular mechanisms of gout through bioinformatics analysis, and collected general data and blood biochemical samples of patients with gout and healthy, then analyzed and compared the expression of factors regulated by key genes.MethodGSE160170 were downloaded from GEO database for analysis. The data were normalized to identify the differentially expressed genes (DEGs), then GO and KEGG enrichment analysis were applied. Protein-protein interaction (PPI) networks and hub genes between DEGs were identified. Then collect general information and blood samples from male patients with acute gout, hyperuricemia and healthy. ELISA method was used to detect pro-ADM levels of different groups, and the data was input into SPSS statistical software for analysis.ResultWe identified 266 DEGs (179 up-regulated and 87 down-regulated) between gout patients and healthy controls. GO analysis results show that DEGs are mostly enriched in inflammatory response, growth factor activity, cytokine activity, chemokine activity, S100 protein binding and CXCR chemokine receptor binding. KEGG pathway analysis showed that DEGs are mainly related to Chemokine signaling pathway and Cytokine-cytokine receptor interaction. ADM, CXCR1, CXCR6, CXCL3, CCL3, CCL18, CCL3L3, CCL4L1, CD69, CD83, AREG, EREG, B7RP1, HBEGF, NAMPT and S100B are the most important hub genes in the PPI network. We found that the expression of pro-ADM in the gout group and hyperuricemia group was higher than that in the healthy group, and the difference was statistically significant.ConclusionIn this study, a series of bioinformatics analyses were performed on DEGs to identify key genes and pathways related to gout. Through clinical verification, we found that pro-ADM can be used as an inflammation-related biomarker for acute attacks of gout, providing new ideas for the diagnosis and treatment of gout.

The Immune Landscape of Hepatitis B Virus-Related Acute Liver Failure by Integration Analysis.

Hepatitis B virus-related acute liver failure (HBV-ALF) is a common type of liver failure, associated with high short-term mortality and morbidity rates. However, the immune landscape of HBV-ALF and its correlation with cell death are currently unknown. Based on 3 Gene Expression Omnibus data sets, infiltrated immune cells were quantified by single-sample gene set enrichment analysis method. The expression levels of immune genes and the abundance of immune cells in liver failure were compared with those in normal liver. The enrichment scores of cell death gene sets from Kyoto Encyclopedia of Genes and Genomes (KEGG) were calculated by gene set variation analysis method, and a protein-protein interaction (PPI) network was constructed using Cytoscape. Besides 21 differentially expressed immune genes, we identified 11 types of differentially infiltrated immune cells in HBV-ALF compared with normal liver. Enriched pathways of these immune genes mainly consisted of chemokine receptors, chemokine binding, interleukin-10 signaling, and TNFs bind their physiological receptors by Reactome pathway analysis. In addition, the enrichment scores of apoptosis and necroptosis pathway instead of autophagy and ferroptosis were increased in liver failure compared with normal liver. PPI network and gene cluster analysis of immune genes and apoptosis and necroptosis genes suggested that hub genes were mainly related to immune response and apoptosis. In summary, our study offers a conceptual framework to understand the immune landscape of HBV-ALF, which might help to improve prognosis.

Blocking of inflammatory heparan sulfate domains by specific antibodies is not protective in experimental glomerulonephritis.

Glomerulonephritis is an acquired serious glomerular disease, which involves the interplay of many factors such as cytokines, chemokines, inflammatory cells, and heparan sulfate (HS). We previously showed that blocking of inflammatory heparan sulfate domains on cultured glomerular endothelium by specific anti-HS single chain antibodies reduced polymorphonuclear cell (PMN) adhesion and chemokine binding. We hypothesized that injection of anti-HS antibodies in PMN-driven experimental glomerulonephritis should reduce glomerular influx of PMNs and thereby lead to a better renal outcome. In contrast to our hypothesis, co-injection of anti-HS antibodies did not alter the final outcome of anti-glomerular basement membrane (anti-GBM)-induced glomerulonephritis. Glomerular PMN influx, normally peaking 2 hours after induction of glomerulonephritis with anti-GBM IgG was not reduced by co-injection of anti-HS antibodies. Four days after induction of glomerulonephritis, albuminuria, renal function, glomerular hyalinosis and fibrin deposition were similar in mice treated and not treated with anti-HS antibodies. Interestingly, we observed transient effects in mice co-injected with anti-HS antibodies compared to mice that did not receive anti-HS antibodies: (i) a decreased renal function 2 hours and 1 day after induction of glomerulonephritis; (ii) an increased albuminuria after 2 hours and 1 day; (iii) an increased glomerular fibrin deposition after 1 day; (iv) a reduced glomerular macrophage influx after 1 day; (v) a sustained glomerular presence of PMNs at day 1 and 4, accompanied by an increased renal expression of IL-6, CXCL1, ICAM-1, L-selectin, CD11b and NF-κB. The mechanism underlying these observations induced by anti-HS antibodies remains unclear, but may be explained by a temporarily altered glycocalyx and/or altered function of PMNs due to the binding of anti-HS antibodies. Nevertheless, the evaluated anti-HS antibodies do not show therapeutic potential in anti-GBM-induced glomerulonephritis. Future research should evaluate other strategies to target HS domains involved in inflammatory processes during glomerulonephritis.

