CCR5 Ligands Research Articles

A phase 2 study to assess the safety and efficacy of FLX475 (tivumecirnon) combined with pembrolizumab in patients with advanced or metastatic gastric cancer.

426 Background: Regulatory T-cells (T regs ) suppress anti-tumor immunity by migrating into tumors via CCL17/CCL22 chemokines binding to CCR4. FLX475 (tivumecirnon), a selective CCR4 antagonist, inhibits T reg recruitment, enhancing the effector T-cell (T eff ) response in the tumor microenvironment. Preclinical studies demonstrate that FLX475 increases the T eff /T reg ratio and boosts anti-tumor efficacy, both as monotherapy and in combination with checkpoint inhibitors (CPI). Here, we present findings on the safety and efficacy of FLX475 with pembrolizumab in a Phase 2 trial, aimed at improving therapeutic outcomes in advanced or metastatic gastric cancer (GC) patients. Methods: This Phase 2, open-label study (NCT04768686) assesses the safety and efficacy of FLX475 combined with pembrolizumab in advanced or metastatic GC patients. Patients received FLX475 100 mg PO QD and pembrolizumab 200 mg IV Q3W. Cohort 1 included CPI-naïve Epstein-Barr Virus (EBV)-negative GC patients who had progressed on at least two prior treatments. Cohort 2 included CPI-naïve EBV-positive GC patients who had received at least one prior treatment. Both cohorts were selected based on high CCR4 ligand expression and T reg enrichment, expected to enhance treatment response. The primary objective of the study was to assess the anti-tumor efficacy of the study treatment, as assessed by the investigator using RECIST 1.1. Results: A total of 20 patients were enrolled in the study, with 10 patients in each cohort. The combination of FLX475 and pembrolizumab was well-tolerated across both cohorts. The most common treatment-related adverse events occurred in more than 10% of patients across cohorts were QTc prolongation (35.0%), and pruritus (15.0%), which were manageable and consistent with the known profiles of the individual agents. In Cohort 2, the confirmed objective response rate (ORR) was 60.0% (95% CI: 26.2 to 87.8). The median time to response (mTTR) was 2.7 months, with a median duration of response (mDOR) of 17.3 months. The median progression-free survival (mPFS) for this cohort was 10.4 months, while the median overall survival was not reached. In Cohort 1, no objective responses were observed, and the best response achieved was stable disease. The relationship between treatment outcomes and biomarkers including CCR4 expression levels and the T eff :T reg ratio was confirmed in exploratory analyses. Conclusions: The combination of FLX475 with pembrolizumab was well-tolerated and showed promising anti-tumor activity and durable responses in patients with advanced or metastatic EBV-positive GC. Clinical trial information: NCT04768686 . Cohort 1 (EBV (-) GC)N=10 Cohort 2 (EBV (+) GC)N=10 ORR n (%)(95% CI) 0 (0.0)(-) 6 (60.0)(26.2, 87.8) mTTR Months (95% CI) - 2.7 (1.2, -) mDOR Months (95% CI) - 17.3 (2.7, -) mPFS Months (95% CI) 1.4 (1.1, 1.5) 10.4 (1.0, -)

Targeting heterogeneous tumor microenvironments in pancreatic cancer mouse models of metastasis by TGF-β depletion.

The dual tumor-suppressive and -promoting functions of TGF-β signaling has made its targeting challenging. We examined the effects of TGF-β depletion by AVID200/BMS-986416 (TGF-β-TRAP), a TGF-β ligand trap, on the tumor microenvironment of pancreatic ductal adenocarcinoma (PDAC) murine models with different organ-specific metastasis. Our study demonstrated that TGF-β-TRAP potentiates the efficacy of anti-programmed cell death 1 (anti-PD-1) in a PDAC orthotopic murine model with liver metastasis tropism, significantly reducing liver metastases. We further demonstrated the heterogeneous response of cytotoxic effector T cells to combination TGF-β-TRAP and anti-PD-1 treatment across several tumor models. Single-nuclear RNA sequencing suggested that TGF-β-TRAP modulates cancer-associated fibroblast (CAF) heterogeneity and suppresses neutrophil degranulation and CD4+ T cell response to neutrophil degranulation. Ligand-receptor analysis indicated that TGF-β-TRAP may modulate the CCL5/CCR5 axis as well as costimulatory and checkpoint signaling from CAFs and myeloid cells. Notably, the most highly expressed ligands of CCR5 shifted from the immunosuppressive CCL5 to CCL7 and CCL8, which may mediate the immune agonist activity of CCR5 following TGF-β-TRAP and anti-PD-1 combination treatment. This study suggested that TGF-β depletion modulates CAF heterogeneity and potentially reprograms CAFs and myeloid cells into antitumor immune agonists in PDAC, supporting the validation of such effects in human specimens.

