CCR5 Expression Research Articles

Circulating Levels of T-Cell Traits and the Risk of Amyotrophic Lateral Sclerosis: A Mendelian Randomization Study.

Amyotrophic lateral sclerosis (ALS) represents a rare and potentially fatal neurodegenerative disease. Diverse T-cell subsets could potentially exert diametrically opposite impacts upon ALS development. A two-sample Mendelian randomization (MR) analysis was performed to investigate the correlation between 244 T-cell subsets and ALS risk. Genetic instrumental variables were procured from a standard genome-wide association study (GWAS) that encompassed 244 T-cell subsets in 3757 individuals of European lineage. ALS-related data were collected from a GWAS comprising 20,806 ALS instances and 59,804 European control participants. Multiple sensitivity analyses were performed to verify the robustness of the significant results. Reverse MR analysis was used for delineating the effects of ALS on the characteristics of T-cells. After multiple comparison corrections, 24 out of the 244 subtypes demonstrated a potential association with ALS risk. Significantly, 75% of these associations encompassed the expression of the CD3 on diverse T-cell subtypes, revealing a highly consistent inverse relation to ALS risk. The proportion of T regulatory cells (Tregs) in CD4+ T cells and secreting Tregs in CD4+ T cells demonstrated negative associations with the risk of ALS. CCR7 expression on naive CD4+ T cells and CCR7 expression on naive CD8+ T cells showed positive associations with ALS risk. Certain T-cell subsets, particularly those identified by CD3 expression on terminally differentiated CD8+ T cells, proportions of Tregs, and CCR7 expression, indicated an association with ALS risk. These findings harmonize with and extend previous observational studies investigating the involvement of T lymphocyte subset-induced immunological processes in ALS.

Discrepant Phenotyping of Monocytes Based on CX3CR1 and CCR2 Using Fluorescent Reporters and Antibodies.

Monocytes, as well as downstream macrophages and dendritic cells, are essential players in the immune system, fulfilling key roles in homeostasis as well as in inflammatory conditions. Conventionally, driven by studies on reporter models, mouse monocytes are categorized into a classical and a non-classical subset based on their inversely correlated surface expression of Ly6C/CCR2 and CX3CR1. Here, we aimed to challenge this concept by antibody staining and reporter mouse models. Therefore, we took advantage of Cx3cr1GFP and Ccr2RFP reporter mice, in which the respective gene was replaced by a fluorescent reporter protein gene. We analyzed the expression of CX3CR1 and CCR2 by flow cytometry using several validated fluorochrome-coupled antibodies and compared them with the reporter gene signal in these reporter mouse strains. Although we were able to validate the specificity of the fluorochrome-coupled flow cytometry antibodies, mouse Ly6Chigh classical and Ly6Clow non-classical monocytes showed no differences in CX3CR1 expression levels in the peripheral blood and spleen when stained with these antibodies. On the contrary, in Cx3cr1GFP reporter mice, we were able to reproduce the inverse correlation of the CX3CR1 reporter gene signal and Ly6C surface expression. Furthermore, differential CCR2 surface expression correlating with the expression of Ly6C was observed by antibody staining, but not in Ccr2RFP reporter mice. In conclusion, our data suggest that phenotyping strategies for mouse monocyte subsets should be carefully selected. In accordance with the literature, the suitability of CX3CR1 antibody staining is limited, whereas for CCR2, caution should be applied when using reporter mice.

Interstitial macrophage phenotypes in Schistosoma-induced pulmonary hypertension.

Schistosomiasis is a common cause of pulmonary hypertension (PH) worldwide. Type 2 inflammation contributes to the development of Schistosoma-induced PH. Specifically, interstitial macrophages (IMs) derived from monocytes play a pivotal role by producing thrombospondin-1 (TSP-1), which in turn activates TGF-β, thereby driving the pathology of PH. Resident and recruited IM subpopulations have recently been identified. We hypothesized that in Schistosoma-PH, one IM subpopulation expresses monocyte recruitment factors, whereas recruited monocytes become a separate IM subpopulation that expresses TSP-1. Mice were intraperitoneally sensitized and then intravenously challenged with S. mansoni eggs. Flow cytometry on lungs and blood was performed on wildtype and reporter mice to identify IM subpopulations and protein expression. Single-cell RNA sequencing (scRNAseq) was performed on flow-sorted IMs from unexposed and at day 1, 3 and 7 following Schistosoma exposure to complement flow cytometry based IM characterization and identify gene expression. Flow cytometry and scRNAseq both identified 3 IM subpopulations, characterized by CCR2, MHCII, and FOLR2 expression. Following Schistosoma exposure, the CCR2+ IM subpopulation expanded, suggestive of circulating monocyte recruitment. Schistosoma exposure caused increased monocyte-recruitment ligand CCL2 expression in the resident FOLR2+ IM subpopulation. In contrast, the vascular pathology-driving protein TSP-1 was greatest in the CCR2+ IM subpopulation. Schistosoma-induced PH involves crosstalk between IM subpopulations, with increased expression of monocyte recruitment ligands by resident FOLR2+ IMs, and the recruitment of CCR2+ IMs which express TSP-1 that activates TGF-β and causes PH.

