Chemokine Receptor CCR5 Research Articles

Cyclin Dependent Kinase Inhibitor 2A Genetic and Epigenetic Alterations Interfere with Several Immune Components and Predict Poor Clinical Outcome.

Cyclin dependent kinase inhibitor 2A (CDKN2A) is a well-known tumor suppressor gene as it functions as a cell cycle regulator. While several reports correlate the malfunction of CDKN2A with the initiation and progression of several types of human tumors, there is a lack of a comprehensive study that analyzes the potential effect of CDKN2A genetic alterations on the human immune components and the consequences of that effect on tumor progression and patient survival in a pan-cancer model. The first stage of the current study was the analysis of CDKN2A differential expression in tumor tissues and the corresponding normal ones and correlating that with tumor stage, grade, metastasis, and clinical outcome. Next, a detailed profile of CDKN2A genetic alteration under tumor conditions was described and assessed for its effect on the status of different human immune components. CDKN2A was found to be upregulated in cancerous tissues versus normal ones and that predicted the progression of tumor stage, grade, and metastasis in addition to poor prognosis under different forms of tumors. Additionally, CDKN2A experienced different forms of genetic alteration under tumor conditions, a characteristic that influenced the infiltration and the status of CD8, the chemokine CCL4, and the chemokine receptor CCR6. Collectively, the current study demonstrates the potential employment of CDKN2A genetic alteration as a prognostic and immunological biomarker under several types of human cancers.

Evaluation of the novel therapeutic anti-CCR7 antibody CAP-100 as an add-on therapy in chronic lymphocytic leukemia patients receiving venetoclax.

The Bruton's tyrosine kinase inhibitor ibrutinib and the B-cell lymphoma 2 anti-apoptotic protein inhibitor venetoclax provide high response rates in chronic lymphocytic leukemia (CLL). However, there is a growing number of patients that relapse after treatment or show refractory disease, thus new targets and agents are still needed. We have previously reported the chemokine receptor CCR7 as a relevant deregulated target in CLL and have developed CAP-100, a novel therapeutic anti-CCR7 antibody that is under evaluation for relapse/refractory CLL (NCT04704323). While CCR7 expression has been shown to be down-modulated in CLL patients treated with ibrutinib, whether venetoclax acts in a similar manner remains unaddressed. Here, we aimed to document the impact of venetoclax on CCR7 expression in CLL cells, as well as on the pre-clinical activity of CAP-100. To this end, we addressed CCR7 expression by flow cytometry and the antibody efficacy by means of in vitro chemotactic and antibody-dependent cell-mediated cytotoxicity (ADCC) assays. Our data indicate that venetoclax treatment did not significantly modify CCR7 expression pattern nor CAP-100 mechanisms of action. Together, these findings support CAP-100 as an adjuvant therapy to venetoclax that may introduce additional modes of action in CLL therapy.

Persistence of a proinflammatory status after treatment of the acute myocardial infarction.

To evaluate the lipid-lowering and antiplatelet combined strategies on the expression of the receptors CCR2, CCR5, and CX3CR1 and the percentage of CCR2, CCR5, and CX3CR1 cells in monocyte subtypes after acute myocardial infarction. Prospective, randomized, open-label study, with blinded analyses of endpoints (PROBE, ClinicalTrials.gov Identifier: NCT02428374, registration date: April 28, 2015). Participants were treated with rosuvastatin 20 mg or simvastatin 40 mg plus ezetimibe 10 mg, as well as ticagrelor 90 mg or clopidogrel 75 mg. The chemokine receptors CCR2, CCR5, and CX3CR1 were analyzed by real-time polymerase chain reaction as well as the percentages of CCR2, CCR5, and CX3CR1 cells in the monocyte subtypes (classical, intermediate, and non-classical), which were quantified by flow cytometry, at baseline, and after 1 and 6 months of treatment. After comparisons between the three visits, regardless of the treatment arm, there was an increase in CCR2 expression after treatment, as well as an increase in intermediate monocytes CCR2+ and a reduction in non-classical monocytes CCR2+ at the end of treatment. There was also a lower expression of CCR5 after treatment and an increase in classical and non-classical monocytes CCR5+. Concerning CX3CR1, there were no differences in the expression after treatment; however, there were reductions in the percentage of intermediate and non-classical monocytes CX3CR1+ at the end of treatment. The results suggest the persistence of the inflammatory phenotype, known as trained immunity, even with the highly-effective lipid-lowering and antiplatelet therapies. Geriatr Gerontol Int 2023; 23: 700-707.

Clinical Significance of Evaluation of Monocytic Receptors in Patients with Hepatitis C Virus Infection.