Blockade of CCR5+ T Cell Accumulation in the Tumor Microenvironment Optimizes Anti-TGF-β/PD-L1 Bispecific Antibody.

In the previous studies, anti-TGF-β/PD-L1 bispecific antibody YM101 is demonstrated, with superior efficacy to anti-PD-L1 monotherapy in multiple tumor models. However, YM101 therapy can not achieve complete regression in most tumor-bearing mice, suggesting the presence of other immunosuppressive elements in the tumor microenvironment (TME) beyond TGF-β and PD-L1. Thoroughly exploring the TME is imperative to pave the way for the successful translation of anti-TGF-β/PD-L1 BsAb into clinical practice. In this work, scRNA-seq is employed to comprehensively profile the TME changes induced by YM101. The scRNA-seq analysis reveals an increase in immune cell populations associated with antitumor immunity and enhances cell-killing pathways. However, the analysis also uncovers the presence of immunosuppressive CCR5+ T cells in the TME after YM101 treatment. To overcome this hurdle, YM101 is combined with Maraviroc, a widely used CCR5 antagonist for treating HIV infection, suppressing CCR5+ T cell accumulation, and optimizing the immune response. Mechanistically, YM101-induced neutrophil activation recruits immunosuppressive CCR5+ T cells via CCR5 ligand secretion, creating a feedback loop that diminishes the antitumor response. Maraviroc then cleared these infiltrating cells and offset YM101-mediated immunosuppressive effects, further unleashing the antitumor immunity. These findings suggest selectively targeting CCR5 signaling with Maraviroc represents a promising and strategic approach to enhance YM101 efficacy.

Abstract Mo049: Loss of Y chromosome and cardiovascular events in chronic kidney disease

Background: Chronic kidney disease represents one of the strongest risk factors for cardiovascular diseases, and particularly for heart failure. Despite improved pharmaceutical treatments mortality remains high. Recently, experimental studies demonstrated that mosaic loss of Y chromosome (LOY) associates with cardiac fibrosis in mice. Since diffuse cardiac fibrosis is the common denominator for progression of all forms of heart failure, we determined the association of LOY on mortality and cardiovascular disease outcomes in patients with chronic kidney disease. Methods: LOY was quantified in patients with stable CKD (CARE for HOMe study, derivation cohort, N=279) and dialysis patients (4D study, validation cohort, N=544). The association between LOY and mortality, combined cardiovascular and heart failure-specific endpoints, and echocardiographic measures was assessed. Results: In CARE for HOMe, mortality was higher in men as compared to women across the entire range of impaired renal function. The frequency of LOY increased with age. LOY was associated with increased mortality (HR 2.58, 95%CI 1.33-5.03) and risk for cardiac decompensation or death (HR 2.30, 95%CI 1.23-4.27). Patients with LOY >17 % showed a significant decline of ejection fraction and an increase of E/E’ within five years. Consistently, validation in the 4D study revealed that LOY was associated with increased mortality (HR 3.13, 95%CI 1.97-4.99), higher risk of death due to heart failure and sudden cardiac death (HR 4.14, 95%CI 2.00-8.57), but not atherosclerotic events (see figure). Patients with LOY >17 % showed significantly higher plasma levels of soluble ST2, a biomarker for myocardial fibrosis. Mechanistically, intermediate monocytes from patients with LOY >17 % showed significantly higher CCR2 expression and higher plasma levels of the CCR2 ligand CCL2, which may have contributed to increased heart failure events. Conclusions: LOY identifies male patients with chronic kidney disease at high risk for mortality and heart failure events.

Enhanced CCR2 expression by ACKR2-deficient NK cells increases tumoricidal cell therapy efficacy.

Chemokines regulate leukocyte navigation to inflamed sites and specific tissue locales and may therefore be useful for ensuring accurate homing of cell therapeutic products. We, and others, have shown that atypical chemokine receptor 2 (ACKR2)-deficient mice (ACKR2-/-) are protected from metastasis development in cell line and spontaneous mouse models. We have shown that this relates to enhanced CCR2 expression on ACKR2-/- natural killer cells, allowing them to home more effectively to CCR2 ligand-expressing metastatic deposits. Here we demonstrate that the metastatic-suppression phenotype in ACKR2-/- mice is not a direct effect of the absence of ACKR2. Instead, enhanced natural killer cell CCR2 expression is caused by passenger mutations that originate from the creation of the ACKR2-/- mouse strain in 129 embryonic stem cells. We further demonstrate that simple selection of CCR2+ natural killer cells enriches for a population of cells with enhanced antimetastatic capabilities. Given the widespread expression of CCR2 ligands by tumors, our study highlights CCR2 as a potentially important contributor to natural killer cell tumoricidal cell therapy.