Chemokine CCL19 and Its Receptors CCR7 and CCRL1 in Chronic Rhinosinusitis.

CCL19 has been shown to predict disease severity in COVID-19 and treatment response in rheumatoid arthritis. CCL19 can exert both pro- and anti-inflammatory effects and is elevated in chronic rhinosinusitis (CRS). However, its role in CRS remains unknown. This study sought to determine the transcriptional changes in CCL19, its receptors, and associated cytokines and their association with disease severity in CRS. A clinical database of control subjects and patients with CRS was examined. Lund-Kennedy, Lund-Mackay, Sinonasal Outcomes Test 22 (SNOT-22), and rhinosinusitis disability index (RSDI) scores were collected at enrollment. mRNA was extracted from sinonasal tissues and subjected to multiplex gene expression analysis. Gene transcript differences between patients with CRS and controls were compared and correlated with disease severity metrics. Immunohistochemical analyses of CCL19, CCR7, and CCRL1 were conducted to compare differences in protein expression between cohorts. A subgroup analysis was performed to compare transcriptional and protein expression difference between patients with (CRSwNP) and without (CRSsNP) nasal polyps and controls. Thirty-eight subjects (control group, n=7; CRS group, n=31) were included in this study. CCRL1 (p=0.0093) and CCR7 (p=0.017) levels were significantly elevated in CRS compared to those in controls. CCL19 (p=0.038) and CCR7 (p=0.0097) levels were elevated in CRSwNP and CCRL1 was elevated in CRSsNP (p=0.0004). CCR7 expression was significantly elevated in sinonasal epithelial cells in CRSwNP (p=0.04). CCL19 expression was positively correlated with TNFA expression (p<0.0002). CCL19 and CCR7 expression was positively correlated with SNOT-22 and RSDI scores (p<0.05). CCL19 and CCR7 may modulate TNF-α-driven pro-inflammatory signaling and contribute to increased disease severity in CRS. Mechanistic studies are required to further elucidate the role of CCRL1 in CRS.

CCR2, CCR4 and CCR9 expression on T cells is required for homing to colon cancer as demonstrated by Zr-89 T cell labeling

Abstract Adoptive T cell therapy relies on trafficking of infused T cells to the tumor. Chemokine gradients and chemokine receptors expressed on T cells mediate this recruitment of T cells. However, due to the high complexity of the tumor microenvironment, regulation of T cell homing to tumors remains poorly understood. We examined how various chemokine receptors expressed on T cells impact their ability to home to cancer using zirconium-89 (89Zr)-oxine cell labeling. Ex vivo activated mouse T cells, wild type (WT) or deficient (KO) for a chemokine receptor, were labeled with 89Zr-oxine and intravenously infused into WT mice bearing subcutaneous syngeneic MC38 colon cancer. Bulk T cells, not tumor antigen specific T cells, were used to determine homing capability of the cells independent of their antigen interaction. Serial PET imaging demonstrated initial lung accumulation of the T cells followed by trafficking to the liver, spleen, and to a much lesser extent other tissues, including the tumor. Ex vivo biodistribution analyses on Day 2 showed CCR2KO, CCR4KO and CCR9KO T cells homed significantly less to the tumor compared to WT T cells, indicating that CCR2, CCR4 and CCR9 expression on T cells is important for homing to colon cancer. Augmenting expression of these chemokine receptors on T cells, or the relevant ligands in the tumor, could enhance T cell homing to colon cancer thereby improving efficacy of T cell therapy.

The Value of Chemokine and Chemokine Receptors in Diagnosis, Prognosis, and Immunotherapy of Hepatocellular Carcinoma.