Monocytes in hepatitis C virus (HCV) infection play a critical role in chronic liver inflammation and fibrosis. We studied circulating monocytes and monocyte receptors in patients with HCV infection who were naive to treatment and those who received direct acting antiviral therapy and achieved sustained virological response. CD64+ CCR2+ (M1-like) and CD206+ CD163+ CX3CR1+ (M2-like) monocyte numbers and receptor expression were evaluated by flow cytometry. Higher expression of the monocyte chemokine receptor CCR2 predicted the severity of liver fibrosis, independent of successful treatment and viral clearance (R2 = 0.235, p = 0.002), whereas monocyte CX3CR1 expression was lower in both treated and untreated patients compared with controls (p = 0.011). The expression of the scavenger receptor CD163 was lower in patients with successful treatment (p = 0.005), supporting its role as a marker of treatment response. CD64+ CCR2+ (M1-like) and CD206+ CD163+ CX3CR1+ (M2-like) monocyte numbers were not altered with fibrosis progression or treatment response. Our findings reflect the diverse functions of monocytes in liver inflammation, fibrosis, and therapy. However, HCV clearance did not lead to complete monocyte reconstitution. Targeting monocytes and their chemokine receptors bears therapeutic potential to reduce liver fibrosis and improve disease outcome.

Strategies for acquisition of resonance assignment spectra of highly dynamic membrane proteins: a GPCR case study.

In protein nuclear magnetic resonance (NMR), chemical shift assignment provides a wealth of information. However, acquisition of high-quality solid-state NMR spectra depends on protein-specific dynamics. For membrane proteins, bilayer heterogeneity further complicates this observation. Since the efficiency of cross-polarization transfer is strongly entwined with protein dynamics, optimal temperatures for spectral sensitivity and resolution will depend not only on inherent protein dynamics, but temperature-dependent phase properties of the bilayer environment. We acquired 1-, 2-, and 3D homo- and heteronuclear experiments of the chemokine receptor CCR3 in a 7:3 phosphatidylcholine:cholesterol lipid environment. 1D direct polarization, cross polarization (CP), and T2' experiments indicate sample temperatures below - 25°C facilitate higher CP enhancement and longer-lived transverse relaxation times. T1rho experiments indicate intermediate timescales are minimized below a sample temperature of - 20°C. 2D DCP NCA experiments indicated optimal CP efficiency and resolution at a sample temperature of - 30°C, corroborated by linewidth analysis in 3D NCACX at - 30°C compared to - 5°C. This optimal temperature is concluded to be directly related the lipid phase transition, measured to be between - 20 and 15°C based on rINEPT signal of all-trans and trans-gauche lipid acyl conformations. Our results have critical implications in acquisition of SSNMR membrane protein assignment spectra, as we hypothesize that different lipid compositions with different phase transition properties influence protein dynamics and therefore the optimal acquisition temperature.

Food sensitized pediatric patients show colonic cow's milk protein-specific Th2 cells.

Food allergies have become a health concern worldwide. Around 6-10% of children are allergic to cow´s milk proteins. We have previously characterized colorectal polyps in patients sensitized to food allergens. These polyps are classified as inflammatory and present a Th2 environment, with elevated IL-13 and IL-4 and are a site of IgE synthesis. In this study, we characterized and isolated cow´s milk protein-specific T cell lines and T cell clones from the lamina propria of polyps from patients sensitized to these proteins. Isolated T cells responded to cow´s milk proteins similarly to peripheral blood T cells, showing antigen-specific cell proliferation and Th2 cytokines release in vitro. T cell clones obtained were all CD4+ T cells and expressed the membrane TCRαβ receptor and secreted higher IL-4, IL-5 and IL-13 amounts than unstimulated cells, whereas IFN-γ secretion remained unchanged. Remarkably, the gut homing chemokine receptor CCR9 was augmented in cow's milk-specific peripheral and lamina propria T cells, and CCL25 was found to be expressed in the inflammatory polyp tissue and not in the adjacent mucosa. In conclusion, we isolated and characterized cow´s milk-specific lamina propria CD4+ Th2 cells from colonic inflammatory polyps. CCR9 expression on these cells, along with increase secretion of CCL25 in the polyp, favor recruitment and cow's milk specific allergic response within the inflammatory polyp tissue. Our findings may be critical to understand the underlying mechanism that promotes IgE synthesis in the colon of cow´s milk proteins allergic patients, contributing to the development of novel T cell-targeted immunotherapies.

Tumor cell-intrinsic c-Myb upregulation stimulates antitumor immunity in a murine colorectal cancer model.