Comprehensive Analysis of TP53 Mutation Characteristics and Identification of Patients with Inferior Prognosis and Enhanced Immune Escape in Diffuse Large B Cell Lymphoma

Background: Diffuse large B-cell lymphoma (DLBCL) is a highly heterogeneous lymphatic malignancy from both the clinical and molecular standpoints. TP53 mutations have been observed in DLBCL with high frequency mutations, which play a crucial role in lymphomagenesis. Considering the complex pathophysiological mechanisms involved in DLBCL, we herein assume that the interaction of TP53 with other genetic variants together promote the development of DLBCL. We also explore that these genetic interactions shape the discrepant immune landscape in DLBCL.Methods: We performed a comprehensive analysis of TP53 mutations in the DNA-binding domain (DBD) region. Genome alterations that were mutually exclusive or co-occurring with TP53 mutations were identified, and their potential value in prognosis and possible relationships with tumor microenvironment (TME) were further examined using targeted next-generation sequencing (n=176), transcriptome sequencing (n=152) and circulating tumor DNA sequencing (n=38).Results: TP53 was frequently mutated in DLBCL with a rate of 30% (53 of 176). The occurrence of TP53 mutations was skewed toward the early stages of DLBCL pathogenesis. Among these variants of TP53, 74% were missense mutations, and the remaining were inactivating frameshift indels, nonsense mutations, coding sequencing indels and splicing mutations. Importantly, most mutations (87.5%) occurred in exons 5-8, which encoded the DNA-binding domain (DBD) region of TP53. Codons 175, 273, and 248 of the p53 protein had the highest mutation frequency, which are also the hot spots of TP53 mutation found in most human cancers. However, TP53 alone is insufficient to effectively differentiate the risk of DLBCL, even when only considering mutations in the DBD region. CD58 mutations, which are mutually exclusive from TP53 mutations, in combination with TP53 mutations, could significantly differentiate the prognosis of DLBCL. The survival of patients with either one of the mutually exclusive mutation patterns, namely, TP53MUT&CD58WT or TP53WT&CD58MUT, was inferior to those harboring both wild-type TP53 and CD58. Notably, patients with TP53WT&CD58MUT mutation pattern had the worst outcome and were characterized by a higher overall tumor mutation burden (TMB) and immune score. An enhanced immune escape feature, including the abundant infiltration of inflammatory cells and upregulation of inhibitory immunomodulatory molecules such as PD-1, TIM3, LAG3, KLRC1, KLRC3, KLRD1, IDO1, IDO2 and TDO2, was observed in these patients with TP53WT&CD58MUT mutation pattern. A larger number of terms related to cytokine and chemokine and inflammatory pathways were enriched in the TP53WT&CD58MUT group, including cytokine and chemokine production, binding and activity, and interferon-γ pathway. Additionally, GSEA demonstrated that interferon-α and -γ responses and IL-6/JAK/STAT3 signaling enriched in the TP53WT&CD58MUT group. These patients with TP53WT&CD58MUT mutation pattern represent the candidate populations for immune therapy.Conclusions: Our findings indicated that the mutation patterns of TP53 and CD58 accurately stratified patients with DLBCL to permit the optional immunotherapy. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

The C-terminal peptide of CCL21 drastically augments CCL21 activity through the dendritic cell lymph node homing receptor CCR7 by interaction with the receptor N-terminus