CD4+CCR5+ T cells and CCL3+ mast cells are increased in the skin of patients with chronic spontaneous urticaria.

The proximity of activated T cells and mast cells in the lesional skin of patients with chronic spontaneous urticaria (CSU) is held to contribute to the development of wheals and angioedema. In a previous study, we demonstrated that increased IL-17 expression in T cells and mast cells in skin lesions of patients with CSU is associated with T/mast cell proximity, but the mechanisms that drive T cell/mast cell co-localization remain unknown. To assess if chemokines expressed in lesional CSU skin contribute to T cell/mast cell proximity. Biopsies from lesional CSU skin were compared to biopsies from healthy skin for expression of CCR5 and its ligand CCL3 by CD4+ T cells and mast cells, respectively. Numbers of CCR5-positive CD4+ T cells in lesional CSU skin were significantly increased as compared to healthy normal skin (p < 0.0001). The number of mast cells expressing CCL3 (ligand for CCR5) in CSU skin was also increased (p < 0.0002) and significant association with T-cell close proximity (p < 0.0001) is noticed. The close proximity of T cells and mast cells in the skin of severe CSU may be driven, at least in part by increased CCR5 and CCL3 expression. Therapies that target CCL3 interaction with CCR5 should be assessed for their effects in CSU.

Flagellin Restricts HIV-1 Infection of Macrophages through Modulation of Viral Entry Receptors and CC Chemokines.

Both bacteria product flagellin and macrophages are implicated in HIV-1 infection/disease progression. However, the impact of their interaction on HIV-1 infection and the associated mechanisms remain to be determined. We thus examined the effect of the flagellins on HIV-1 infection of primary human macrophages. We observed that the pretreatment of macrophages with the flagellins from the different bacteria significantly inhibited HIV-1 infection. The mechanistic investigation showed that the flagellin treatment of macrophages downregulated the major HIV-1 entry receptors (CD4 and CCR5) and upregulated the CC chemokines (MIP-1α, MIP-1β and RANTES), the ligands of CCR5. These effects of the flagellin could be compromised by a toll-like receptor 5 (TLR5) antagonist. Given the important role of flagellin as a vaccine adjuvant in TLR5 activation-mediated immune regulation and in HIV-1 infection of macrophages, future investigations are necessary to determine the in vivo impact of flagellin-TLR5 interaction on macrophage-mediated innate immunity against HIV-1 infection and the effectiveness of flagellin adjuvant-based vaccines studies.

Topical application of a CCL22-binding aptamer suppresses contact allergy

Allergic contact dermatitis is a prevalent occupational disease with limited therapeutic options. The chemokine CCL22, a ligand of the chemokine receptor CCR4, directs the migration of immune cells. Here, it is shown that genetic deficiency of CCL22 effectively ameliorated allergic reactions in contact hypersensitivity (CHS), a commonly used mouse model of allergic contact dermatitis. For pharmacological inhibition of CCL22, DNA aptamers specific for murine CCL22 were generated by systematic evolution of ligands by exponential enrichment (SELEX). Nine CCL22-binding aptamers were initially selected and functionally tested in vitro. The 29-nucleotide DNA aptamer AJ102.29m profoundly inhibited CCL22-dependent T cell migration and did not elicit undesired Toll-like receptor-dependent immune activation. AJ102.29m efficiently ameliorated CHS in vivo after systemic application. Moreover, CHS-associated allergic symptoms were also reduced following topical application of the aptamer on the skin. Microscopic analysis of skin treated with AJ102.29m ex vivo demonstrated that the aptamer could penetrate into the epidermis and dermis. The finding that epicutaneous application of the aptamer AJ102.29m in a cream was as effective in suppressing the allergic reaction as intraperitoneal injection paves the way for therapeutic use of aptamers beyond the current routes of systemic administration.

Developmental and therapeutic implications of IL4ra expression for rhabdomyosarcoma.