The transcription factor c-Myb is overexpressed in many different types of solid tumors, including colorectal cancer. However, its exact role in tumorigenesis is unclear. In this study, we show that tumor-intrinsic c-Myb expression in mouse models of colon cancer and melanoma suppresses tumor growth. While no differences in proliferation, apoptosis, and angiogenesis of tumors were evident tumors with distinct levels of c-Myb expression, we observed changes in intratumoral immune cell infiltrates. MC38 tumors with upregulated c-Myb expression showed increased numbers of CD103+ dendritic cells and eosinophils, but decreased tumor-associated macrophages (TAMs). Concomitantly, an increase in the number of activated cytotoxic CD8+ T cells upon c-Myb upregulation was observed, which correlated with a pro-inflammatory tumor microenvironment and increased numbers of M1 polarized TAMs. Mechanistically, c-Myb upregulation in immunogenic MC38 colon cancer cells resulted in enhanced expression of immunomodulatory genes, including those encoding 2-microglobulin and IFNβ, and decreased expression of the gene encoding the chemokine receptor CCR2. The increased numbers of activated cytotoxic CD8+ T cells contributed to tumor growth attenuation. In poorly immunogenic CT26, LLC, and B16-BL6 tumor cells, c-Myb upregulation did not affect the immunomodulatory gene expression. Despite this, c-Myb upregulation led to reduced B16-BL6 tumor growth but it did not affect tumor growth of CT26 and LLC tumors. Altogether, we postulate that c-Myb functions as a tumor suppressor in a tumor cell-type specific manner and modulates antitumor immunity.

The effect of the Litcubanine A on the treatment of murine experimental periodontitis by inhibiting monocyte-macrophage chemotaxis and osteoclast differentiation.

Periodontal disease is an inflammatory disease of periodontal tissues that is closely connected with systemic diseases. During periodontitis, the inappropriate recruitment and activation of monocytes-macrophages causes an increase in osteoclast activity and disrupts bone homeostasis. Therefore, it is a promising therapeutic strategy to treat periodontitis by regulating the functions of monocytes-macrophages. Litcubanine A (LA) is an isoquinoline alkaloid extracted from the traditional Chinese medicine Litsea cubeba, which was proven to have reproducible anti-inflammatory effects, but its regulatory role on bone homeostasis in periodontitis is still not clear. In this study, zebrafish experiments and a mouse ligature-induced periodontitis model were performed, and histological analysis was used to investigate the effect of LA on macrophage chemotaxis under the inflammatory environment. Real-time PCR was used to detect the regulatory effect of LA (100 nM ~ 100 μM) on the chemotaxis function of macrophages induced by LPS. Apoptosis assay and flow cytometry were used to elucidate the influence of LA on macrophage apoptosis and proliferation. To further clarify the regulatory role of LA on macrophage osteoclast differentiation, real-time PCR, histological analysis, western blot, and micro-computed tomography (micro-CT) were performed in vivo and in vitro to verify the impact of LA on bone homeostasis. Compared with the control group, the chemotaxis function of macrophage was significantly attenuated by LA in vivo. LA could significantly inhibit the expression of genes encoding the chemokine receptors Ccr1 and Cxcr4, and its ligand chemokine Cxcl12 in macrophages, and suppresses the differentiation of osteoclastic precursors to osteoclasts through the MAPK signaling pathway. There were significantly lower osteoclast differentiation and bone loss in the LA group compared with the control in the ligature-induced periodontitis model. LA is a promising candidate for the treatment of periodontitis through its reproducible functions of inhibiting monocyte-macrophage chemotaxis and osteoclast differentiation.

Neutrophil-specific STAT4 deficiency attenuates atherosclerotic burden and improves plaque stability via reduction in neutrophil activation and recruitment into aortas of Ldlr-/- mice.