The endogenous chemokines CCL19 and CCL21 signal via their common receptor CCR7. CCL21 is the main lymph node homing chemokine, but a weak chemo-attractant compared to CCL19. Here we show that the 41-amino acid positively charged peptide, released through C-terminal cleavage of CCL21, C21TP, boosts the immune cell recruiting activity of CCL21 by up to 25-fold and the signaling activity via CCR7 by ~ 100-fold. Such boosting is unprecedented. Despite the presence of multiple basic glycosaminoglycan (GAG) binding motifs, C21TP boosting of CCL21 signaling does not involve interference with GAG mediated cell-surface retention. Instead, boosting is directly dependent on O-glycosylations in the CCR7 N-terminus. As dictated by the two-step binding model, the initial chemokine binding involves interaction of the chemokine fold with the receptor N-terminus, followed by insertion of the chemokine N-terminus deep into the receptor binding pocket. Our data suggest that apart from a role in initial chemokine binding, the receptor N-terminus also partakes in a gating mechanism, which could give rise to a reduced ligand activity, presumably through affecting the ligand positioning. Based on experiments that support a direct interaction of C21TP with the glycosylated CCR7 N-terminus, we propose that electrostatic interactions between the positively charged peptide and sialylated O-glycans in CCR7 N-terminus may create a more accessible version of the receptor and thus guide chemokine docking to generate a more favorable chemokine-receptor interaction, giving rise to the peptide boosting effect.

Optimisation of Neuraminidase Expression for Use in Drug Discovery by Using HEK293-6E Cells.

Influenza virus is a highly contagious virus that causes significant human mortality and morbidity annually. The most effective drugs for treating influenza are the neuraminidase inhibitors, but resistance to these inhibitors has emerged, and additional drug discovery research on neuraminidase and other targets is needed. Traditional methods of neuraminidase production from embryonated eggs are cumbersome, while insect cell derived protein is less reflective of neuraminidase produced during human infection. Herein we describe a method for producing neuraminidase from a human cell line, HEK293-6E, and demonstrate the method by producing the neuraminidase from the 1918 H1N1 pandemic influenza strain. This method produced high levels of soluble neuraminidase expression (>3000 EU/mL), was enhanced by including a secretion signal from a viral chemokine binding protein, and does not require co-expression of additional proteins. The neuraminidase produced was of sufficient quantity and purity to support high resolution crystal structure determination. The structure solved using this protein conformed to the previously reported structure. Notably the glycosylation at three asparagine residues was superior in quality to that from insect cell derived neuraminidase. This method of production of neuraminidase should prove useful in further studies, such as the characterisation of inhibitor binding.

Isolation and Characterization of Heparan Sulfate from Human Lung Tissues.

Glycosaminoglycans are a class of linear, highly negatively charged, O-linked polysaccharides that are involved in many (patho)physiological processes. In vitro experimental investigations of such processes typically involve porcine-derived heparan sulfate (HS). Structural information about human, particularly organ-specific heparan sulfate, and how it compares with HS from other organisms, is very limited. In this study, heparan sulfate was isolated from human lung tissues derived from five donors and was characterized for their overall size distribution and disaccharide composition. The expression profiles of proteoglycans and HS-modifying enzymes was quantified in order to identify the major core proteins for HS. In addition, the binding affinities of human HS to two chemokines—CXCL8 and CCL2—were investigated, which represent important inflammatory mediators in lung pathologies. Our data revealed that syndecans are the predominant proteoglycan class in human lungs and that the disaccharide composition varies among individuals according to sex, age, and health stage (one of the donor lungs was accidentally discovered to contain a solid tumor). The compositional difference of the five human lung HS preparations affected chemokine binding affinities to various degrees, indicating selective immune cell responses depending on the relative chemokine–glycan affinities. This represents important new insights that could be translated into novel therapeutic concepts for individually treating lung immunological disorders via HS targets.

The Role of Post-Translational Modifications of Chemokines by CD26 in Cancer.