Rhabdomyosarcoma (RMS) is a solid tumor whose metastatic progression can be accelerated through interleukin-4 receptor alpha (Il4ra) mediated interaction with normal muscle stem cells (satellite cells). To understand the function of Il4ra in this tumor initiation phase of RMS, we conditionally deleted Il4ra in genetically-engineered RMS mouse models. Nullizygosity of Il4ra altered the latency, site and/or stage distribution of RMS tumors compared to IL4RA intact models. Primary tumor cell cultures taken from the genetically-engineered models then used in orthotopic allografts further defined the interaction of satellite cells and RMS tumor cells in the context of tumor initiation: in alveolar rhabdomyosarcoma (ARMS), satellite cell co-injection was necessary for Il4ra null tumor cells engraftment, whereas in embryonal rhabdomyosarcoma (ERMS), satellite cell co-injection decreased latency of engraftment of Il4ra wildtype tumor cells but not Il4ra null tumor cells. When refocusing on Il4ra wildtype tumors by single cell sequencing and cytokine studies, we have uncovered a putative signaling interplay of Il4 from T-lymphocytes being received by Il4ra + rhabdomyosarcoma tumor cells, which in turn express Ccl2, the ligand for Ccr2 and Ccr5. Taken together, these results suggest that mutations imposed during tumor initiation have different effects than genetic or therapeutic intervention imposed once tumors are already formed. We also propose that CCL2 and its cognate receptors CCR2 and/or CCR5 are potential therapeutic targets in Il4ra mediated RMS progression.

Single-Cell Profiling Reveals Immune Aberrations in Progressive Idiopathic Pulmonary Fibrosis.

Rationale: Changes in peripheral blood cell populations have been observed, but not detailed, at single-cell resolution in idiopathic pulmonary fibrosis (IPF). Objectives: We sought to provide an atlas of the changes in the peripheral immune system in stable and progressive IPF. Methods: Peripheral blood mononuclear cells (PBMCs) from patients with IPF and control subjects were profiled using 10× chromium 5' single-cell RNA sequencing. Flow cytometry was used for validation. Protein concentrations of regulatory T cells (Tregs) and monocyte chemoattractants were measured in plasma and lung homogenates from patients with IPF and control subjects. Measurements and Main Results: Thirty-eight PBMC samples from 25 patients with IPF and 13 matched control subjects yielded 149,564 cells that segregated into 23 subpopulations. Classical monocytes were increased in patients with progressive and stable IPF compared with control subjects (32.1%, 25.2%, and 17.9%, respectively; P < 0.05). Total lymphocytes were decreased in patients with IPF versus control subjects and in progressive versus stable IPF (52.6% vs. 62.6%, P = 0.035). Tregs were increased in progressive versus stable IPF (1.8% vs. 1.1% of all PBMCs, P = 0.007), although not different than controls, and may be associated with decreased survival (P = 0.009 in Kaplan-Meier analysis; and P = 0.069 after adjusting for age, sex, and baseline FVC). Flow cytometry analysis confirmed this finding in an independent cohort of patients with IPF. The fraction of Tregs out of all T cells was also increased in two cohorts of lung single-cell RNA sequencing. CCL22 and CCL18, ligands for CCR4 and CCR8 Treg chemotaxis receptors, were increased in IPF. Conclusions: The single-cell atlas of the peripheral immune system in IPF reveals an outcome-predictive increase in classical monocytes and Tregs, as well as evidence for a lung-blood immune recruitment axis involving CCL7 (for classical monocytes) and CCL18/CCL22 (for Tregs).

CCR5 promotes the migration of pathological CD8+ T cells to the leishmanial lesions.

Cytolytic CD8+ T cells mediate immunopathology in cutaneous leishmaniasis without controlling parasites. Here, we identify factors involved in CD8+ T cell migration to the lesion that could be targeted to ameliorate disease severity. CCR5 was the most highly expressed chemokine receptor in patient lesions, and the high expression of CCL3 and CCL4, CCR5 ligands, was associated with delayed healing of lesions. To test the requirement for CCR5, Leishmania-infected Rag1-/- mice were reconstituted with CCR5-/- CD8+ T cells. We found that these mice developed smaller lesions accompanied by a reduction in CD8+ T cell numbers compared to controls. We confirmed these findings by showing that the inhibition of CCR5 with maraviroc, a selective inhibitor of CCR5, reduced lesion development without affecting the parasite burden. Together, these results reveal that CD8+ T cells migrate to leishmanial lesions in a CCR5-dependent manner and that blocking CCR5 prevents CD8+ T cell-mediated pathology.