Neutrophils drive atheroprogression and directly contribute to plaque instability. We recently identified signal transducer and activator of transcription 4 (STAT4) as a critical component for bacterial host defense in neutrophils. The STAT4-dependent functions of neutrophils in atherogenesis are unknown. Therefore, we investigated a contributory role of STAT4 in neutrophils during advanced atherosclerosis. We generated myeloid-specific Stat4ΔLysMLdlr-/-, neutrophil-specific Stat4ΔS100A8Ldlr-/-, and control Stat4fl/flLdlr-/- mice. All groups were fed a high-fat/cholesterol diet (HFD-C) for 28 weeks to establish advanced atherosclerosis. Aortic root plaque burden and stability were assessed histologically by Movat pentachrome staining. Nanostring gene expression analysis was performed on isolated blood neutrophils. Flow cytometry was utilized to analyze hematopoiesis and blood neutrophil activation. In vivo homing of neutrophils to atherosclerotic plaques was performed by adoptively transferring prelabeled Stat4ΔLysMLdlr-/- and Stat4fl/flLdlr-/- bone marrow cells into aged atherosclerotic Apoe-/- mice and detected by flow cytometry. STAT4 deficiency in both myeloid-specific and neutrophil-specific mice provided similar reductions in aortic root plaque burden and improvements in plaque stability via reduction in necrotic core size, improved fibrous cap area, and increased vascular smooth muscle cell content within the fibrous cap. Myeloid-specific STAT4 deficiency resulted in decreased circulating neutrophils via reduced production of granulocyte-monocyte progenitors in the bone marrow. Neutrophil activation was dampened in HFD-C fed Stat4ΔLysMLdlr-/- mice via reduced mitochondrial superoxide production, attenuated surface expression of degranulation marker CD63, and reduced frequency of neutrophil-platelet aggregates. Myeloid-specific STAT4 deficiency diminished expression of chemokine receptors CCR1 and CCR2 and impaired in vivo neutrophil trafficking to atherosclerotic aorta. Our work indicates a pro-atherogenic role for STAT4-dependent neutrophil activation and how it contributes to multiple factors of plaque instability during advanced atherosclerosis in mice.

Development of a CCR8 monoclonal antibody to block CCR8 signaling and to abolish the immunosuppressive function of Treg for the treatment of cancers.

e14506 Background: The tumor microenvironment (TME) contains regulatory T (Treg) cells, which play a critical role in immunosuppression and are thus an attractive target for cancer therapy. The ideal approach would be to inhibit Tregs within tumors while sparing those in peripheral and normal tissues. Tumor-associated Tregs express the chemokine receptor CCR8, which is involved in the induction, expansion, migration, and immunosuppression of Tregs, making targeted inhibition of CCR8 signaling a potential therapeutic strategy against cancer. Methods: We have developed IPG0521, a monoclonal antibody antagonist of CCR8, and characterized its properties, including signaling blocking, chemotaxis, species cross-reactivity, anti-tumor efficacy, and synergistic antitumor effect with PD1 antibody. Single-cell RNA sequencing with CD45+ cells isolated from tumors treated with or without IPG0521 was also employed for profiling the mechanism of the antitumor effect of IPG0521 in the murine H22 cancer model. Results: IPG0521 inhibits receptor-mediated signaling and chemotaxis in Treg cells with a single-digit nanomolar IC50. It is cross-reactive in five species: mouse, rat, dog, monkey, and human. IPG0521 has shown excellent anti-tumor efficacy in several murine syngeneic cancer models, and its anti-tumor effect is enhanced when combined with a PD-1 antibody. Its sustained anti-tumor efficacy has been demonstrated in the murine H22 cancer model via even withdrawing the antibody during the study. The results of deep single-cell RNA sequencing on tumor-infiltrating lymphocytes (TILs) showed that the transcriptional profiles of these Treg cells could be distinguished into three distinct subsets based on molecular, functional, and immunosuppressive properties. These subsets were defined as the Treg_Foxp3 high subset, which exhibited low immunosuppression, the Treg_Foxp3 mild subset, which demonstrated mild immunosuppression, and the Treg_Foxp3 low subset, which had high proliferation. IPG0521 treatment resulted in a significant increase in the Treg_ Foxp3 high subset and a significant decrease in the Treg _ Foxp3 mild subset while no change in the Treg_ Foxp3 low subset, indicating a reduction in Treg immunosuppression. This change in Treg cells also altered the composition of other immune cells, including a significant increase in neutrophils and natural killer (NK) cells, which ultimately inhibited tumor growth. Conclusions: IPG0521 has potent anti-tumor effects in multiple tumor types, including lung, liver, and breast cancers, in both syngeneic and immune-humanized mouse models. It inhibits Tregs immunosuppression and activates CD8+ T cells to achieve its anti-tumor effects. These results support the development of IPG0521, which is currently under IND filing, as a novel therapeutic agent for the treatment of cancers.

A transcriptomic landscape analysis of human necrotizing enterocolitis: Important roles of immune infiltration

AbstractNecrotizing enterocolitis (NEC) is one of the most common and destructive diseases in neonates and an unpredictable surgical emergency. However, the molecular pathological mechanism of NEC is still not well understood. This study was designed to provide a molecular basis for the pathogenesis of human NEC through bioinformatics analysis and immune infiltration. For RNA‐Seq, DEseq2 algorithm was used to identify differentially expressed genes (DEGs) and to perform functional enrichment analysis. Immune infiltration was analyzed by CIBERSORT algorithm. A total of 34,712 genes were detected and 7463 DEGs were identified in this study. Gene Ontology analysis revealed that DEGs were mainly involved in CCR1 chemokine receptor binding, transporter activity, growth factor binding, etc. KEGG pathway analysis showed that the DEGs were significantly enriched in the toll‐like receptor signaling pathway, Th17 cell differentiation, and cytokine–cytokine receptor interaction. The immune infiltration profiles varied significantly between NEC, NEC self‐control, and normal intestinal tissues. Finally, the expression levels of 21 DEGs were verified by reverse transcription quantitative real‐time PCR. Our findings may provide new insights into the development of NEC.