Simple SummaryChemokines are central players in cancer and can be post-translationally modified by dipeptidyl peptidase IV (DPPIV)/CD26. This can have different effects on chemokine function, ranging from reduced, unchanged to enhanced activity. CD26 is differentially expressed in tumors, which affects the dominant chemokine isoform present in the tumor microenvironment. In this review, we aim to recapitulate the current knowledge on the interplay between CD26 and chemokine activity in cancer.Chemokines are a large family of small chemotactic cytokines that fulfill a central function in cancer. Both tumor-promoting and -impeding roles have been ascribed to chemokines, which they exert in a direct or indirect manner. An important post-translational modification that regulates chemokine activity is the NH2-terminal truncation by peptidases. CD26 is a dipeptidyl peptidase (DPPIV), which typically clips a NH2-terminal dipeptide from the chemokine. With a certain degree of selectivity in terms of chemokine substrate, CD26 only recognizes chemokines with a penultimate proline or alanine. Chemokines can be protected against CD26 recognition by specific amino acid residues within the chemokine structure, by oligomerization or by binding to cellular glycosaminoglycans (GAGs). Upon truncation, the binding affinity for receptors and GAGs is altered, which influences chemokine function. The consequences of CD26-mediated clipping vary, as unchanged, enhanced, and reduced activities are reported. In tumors, CD26 most likely has the most profound effect on CXCL12 and the interferon (IFN)-inducible CXCR3 ligands, which are converted into receptor antagonists upon truncation. Depending on the tumor type, expression of CD26 is upregulated or downregulated and often results in the preferential generation of the chemokine isoform most favorable for tumor progression. Considering the tight relationship between chemokine sequence and chemokine binding specificity, molecules with the appropriate characteristics can be chemically engineered to provide innovative therapeutic strategies in a cancer setting.

CCL3/CCR5 and CX3CL1/CX3CR1 chemokine interactions are involved in T-cell accumulation in human atherosclerotic plaques

Background and Aims: Immune cells, particularly T-lymphocytes, play a critical role in atherosclerotic plaques’ progression and destabilization leading to myocardial infarctions and strokes. Chemokine binding was proposed to drive T-cell accumulation in plaques. We analysed the chemokine receptor signatures of T-cells isolated from 17 atherosclerotic plaques from 13 patients and compared them with autologous peripheral blood T-cells. Also, the chemokine expression levels in plaque tissue were compared with those in peripheral blood cells.

Genetic mechanisms of COVID-19 and its association with smoking and alcohol consumption.

We aimed to investigate the genetic mechanisms associated with coronavirus disease of 2019 (COVID-19) outcomes in the host and to evaluate the possible associations between smoking and drinking behavior and three COVID-19 outcomes: severe COVID-19, hospitalized COVID-19 and COVID-19 infection. We described the genomic loci and risk genes associated with the COVID-19 outcomes, followed by functional analyses of the risk genes. Then, a summary data-based Mendelian randomization (SMR) analysis, and a transcriptome-wide association study (TWAS) were performed for the severe COVID-19 dataset. A two-sample Mendelian randomization (MR) analysis was used to evaluate the causal associations between various measures of smoking and alcohol consumption and the COVID-19 outcomes. A total of 26 protein-coding genes, enriched in chemokine binding, cytokine binding and senescence-related functions, were associated with either severe COVID-19 or hospitalized COVID-19. The SMR and the TWAS analyses highlighted functional implications of some GWAS hits and identified seven novel genes for severe COVID-19, including CCR5, CCR5AS, IL10RB, TAC4, RMI1 and TNFSF15, some of which are targets of approved or experimental drugs. According to our studies, increasing consumption of cigarettes per day by 1 standard deviation is related to a 2.3-fold increase in susceptibility to severe COVID-19 and a 1.6-fold increase in COVID-19-induced hospitalization. Contrarily, no significant links were found between alcohol consumption or binary smoking status and COVID-19 outcomes. Our study revealed some novel COVID-19 related genes and suggested that genetic liability to smoking may quantitatively contribute to an increased risk for a severe course of COVID-19.

Identification of Functional Domains of CXCL14 Involved in High-Affinity Binding and Intracellular Transport of CpG DNA