CCR10 Deficiency Reduces Blood Pressure Elevations, End-Organ Damage, and Cutaneous Infiltration of Regulatory T Cells in a Murine Model of Hypertension

Emerging evidence suggests a key role for inflammation and immune cell activation in the pathogenesis of hypertension and its associated end-organ damage. Regulatory T cells (Tregs) function primarily to limit inflammation and are generally considered protective in hypertension. However, Treg subsets can also have tissue-specific functions and play pathogenic roles in some inflammatory diseases through mechanisms, such as limiting angiogenesis. As such, the role of Tregs and its subsets in hypertension remain unclear. Recently, we demonstrated that CCR10+ Tregs are selectively decreased in the circulation of hypertensive patients. CCR10 is a key chemokine receptor for recruitment of immune cells to the skin. Although the skin has not been well studied in hypertension, prior reports in humans demonstrate that loss of skin microvessels (microvascular rarefaction) is a putative mechanism for blood pressure (BP) elevations in hypertension. Recent evidence also suggests that Tregs can promote apoptosis of vascular smooth muscle cells and endothelial cells (ECs). Thus, we hypothesized that CCR10+ Tregs promote hypertension through skin infiltration leading to enhanced microvascular rarefaction. Herein, we demonstrate that, compared to normotensive littermates, male mice with angiotensin II (Ang II)-induced hypertension have selectively increased abundance of CCR10+ Tregs in the skin (p=0.0004) with a corresponding decrease in the circulation (p=0.003). These mice also displayed increased cutaneous levels of the CCR10 ligand CCL27 (p=0.0007), suggesting a mechanism for increased CCR10+ Treg infiltration. To test a causal role of CCR10 in hypertension, we infused Ang II in control CCR10+/+ or CCR10-deficient (CCR10−/−) mice for 4 weeks. Our results demonstrate that CCR10−/− mice exhibit significant reductions in systolic blood pressure (p=0.004), albuminuria (p=0.01), and cardiac fibrosis (p=0.04) compared to wild type mice after Ang II infusion. In addition, CCR10−/− mice had a selective decrease in Tregs in the skin (p=0.02) without change in abundance of conventional CD4+ or CD8+ T cells after Ang II. CCR10−/− mice also exhibited greater cutaneous arteriolar density (p=0.02) after Ang II, consistent with reduced skin microvascular rarefaction. Finally, using PrediXcan, we demonstrate that higher genetically predicted levels of CCR10 are associated with increased risk of hypertension in humans. Taken together, our data suggest that CCR10 promotes Ang II-induced BP elevations and associated end-organ injury, at least in part by promoting Treg recruitment to the skin leading to microvascular rarefaction. K08HL153786, 5T32HL144446-05. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Abstract CT162: Trial in progress: A phase I, open-label, multicenter study of ZL-1218, an anti-CCR8 IgG1, as a single agent and as combination therapy with anti-PD-1 antibody to evaluate the safety, tolerability, and pharmacokinetics in subjects with advanced solid tumor

Abstract Background ZL-1218 is an anti-chemokine receptor 8 (CCR8) humanized IgG1 antibody with enhanced antibody-dependent cellular cytotoxicity (ADCC) activity. CCR8 demonstrates a preferential expression in tumor-associated Tregs over peripheral Tregs. Thus, targeting CCR8 may allow for specific depletion of intratumoral Tregs without impacting peripheral Tregs/other immune cells. ZL-1218 is intended to decrease the intratumoral Tregs by binding to both high- and low-expressing CCR8+ cells and eliciting potent ADCC activity. ZL-1218 blocked CCR8 ligand CCL1 from binding to CCR8, and reduced Treg recruitment by blocking chemotaxis of CCR8+ cells (unpublished data). Additionally, ZL-1218 demonstrated enhanced antitumor activity in preclinical models in combination with anti-PD-1. Methods This is a Phase I study of ZL-1218 as a single agent and in combination with pembrolizumab (P) to evaluate the safety, tolerability, pharmacokinetics/pharmacodynamics, and preliminary antitumor activity in subjects with advanced solid tumor malignancies. The study consists of Part 1 Dose Escalation and Part 2 Cohort Expansion. In Part 1, subjects will be treated with escalating doses of ZL-1218 alone and in combination with a fixed dose of P to identify the maximum tolerated dose (MTD) and/or the recommended expansion dose (RD). The Bayesian optimal interval (BOIN) design is used and adopted with overdose control. Upon progression in the single agent group, a subject may enroll into combination therapy of ZL-1218 with P. Subjects will start at their respective single agent dose or the cleared combination dose level, whichever is lower. Part 2 will explore the combination with pembrolizumab in subjects with selected tumor types, with or without prior CPI therapy. Major inclusion criteria include: (1) Subjects must have histologically confirmed and documented diagnosis of locally advanced unresectable/metastatic advanced solid tumor that is refractory/intolerant to standard treatment, or for which no standard treatment exists, and no greater than 3 prior lines of therapy in the relapse/refractory setting (2). Subjects must have at least one target lesion as defined by RECIST v1.1 on CT, PET/CT, or MRI scan. (3) Subjects must have a site of disease which is not previously irradiated and is safe and amenable to biopsy. Major exclusion criteria include: (1) Prior exposure to CCR8 inhibitor, or severe hypersensitivity (≥ Grade 3) to P and/or any of its excipients. (2) Subjects with symptomatic or uncontrolled brain metastasis requiring concurrent treatment, impaired major organ functions, active autoimmune disease, or active infections. Part 1 Dose Escalation is ongoing in the US and EU. Citation Format: Elena Garralda, Valentina Boni, David Vincente Baz, Arvind Chaudhry, Ignacio Gil Bazo, Martin Gutierrez, Jyoti Malhotra, Casal Guzman Alonso, Valentina Gambardella, Michael Chisamore, Renke Zhou, Maria Tea, Deborah Doroshow. Trial in progress: A phase I, open-label, multicenter study of ZL-1218, an anti-CCR8 IgG1, as a single agent and as combination therapy with anti-PD-1 antibody to evaluate the safety, tolerability, and pharmacokinetics in subjects with advanced solid tumor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr CT162.