EPCR deficiency ameliorates inflammatory arthritis in mice by suppressing the activation and migration of T cells and dendritic cells.

Endothelial protein C receptor (EPCR) is highly expressed in synovial tissues of patients with RA, but the function of this receptor remains unknown in RA. This study investigated the effect of EPCR on the onset and development of inflammatory arthritis and its underlying mechanisms. CIA was induced in EPCR gene knockout (KO) and matched wild-type (WT) mice. The onset and development of arthritis was monitored clinically and histologically. T cells, dendritic cells (DCs), EPCR and cytokines from EPCR KO and WT mice, RA patients and healthy controls (HCs) were detected by flow cytometry and ELISA. EPCR KO mice displayed >40% lower arthritis incidence and 50% less disease severity than WT mice. EPCR KO mice also had significantly fewer Th1/Th17 cells in synovial tissues with more DCs in circulation. Lymph nodes and synovial CD4 T cells from EPCR KO mice expressed fewer chemokine receptors CXCR3, CXCR5 and CCR6 than WT mice. In vitro, EPCR KO spleen cells contained fewer Th1 and more Th2 and Th17 cells than WT and, in concordance, blocking EPCR in WT cells stimulated Th2 and Th17 cells. DCs generated from EPCR KO bone marrow were less mature and produced less MMP-9. Circulating T cells from RA patients expressed higher levels of EPCR than HC cells; blocking EPCR stimulated Th2 and Treg cells in vitro. Deficiency of EPCR ameliorates arthritis in CIA via inhibition of the activation and migration of pathogenic Th cells and DCs. Targeting EPCR may constitute a novel strategy for future RA treatment.

Differential immunophenotype of circulating monocytes from pregnant women in response to viral ligands

BackgroundViral infections during pregnancy can have deleterious effects on mothers and their offspring. Monocytes participate in the maternal host defense against invading viruses; however, whether pregnancy alters monocyte responses is still under investigation. Herein, we undertook a comprehensive in vitro study of peripheral monocytes to characterize the differences in phenotype and interferon release driven by viral ligands between pregnant and non-pregnant women.MethodsPeripheral blood was collected from third-trimester pregnant (n = 20) or non-pregnant (n = 20, controls) women. Peripheral blood mononuclear cells were isolated and exposed to R848 (TLR7/TLR8 agonist), Gardiquimod (TLR7 agonist), Poly(I:C) (HMW) VacciGrade™ (TLR3 agonist), Poly(I:C) (HMW) LyoVec™ (RIG-I/MDA-5 agonist), or ODN2216 (TLR9 agonist) for 24 h. Cells and supernatants were collected for monocyte phenotyping and immunoassays to detect specific interferons, respectively.ResultsThe proportions of classical (CD14hiCD16−), intermediate (CD14hiCD16+), non-classical (CD14loCD16+), and CD14loCD16− monocytes were differentially affected between pregnant and non-pregnant women in response to TLR3 stimulation. The proportions of pregnancy-derived monocytes expressing adhesion molecules (Basigin and PSGL-1) or the chemokine receptors CCR5 and CCR2 were diminished in response to TLR7/TLR8 stimulation, while the proportions of CCR5− monocytes were increased. Such differences were found to be primarily driven by TLR8 signaling, rather than TLR7. Moreover, the proportions of monocytes expressing the chemokine receptor CXCR1 were increased during pregnancy in response to poly(I:C) stimulation through TLR3, but not RIG-I/MDA-5. By contrast, pregnancy-specific changes in the monocyte response to TLR9 stimulation were not observed. Notably, the soluble interferon response to viral stimulation by mononuclear cells was not diminished in pregnancy.ConclusionsOur data provide insight into the differential responsiveness of pregnancy-derived monocytes to ssRNA and dsRNA, mainly driven by TLR8 and membrane-bound TLR3, which may help to explain the increased susceptibility of pregnant women to adverse outcomes resulting from viral infection as observed during recent and historic pandemics.