Abstract Some CXC chemokines, including CXCL14, transport CpG oligodeoxynucleotides (ODNs) into dendritic cells (DCs), thereby activating TLR9. The molecular basis of this noncanonical function of CXC chemokines is not well understood. In this study, we investigated the CpG ODN binding and intracellular transport activities of various CXC chemokines and partial peptides of CXCL14 in mouse bone marrow–derived dendritic cells. CXCL14, CXCL4, and CXCL12 specifically bound CpG ODN, but CXCL12 failed to transport it into cells at low dose. CXCL14 N-terminal peptides 1–47, but not 1–40, was capable of transporting CpG ODN into the cell, resulting in an increase in cytokine production. However, both the 1–47 and 1–40 peptides bound CpG ODN. By contrast, CXCL14 peptides 13–50 did not possess CpG ODN binding capacity or transport activity. The chimeric peptides CXCL12 (1–22)–CXCL14 (13–47) bound CpG ODN but failed to transport it. These results suggest that amino acids 1–12 and 41–47 of CXCL14 are required for binding and intracellular transport of CpG ODN, respectively. We found that an anti-CXCL14 Ab blocked cell-surface binding and internalization of the CpG ODN/CXCL14 complex. On the basis of these findings, we propose that CXCL14 has two functional domains, one involved in DNA recognition and the other in internalization of CXCL14-CpG DNA complex via an unidentified CXCL14 receptor, which together are responsible for eliciting the CXCL14/CpG ODN-mediated TLR9 activation. These domains could play roles in CXCL14-related diseases such as arthritis, obesity-induced diabetes, and various types of carcinoma.

Contribution of Heparan Sulphate Binding in CCL21-Mediated Migration of Breast Cancer Cells.

Simple SummaryBreast cancer is a leading cause of cancer-related deaths worldwide, predominantly caused by metastasis. Chemokine receptor CCR7 and its ligand CCL21 are implicated in the metastasis of breast cancer to the lymph nodes. Chemokine function is dependent upon binding to their specific chemokine receptors and negatively charged molecules on the cell surface (heparan sulphate). The role of heparan sulphate in CCR7-mediated lymph node metastasis was investigated by creating a non-heparan sulphate binding mutant chemokine CCL21. Mutant-CCL21 was tested in vitro in a range of assays, including cell migration, calcium flux and surface plasmon resonance spectroscopy. Mutant-CCL21 induced leukocyte chemotaxis in diffusion gradients but did not stimulate trans-endothelial migration of breast cancer cells. A murine model was used to assess the potential of mutant-CCL21 to prevent lymph node metastasis in vivo. Lymph node metastasis was significantly reduced by the administration of mutant-CCL21 compared to the control. Targeting chemokine–heparan sulphate interactions may be a promising approach to inhibit chemokine activity and metastasis.Chemokine receptor CCR7 is implicated in the metastasis of breast cancer to the lymph nodes. Chemokine function is dependent upon their binding to both cell-surface heparan sulphate (HS) and to their specific receptors; thus, the role of HS in CCR7-mediated lymph node metastasis was investigated by creating a non-HS binding chemokine CCL21 (mut-CCL21). Mut-CCL21 (Δ103–134) induced leukocyte chemotaxis in diffusion gradients but did not stimulate trans-endothelial migration of PBMCs (p < 0.001) and 4T1-Luc cells (p < 0.01). Furthermore, the effect of heparin and HS on the chemotactic properties of wild-type (WT) and mut-CCL21 was examined. Interestingly, heparin and HS completely inhibit the chemotaxis mediated by WT-CCL21 at 250 and 500 µg/mL, whereas minimal effect was seen with mut-CCL21. This difference could potentially be attributed to reduced HS binding, as surface plasmon resonance spectroscopy showed that mut-CCL21 did not significantly bind HS compared to WT-CCL21. A murine model was used to assess the potential of mut-CCL21 to prevent lymph node metastasis in vivo. Mice were injected with 4T1-Luc cells in the mammary fat pad and treated daily for a week with 20 µg mut-CCL21. Mice were imaged weekly with IVIS and sacrificed on day 18. Luciferase expression was significantly reduced in lymph nodes from mice that had been treated with mut-CCL21 compared to the control (p = 0.0148), suggesting the potential to target chemokine binding to HS as a therapeutic option.

Structural basis for the constitutive activity and immunomodulatory properties of the Epstein-Barr virus-encoded G protein-coupled receptor BILF1

Epstein-Barr virus (EBV) encodes a G protein-coupled receptor (GPCR) termed BILF1 that is essential for EBV-mediated immunosuppression and oncogenesis. BILF1 couples with inhibitory G protein (Gi), the major intracellular signaling effector for human chemokine receptors, and exhibits constitutive signaling activity; the ligand(s) for BILF1 are unknown. We studied the origins of BILF1's constitutive activity through structure determination of BILF1 bound to the inhibitory G protein (Gi) heterotrimer. The 3.2-Å resolution cryo-electron microscopy structure revealed an extracellular loop within BILF1 that blocked the typical chemokine binding site, suggesting ligand-autonomous receptor activation. Rather, amino acid substitutions within BILF1 transmembrane regions at hallmark ligand-activated class A GPCR "microswitches" stabilized a constitutively active BILF1 conformation for Gi coupling in a ligand-independent fashion. Thus, the constitutive activity of BILF1 promotes immunosuppression and virulence independent of ligand availability, with implications for the function of GPCRs encoded by related viruses and for therapeutic targeting of EBV.