DA-6034 ameliorates hepatic steatosis and inflammation in high fat diet-induced obese mice.

Nonalcoholic fatty liver disease (NAFLD) is characterized by an increase in hepatic triglyceride content and increased inflammatory macrophage infiltration through the C-C motif chemokine receptor (CCR) 5 pathway in the liver. DA-6034 (7-carboxymethyloxy-3',4',5-trimethoxy flavone), is a synthetic derivative of eupatilin that exhibits anti-inflammatory activity in inflammatory bowel disease. However, the effect of DA-6034 on the inflammatory response in NAFLD is not well elucidated. Therefore, we aimed to determine the effect of DA-6034 on hepatic steatosis and inflammation. Forty male C57BL/6J mice were divided into the following four groups: (1) regular diet (RD), (2) RD with DA-6034, (3) high fat diet (HFD), and (4) HFD with DA-6034. All mice were sacrificed 12 weeks after the start of the experiment. The effects of DA-6034 on macrophages were assessed using RAW264.7 cells. DA-6034 not only reduced hepatic triglyceride levels and lipid accumulation but also macrophage infiltration and proinflammatory cytokines in HFD-fed mice. According to fluorescence-activated cell sorter analysis, DA-6034 reduced the CD8+ T cell fraction in the liver of HFD-fed mice. DA-6034 also reduced CCR5 expression and the migration of liver macrophages in HFD-fed mice and inhibited CCR2 ligand and CCR4 ligand, which stimulated the migration of macrophages. Overall, DA-6034 attenuates hepatic steatosis and inflammation in obesity by regulating CCR5 expression in macrophages.

Expression of the chemokine receptor CCR1 decreases sensitivity to bortezomib in multiple myeloma cell lines

BackgroundThe proteasome inhibitor bortezomib is one of the primary therapies used for the haematological malignancy multiple myeloma (MM). However, intrinsic or acquired resistance to bortezomib, via mechanisms that are not fully elucidated, is a barrier to successful treatment in many patients. Our previous studies have shown that elevated expression of the chemokine receptor CCR1 in MM plasma cells in newly diagnosed MM patients is associated with poor prognosis. Here, we hypothesised that the poor prognosis conferred by CCR1 expression is, in part, due to a CCR1-mediated decrease in MM plasma cell sensitivity to bortezomib. MethodsIn order to investigate the role of CCR1 in MM cells, CCR1 was knocked out in human myeloma cell lines OPM2 and U266 using CRISPR-Cas9. Additionally, CCR1 was overexpressed in the mouse MM cell line 5TGM1. The effect of bortezomib on CCR1 knockout or CCR1-overexpressing cells was then assessed by WST-1 assay, with or without CCL3 siRNA knockdown or addition of recombinant human CCL3. NSG mice were inoculated intratibially with OPM2-CCR1KO cells and were treated with 0.7 mg/kg bortezomib or vehicle twice per week for 3 weeks and GFP+ tumour cells in the bone marrow were quantitated by flow cytometry. The effect of CCR1 overexpression or knockout on unfolded protein response pathways was assessed using qPCR for ATF4, HSPA5, XBP1, ERN1 and CHOP and Western blot for IRE1α and p-Jnk. ResultsUsing CCR1 overexpression or CRIPSR-Cas9-mediated CCR1 knockout in MM cell lines, we found that CCR1 expression significantly decreases sensitivity to bortezomib in vitro, independent of the CCR1 ligand CCL3. In addition, CCR1 knockout rendered the human MM cell line OPM2 more sensitive to bortezomib in an intratibial MM model in NSG mice in vivo. Moreover, CCR1 expression negatively regulated the expression of the unfolded protein response receptor IRE1 and downstream target gene XBP1, suggesting this pathway may be responsible for the decreased bortezomib sensitivity of CCR1-expressing cells. ConclusionsTaken together, these studies suggest that CCR1 expression may be associated with decreased response to bortezomib in MM cell lines.