Potent anti-Toxoplasma gondii activity of 4-chlorophenylthioacetone-derived thiosemicarbazones: Involvement of CCR2 and CCR5 receptors and 5-lipoxygenase in the mode of action

Toxoplasmosis is a disease requiring therapeutic innovation, and thiosemicarbazones with antimicrobial activity are candidates to control Toxoplasma gondii infection. Here, the anti-T. gondii activities of (E)-2-(1-(4-chlorophenylthio)propan-2-ylidene)-hydrazinecarbothioamides (Ca and Cb) were investigated. T. gondii-infected macrophages (MOs) or glial cells treated with Ca or Cb showed a decrease in the number of intracellular parasites. A deficiency in the chemokine receptor CCR2, but not CCR5, partially reduced anti-T. gondii activity induced by Ca or Cb. In contrast, a deficiency in 5-lipoxygenase (5-LO) activity potentiated anti-T. gondii activities induced by these compounds. In vivo treatment with Ca increased the survival of T. gondii-infected wild-type mice, and this was associated with increased IFN-γ and IL-12 production. A deficiency in CCR5 or CCR2 abolished the protective effect of Ca treatment in vivo, while a deficiency in 5-LO increased Cb anti-T. gondii effects. Collectively, our data suggest that Ca and Cb are potential therapeutic candidates for the treatment of toxoplasmosis.

The Synthetic Opioid Fentanyl Increases HIV Replication and Chemokine Co-Receptor Expression in Lymphocyte Cell Lines.

In the United States, the illicit use of synthetic opioids such as fentanyl has led to a serious public health crisis. Synthetic opioids are known to enhance viral replication and to suppress immunologic responses, but their effects on HIV pathogenesis remain unclear. Thus, we examined the impact of fentanyl on HIV-susceptible and HIV-infected cell types. TZM-bl and HIV-infected lymphocyte cells were incubated with fentanyl at varying concentrations. Expression levels of the CXCR4 and CCR5 chemokine receptors and HIV p24 antigen were quantified with ELISA. HIV proviral DNA was quantified using SYBR RT-PCR. Cell viability was detected with the MTT assay. RNAseq was performed to characterize cellular gene regulation in the presence of fentanyl. Fentanyl enhanced expression of both chemokine receptor levels in a dose-dependent manner in HIV-susceptible and infected cell lines. Similarly, fentanyl induced viral expression in HIV-exposed TZM-bl cells and in HIV-infected lymphocyte cell lines. Multiple genes associated with apoptosis, antiviral/interferon response, chemokine signaling, and NFκB signaling were differentially regulated. Synthetic opioid fentanyl impacts HIV replication and chemokine co-receptor expression. Increased virus levels suggest that opioid use may increase the likelihood of transmission and accelerate disease progression.

CCL17 and CCL22 chemokines are upregulated in human obesity and play a role in vascular dysfunction.

Chemokines are known to play critical roles mediating inflammation in many pathophysiological processes. The aim of this study was to investigate the role of chemokine receptor CCR4 and its ligands CCL17 and CCL22 in human morbid obesity. Circulating levels of CCL17 and CCL22 were measured in 60 morbidly obese patients (mean age, 45 ± 1 years; body mass index/BMI, 44 ± 1 kg/m2) who had undergone bariatric bypass surgery, and 20 control subjects. Paired subcutaneous (SCAT) and visceral adipose tissue (VCAT) from patients were analysed to measure expression of CCR4 and its ligands by RT-PCR, western blot and immunohistochemical analysis. The effects of CCR4 neutralization ex vivo on leukocyte-endothelial cells were also evaluated. Compared with controls, morbidly obese patients presented higher circulating levels of CCL17 (p=0.029) and CCL22 (p<0.001) and this increase was positively correlated with BMI (p=0.013 and p=0.0016), and HOMA-IR Index (p=0.042 and p< 0.001). Upregulation of CCR4, CCL17 and CCL22 expression was detected in VCAT in comparison with SCAT (p<0.05). Using the parallel-plate flow chamber model, blockade of endothelial CCR4 function with the neutralizing antibody anti-CCR4 in morbidly obese patients significantly reduced leucocyte adhesiveness to dysfunctional endothelium, a key event in atherogenesis. Additionally, CCL17 and CCL22 increased activation of the ERK1/2 mitogen-activated protein kinase signalling pathway in human aortic endothelial cells, which was significantly reduced by CCR4 inhibition (p=0.016 and p<0.05). Based on these findings, pharmacological modulation of the CCR4 axis could represent a new therapeutic approach to prevent adipose tissue dysfunction in obesity.