Chemokines act as phosphatidylserine-bound "find-me" signals in apoptotic cell clearance.

Removal of apoptotic cells is essential for maintenance of tissue homeostasis. Chemotactic cues termed "find-me" signals attract phagocytes toward apoptotic cells, which selectively expose the anionic phospholipid phosphatidylserine (PS) and other "eat-me" signals to distinguish healthy from apoptotic cells for phagocytosis. Blebs released by apoptotic cells can deliver find-me signals; however, the mechanism is poorly understood. Here, we demonstrate that apoptotic blebs generated in vivo from mouse thymus attract phagocytes using endogenous chemokines bound to the bleb surface. We show that chemokine binding to apoptotic cells is mediated by PS and that high affinity binding of PS and other anionic phospholipids is a general property of many but not all chemokines. Chemokines are positively charged proteins that also bind to anionic glycosaminoglycans (GAGs) on cell surfaces for presentation to leukocyte G protein-coupled receptors (GPCRs). We found that apoptotic cells down-regulate GAGs as they up-regulate PS on the cell surface and that PS-bound chemokines, unlike GAG-bound chemokines, are able to directly activate chemokine receptors. Thus, we conclude that PS-bound chemokines may serve as find-me signals on apoptotic vesicles acting at cognate chemokine receptors on leukocytes.

Behavior of Chemokine Receptor 6 (CXCR6) in Complex with CXCL16 Soluble form Chemokine by Molecular Dynamic Simulations: General Protein‒Ligand Interaction Model and 3D-QSAR Studies of Synthetic Antagonists.

The CXCR6‒CXCL16 axis is involved in several pathological processes, and its overexpression has been detected in different types of cancer, such as prostate, breast, ovary, and lung cancer, along with schwannomas, in which it promotes invasion and metastasis. Moreover, this axis is involved in atherosclerosis, type 1 diabetes, primary immune thrombocytopenia, vitiligo, and other autoimmune diseases, in which it is responsible for the infiltration of different immune system cells. The 3D structure of CXCR6 and CXCL16 has not been experimentally resolved; therefore, homology modeling and molecular dynamics simulations could be useful for the study of this signaling axis. In this work, a homology model of CXCR6 and a soluble form of CXCL16 (CXCR6‒CXCL16s) are reported to study the interactions between CXCR6 and CXCL16s through coarse-grained molecular dynamics (CG-MD) simulations. CG-MD simulations showed the two activation steps of CXCR6 through a decrease in the distance between the chemokine and the transmembrane region (TM) of CXCR6 and transmembrane rotational changes and polar interactions between transmembrane segments. The polar interactions between TM3, TM5, and TM6 are fundamental to functional conformation and the meta-active state of CXCR6. The interactions between D77-R280 and T243-TM7 could be related to the functional conformation of CXCR6; alternatively, the interaction between Q195-Q244 and N248 could be related to an inactive state due to the loss of this interaction, and an arginine cage broken in the presence of CXCL16s allows the meta-active state of CXCR6. A general protein‒ligand interaction supports the relevance of TM3‒TM5‒TM6 interactions, presenting three relevant pharmacophoric features: HAc (H-bond acceptor), HDn (H-bond donator), and Hph (hydrophobic), distributed around the space between extracellular loops (ECLs) and TMs. The HDn feature is close to TM3 and TM6; likewise, the HAc and Hph features are close to ECL1 and ECL2 and could block the rotation and interactions between TM3‒TM6 and the interactions of CXCL16s with the ECLs. Tridimensional quantitative structure-activity relationships (3D-QSAR) models show that the positive steric (VdW) and electrostatic fields coincide with the steric and positive electrostatic region of the exo-azabicyclo[3.3.1]nonane scaffold in the best pIC50 ligands. This substructure is close to the E274 residue and therefore relevant to the activity of CXCR6. These data could help with the design of new molecules that inhibit chemokine binding or antagonize the receptor based on the activation mechanism of CXCR6 and provoke a decrease in chemotaxis caused by the CXCR6‒CXCL16 axis.