Arylsulfatases and neuraminidases modulate engagement of CCR5 by chemokines by removing key electrostatic interactions

The chemokine receptor CCR5 is known to exist in cell surface subpopulations that differ in their capacity to engage ligands. One proposed explanation for this phenomenon is the presence of CCR5 species with different levels of post-translational modifications (PTMs). Tyrosine sulfation and O-glycan sialylation are PTMs that add negative charges to the extracellular domain of CCR5 and make strong contributions to chemokine binding but it is not known whether cellular mechanisms to control their levels exist. In this study we used a combination of sulfation-sensitive and sulfation-insensitive CCR5 ligands to show that the rate of turnover of CCR5 tyrosine sulfation is more rapid than the rate of turnover of the receptor itself. This suggests that the steady state level of CCR5 sulfation is maintained through the combination of tyrosine protein sulfotransferase (TPST), the trans-Golgi network (TGN)-resident ‘source enzyme, and a ‘sink’ activity that removes tyrosine sulfation from CCR5. By measuring the effects on ligand binding of knockdown and overexpression experiments, we provided evidence that non-lysosomal cellular arylsulfatases, particularly ARSG, ARSI and ARSJ, are CCR5 sulfation ‘sink’ enzymes. We also used targeted knockdown and sialylation-sensitive and insensitive chemokines to identify the sialidase NEU3 as a candidate ‘sink’ enzyme for CCR5 O-glycan sialylation. This study provides the first experimental evidence of activity of sulfatase and sialidase ‘sink’ enzymes on CCR5, providing a potential mechanism for cells to control steady-state levels of these PTMs and thereby exert dynamic control over receptor-ligand interactions at the cell surface and during receptor desensitization.

The chemokine receptor CCR8 is not a high-affinity receptor for the human chemokine CCL18.

The primate-specific chemokine CCL18 is a potent chemoattractant for T cells and is expressed at elevated levels in several inflammatory diseases. However, the cognate receptor for CCL18 remains unconfirmed. Here, we describe attempts to validate a previous report that the chemokine receptor CCR8 is the human CCL18 receptor (Islam et al. J Exp Med. 2013, 210:1889-98). Two mouse pre-B cell lines (4DE4 and L1.2) exogenously expressing CCR8 exhibited robust migration in response to the known CCR8 ligand CCL1 but not to CCL18. Similarly, CCL1 but not CCL18 induced internalization of CCR8 on 4DE4 cells. CCR8 expressed on Chinese hamster ovarian (CHO) cells mediated robust G protein activation, inhibition of cAMP synthesis and β-arrestin2 recruitment in response to CCL1 but not CCL18. Several N- and C-terminal variants of CCL18 also failed to stimulate CCR8 activation. On the other hand, and as previously reported, CCL18 inhibited CCL11-stimulated migration of 4DE4 cells expressing the receptor CCR3. These data suggest that CCR8, at least in the absence of unidentified cofactors, does not function as a high affinity receptor for CCL18.

Chemokine CCL19 promotes type 2 T-cell differentiation and allergic airway inflammation