Abstract 5958: Analysis of MDSCs as a diagnostic and therapeutic target for gastric cancer

Abstract Background: Gastric cancer (GC) is the second most common cancer in Japan in terms of incidence and the third most common in terms of deaths. The onset and progression of cancer are generally suppressed by the action of immune cells, which is an inflammatory response. MDSCs are thought to promote cancer progression and invasion by suppressing the function of immune cells, making them potential therapeutic and diagnostic targets. We measured MDSCs in GC patients and observed more monocyte-derived MDSCs in peripheral blood in advanced GC than in early GC. However, granulocyte-derived MDSCs showed no difference in GC progression when markers for classical MDSCs were used. We considered that granulocyte-derived MDSCs were much more abundant than monocyte-derived MDSCs in peripheral blood and were involved in cancer progression. We hypothesized that we had a new granulocyte-derived marker for advanced GC that did not show differences in classical markers, but may have different functions, and aimed to identify a novel subset that may be a therapeutic and diagnostic target. Method: For GC patients, we used heparin blood samples provided by patients who consented to Juntendo University. We analyzed the expression of various cell surface proteins against populations of CD33high, CD66bhigh, and HLA-DRlow as classical markers of MDSCs derived from granulocytes of GC patients using FACS (Melody). MDSCs were isolated by a cell sorter and used for experiments such as RNA-Seq. Novel markers and involved cytokines were quantified in plasma components by cytometric bead array. Result: The chemokine receptors CCR2, CCR5, CXCR4, and CXCR5 were highly expressed in MDSCs of advanced GC patients. The ability to suppress T-cell activation was also found to be stronger in MDSCs with advanced GC. In addition, IL-10 produced by MDSCs was also more abundant in the plasma of patients with advanced GC, as well as chemokines according to chemokine receptors. Conclusion: The high expression of chemokine receptors on MDSCs in advanced GC may indicate that chemokines produced at the cancer site facilitate the migration of MDSCs. These novel subsets of MDSCs found in advanced GC suggest that they may be new therapeutic targets. Citation Format: Suguru Yamauchi, Takumi Iwasawa, Tomoaki Ito, Malcom V. Brock, Tetsu Fukunaga, Hajime Orita, Kazunori Kato. Analysis of MDSCs as a diagnostic and therapeutic target for gastric cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5958.

Abstract 2270: Combination of CXCR4 antagonist and anti-PD1 therapy results in significant mobilization and increased infiltration of myeloid cells into the metastatic liver microenvironment of PDAC

Abstract Pancreatic adenocarcinoma (PDAC) is extremely lethal and resistant to checkpoint immunotherapy, characterized by an immunosuppressive tumor microenvironment consisting of stromal and myeloid cells. Prior studies have demonstrated the utility of targeting the chemokine signaling axis CXCR4-CXCL12 (SDF-1), a highlighted feature in PDACs, to overcome the CXCL12-driven immobilization of T cells and thus facilitate their antitumor role within the tumor. Based on these findings, we have conducted a phase 2 trial evaluating the effects of plerixafor, a CXCR4 antagonist, and cemiplimab, a PD1 inhibitor antibody, in patients with metastatic PDAC who have progressed after one line of systemic chemotherapy (NCT04177810). To determine the immunological responses to therapy, blood samples and tissue biopsies were obtained at baseline and during treatment. Hematological assessment confirmed the activity of CXCR4 antagonist in mobilizing hematopoietic precursors (CD34+), immature myeloid cells, and lymphoid cells, as well as monocytic and granulocytic cell populations. Suspension mass cytometry analysis of peripheral blood mononuclear cells revealed that mobilized monocytic subpopulations had high expressions of chemokine receptors CCR2, CCR5, and CXCR2. Histopathologic evaluation of the serial tissue biopsies from the liver metastatic PDAC revealed increased levels of inflammation upon treatment. To further characterize the cellular constituents of the observed inflammation, multiplexed immunohistochemistry by imaging mass cytometry was performed, demonstrating strong trends toward increased infiltration of not only effector T cells but also macrophages and granulocytic cells into the tumor microenvironment. Taken together, these findings suggest that mobilization of myeloid cells by CXCR4 antagonism results in the recruitment of additional myeloid cells from circulation and that alternative chemokine signaling pathways are sufficient for doing so. This implicates a potential mode of resistance against CXCR4-targeted therapies. Furthermore, these observations reinforce the value of ongoing research efforts in the field to subvert the recruitment or immunosuppressive function of myeloid cells, which would be particularly relevant in the setting of CXCR4 antagonism. Citation Format: Sarah M. Shin, Alexei Hernandez, Erin Coyne, Zhehao Zhang, Sarah Mitchell, Jennifer Durham, Xuan Yuan, Hongqui Yang, Elana J. Fertig, Elizabeth M. Jaffee, Katherine M. Bever, Dung T. Le, Won Jin Ho. Combination of CXCR4 antagonist and anti-PD1 therapy results in significant mobilization and increased infiltration of myeloid cells into the metastatic liver microenvironment of PDAC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2270.