Allergic asthma is driven largely by allergen-specific TH2 cells, which develop in regional lymph nodes on the interaction of naive CD4+ T cells with allergen-bearing dendritic cells that migrate from the lung. This migration event is dependent on CCR7 and its chemokine ligand, CCL21. However, is has been unclear whether the other CCR7 ligand, CCL19, has a role in allergic airway disease. This study sought to define the role of CCL19 in TH2 differentiation and allergic airway disease. Ccl19-deficient mice were studied in an animal model of allergic asthma. Dendritic cells or fibroblastic reticular cells from wild-type and Ccl19-deficient mice were cultured with naive CD4+ T cells, and cytokine production was measured by ELISA. Recombinant CCL19 was added to CD4+ T-cell cultures, and gene expression was assessed by RNA-sequencing and quantitative PCR. Transcription factor activation was assessed by flow cytometry. Lungs of Ccl19-deficient mice had less allergic airway inflammation, reduced airway hyperresponsiveness, and less IL-4 and IL-13 production compared with lungs of Ccl19-sufficient animals. Naive CD4+ T cells cocultured with Ccl19-deficient dendritic cells or fibroblastic reticular cells produced lower amounts of type 2 cytokines than did T cells cocultured with their wild-type counterparts. Recombinant CCL19 increased phosphorylation of STAT5 and induced expression of genes associated with TH2 cell and IL-2 signaling pathways. These results reveal a novel, TH2 cell-inducing function of CCL19 in allergic airway disease and suggest that strategies to block this pathway might help to reduce the incidence or severity of allergic asthma.

CD4+CCR8+ Tregs in ovarian cancer: a potential effector Tregs for immune regulation

BackgroundTregs are key drivers of immunosuppression in solid tumors. As an important chemokine receptor on Tregs, the regulatory effect of CCR8 on tumor immunity has received more and more attention. However, the current research on CCR8 in the immune microenvironment of ovarian cancer has not been clear.MethodsBioinformatics analysis was used to compare the transcriptome differences between CD4+ T cells in the peripheral circulation and infiltrated in ovarian tumor tissues. RT-PCR was used to detect the expression levels of chemokine receptor-related differential genes on CD4+ T cells in peripheral blood and ovarian tumor tissues. Multiparameter flow cytometry was used to detect the proportion and phenotypic characteristics of CD4+CCR8+ Tregs and CD4+CCR8− Tregs in different sample types. The expression level of CCR8 ligands was detected at multiple levels. To explore the important role of CCR8-CCL1 and CCR8-CCL18 axis in the migration and invasion of CD4+CCR8+ Tregs into ovarian tumor tissues by establishing a chemotaxis system in vitro.ResultsIn this study, significantly different gene expression profiles were found between peripheral circulating CD4+ T cells and infiltrating CD4+ T cells in ovarian tumor tissues, in which chemokine-chemokine receptor signaling pathway was significantly enriched in all three groups of differential genes. The expression level of CCR8 in infiltrating CD4+ T cells of ovarian cancer tissue was significantly higher than that in peripheral blood of healthy controls and ovarian cancer patients, and high expression of CCR8 was significantly correlated with advanced tumor stage and poor differentiation. CD4+CCR8+ Tregs are the main type of infiltrating CD4+ Tregs in ovarian tumor tissues, which have stronger immunosuppressive phenotypes, secrete more inhibitory cytokines and have stronger proliferation ability. The ligands CCL1 and CCL18 corresponding to CCR8 were significantly overexpressed in ovarian tumor tissues, and the CCR8-CCL1 and CCR8-CCL18 axis played a key role in the migration and infiltration of CD4+CCR8+ Tregs into ovarian tumor tissues.ConclusionsThe results of this study may help to understand the phenotypic characteristics and recruitment process of Tregs in the tumor, and provide new ideas for improving the immunosuppressive status of the ovarian cancer microenvironment.

CCR5 drives NK cell-associated airway damage in pulmonary ischemia-reperfusion injury.

Primary graft dysfunction (PGD) limits clinical benefit after lung transplantation, a life-prolonging therapy for patients with end-stage disease. PGD is the clinical syndrome resulting from pulmonary ischemia-reperfusion injury (IRI), driven by innate immune inflammation. We recently demonstrated a key role for NK cells in the airways of mouse models and human tissue samples of IRI. Here, we used 2 mouse models paired with human lung transplant samples to investigate the mechanisms whereby NK cells migrate to the airways to mediate lung injury. We demonstrate that chemokine receptor ligand transcripts and proteins are increased in mouse and human disease. CCR5 ligand transcripts were correlated with NK cell gene signatures independently of NK cell CCR5 ligand secretion. NK cells expressing CCR5 were increased in the lung and airways during IRI and had increased markers of tissue residency and maturation. Allosteric CCR5 drug blockade reduced the migration of NK cells to the site of injury. CCR5 blockade also blunted quantitative measures of experimental IRI. Additionally, in human lung transplant bronchoalveolar lavage samples, we found that CCR5 ligand was associated with increased patient morbidity and that the CCR5 receptor was increased in expression on human NK cells following PGD. These data support a potential mechanism for NK cell migration during lung injury and identify a plausible preventative treatment for PGD.