Abstract 2340: Development of a CCR8 monoclonal antibody which blocks CCR8 signaling and abolishes the immunosuppressive function of Treg for the treatment of cancer

Abstract Within the tumor microenvironment (TME), regulatory T (Treg) cells are one attractive cell type for targeting as they play a critical role in immunosuppression. Therapeutic strategies that specifically inhibit tumor-infiltrating Tregs while sparing peripheral and normal tissue Tregs are highly desirable. The tumor-associated Tregs express the chemokine receptor CCR8, which plays a role in the induction, expansion, chemotactic migration, and immunosuppression of tumor-associated Tregs. Thus, targeted inhibition of CCR8 is a potential therapeutic strategy against cancer. We have developed a monoclonal antibody antagonist of CCR8, IPG0521, which potently inhibited the receptor-mediated signaling and chemotaxis in Treg cells with single digital nanomolar IC50. RNA sequencing (RNA-seq)analysis indicated that CCL1-induced up-regulation of immunosuppressive genes, including PD-1, CTLA4, IL-10, TIGIT etc., were reversed by IPG0521 in tumor-derived Treg cells. IPG0521 potently inhibited the growth of multiple tumor types in both syngeinic and immune-humanized mouse models, including lung cancer, liver cancer, breast cancer, via inhibiting Treg and activating CD8+ T cells. These data pave the way for the development of IPG0521, which is under IND filing, as a novel therapeutic agent against cancer. Citation Format: Guohuang Fan, Jianfei Wang, Na Wang, Yuanyuan Zhang, Yong Zhang, Binle Tian, Xuebing Jia. Development of a CCR8 monoclonal antibody which blocks CCR8 signaling and abolishes the immunosuppressive function of Treg for the treatment of cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2340.

Abstract 1543: Discovery of CCR8 antibodies targeting regulatory T cells to enable tumor sensitization

Abstract Regulatory T (Treg) cells in the tumor microenvironment have an immune suppressive function, and their presence is associated with poor prognosis. Depletion of Treg cells may be a mechanism to sensitize tumors and represents a novel strategy for cancer immunotherapy. High expression of chemokine receptor CCR8 has been found in Treg cells in microenvironments of numerous tumors but not on Treg cells in systemic lymphoid tissues. The ability to selectively kill Treg cells in tumor environments may provide an opportunity for therapy that leaves Treg function intact elsewhere in the body where they normally mediate tolerance to self-antigen. We sought to identify CCR8 antibodies for therapeutics and designed an antibody discovery strategy to address the challenges of this target. CCR8 is a member of the G protein-coupled receptor (GPCR) family. Like many GPCRs, CCR8 is a valuable therapeutic target but is challenging to target using antibodies due to its poor expression, membrane-dependent structure, small extracellular regions, and poor immunogenicity due to sequence conservation. We developed an antibody discovery platform (MPS) that specifically addresses each of these challenges through use of advanced immunization techniques and evolutionarily divergent host species (chickens) for robust immune responses against conserved targets. We extensively engineered CCR8 antigen to enable high expression for immunization. When sufficient antibody diversity using our standard immunization techniques was not obtained, we developed mRNA immunization techniques for this program. Using these approaches, we were able to identify a panel of high-affinity antibodies that bind in the nanomolar or subnanomolar range with high specificity. We will present data from our antibody discovery campaign including mRNA immunization, binding profiles, GPCR functional data, and the ability of the antibodies to kill Treg cells in vitro using both ADCC and antibody-drug conjugates (ADC). Citation Format: Brad Screnci, Hayley Roth, Sarah Campbell, David Tucker, Colleen Fenn-Ulke, Erin Rosenberg, Meghan Pitts, Naomi Saint-Jean, Jawhara Karam, Paige Murphy, Daniela Reyes, Janae Latta, Kyle Guldner, Samantha Gilman, Tim Phillips, L. Joe Stafford, Breanna Tyrell, Kristen Shema, Chidananda Sulli, Nick Molino, Allison Snyder, Marianne Assogba, Janvi Shah, McKayla Zimmerman, Trevor Barnes, Riley Payne, Alyssa Cunningham, Elisabeth Charrier, Holden Ohl, Kyle Doolan, Ileine Sanchez, Joseph B. Rucker, Ross Chambers, Benjamin Doranz. Discovery of CCR8 antibodies targeting regulatory T cells to enable tumor sensitization [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1543.