Chemokines and chemokine receptors (CCRs) are involved in a variety of anti-tumour and pro-tumour immune processes in vivo, such as angiogenesis, metastasis, proliferation and invasiveness, and influence patient prognosis and response to therapy. CCRs differentially expressed in HCC and associated with prognosis were extracted from TCGA and GEO databases, and the obtained CCRs were then used to construct signature genes, and the signature gene were selected for expression validation as well as functional experiments to explore the role of CCRs in the treatment and prognosis of HCC. We constructed a prognostic model including five CCRs (CCL20, CCL23, CCR3, CCR10, and CXCR3) and validated the expression of signature genes. The model's risk score is an independent prognostic factor for HCC. We have also developed prognostic model nomograms for clinical use. In addition, we validated that CCR3 expression is associated with poor prognosis in HCC, and the proliferation and migration ability of HCC cells was significantly inhibited after interfering with the expression of CCR3 in MHCC-LM3. We also looked at differences in pathway enrichment, immune infiltration and immune checkpoints. Finally, we found that risk scores were also correlated with drug sensitivity, the high-risk group had a better sensitivity to sorafenib. The CCRs-related gene signature may better assess HCC prognosis and response to immunotherapy and tyrosine kinase inhibitors such as sorafenib in HCC, providing prospective solutions for diagnosis and treatment.

Modified Zhenwu Decoction suppresses chronic colitis via targeting macrophage CCR2/Fyn/p38 MAPK signaling axis

BackgroundUlcerative colitis (UC) is associated with intestinal macrophage infiltration due to disruption of the mucosal barrier and bacterial invasion. Therefore, it is crucial to identify therapeutic agents capable of attenuating the macrophage-induced inflammatory response to preserve mucosal homeostasis and immune tolerance. The modified Zhenwu decoction (CDD-2103) is a novel herbal formulation developed based on the principles of Traditional Chinese medicine. To date, there are no clinically approved herbal formulations for UC with a well-known mechanism of action on macrophages. PurposeThe objective of this study was to systematically investigate the inhibitory effect of the active fraction of CDD-2103 in a mouse model of chronic colitis and delineate the mechanisms underlying its inhibitory action. MethodsCDD-2103 was extracted into four fractions using organic solvents with increasing polarity. A chronic 49-day dextran sulfate sodium (DSS)-induced colitis mice model, closely resembling human clinical conditions, was used to examine the effect of CDD-2103 on chronic colitis. To confirm the effect of CDD-2103 on macrophages in this chronic colitis model, adoptive macrophage transfer and CCL2 supplementation were conducted. The mechanisms of action of CDD-2103 were further elucidated utilizing bone marrow-derived macrophages (BMDMs). Transcriptome analysis was conducted to gain insights into the underlying mechanism of action of CDD-2103 in BMDMs. ResultsOur in vitro and in vivo findings demonstrated that the ethanol-enriched fraction of CDD-2103 exhibited significant anti-inflammatory effects, leading to the suppression of colitis severity. This effect was associated with diminished accumulation of colonic macrophages in the lamina propria of CDD-2103-intervened colitis mice. Specifically, CDD-2103 inhibited CCR2/L2-mediated proinflammatory macrophage infiltration into the colon without affecting macrophage proliferation. Mechanistically, CDD-2103 inhibited Fyn expression-mediated p38 MAPK activation and subsequently suppressed CCR2 expression in BMDMs. ConclusionsCollectively, our study supports the potential use of CDD-2103 to limit macrophage infiltration, thereby reducing inflammation during UC treatment. CDD-2103 and the components in the ethanolic fraction are promising candidates for the development of novel drugs for UC management. Additionally, our study underscores Fyn-mediated CCR2 expression as a potential therapeutic target for the management of UC.

Early-life thymectomy upregulates proinflammatory chemokine signaling in visceral adipose tissue

We have previously reported that early-life thymectomy (thymex) induces an aged T cell phenotype (i.e. shift from naïve to memory) and subsequent glucose intolerance at 9mo compared to control mice. To determine potential mechanisms of inflammation and glucose intolerance in these mice, we preformed RNA sequencing (RNAseq) on the perigonadal white adipose tissue (pWAT) from thymex and control mice. We hypothesized that thymex mice would exhibit an upregulation of inflammatory genes compared to controls. This study was approved by the IACUC at the University of Texas Arlington. Male C57BL/6 mice underwent thymectomy (n=4-10) at 3wks of age or were left with their thymus intact (control n=4-10). At 9mo of age, mice were euthanized and both pWAT fat pads were dissected. One fat pad was used to determine T cell phenotype via flow cytometry, RNA was extracted from the other fat pat for RNAseq. Group differences were assessed by a students t test; RNAseq p values were adjusted for false discovery rate. RNAseq data is reported as a fold change (normalized to control group gene expression); T cell data reported as mean ± SEM (control vs thymex). There were no statistical differences in the total weight of the pWAT nor the total number of T cells in the fat pads. Gene ontology and KEGG analysis revealed that the adaptive immune response, chemokine signaling, chemokine binding and chemokine receptor binding pathways, all markers of inflammation, were upregulated in the pWAT of thymex mice compared to controls. Further analysis of these pathways revealed that gene expression for the chemokines ccl2 (MCP-1) (3.2 times greater than control; p=&lt;0.01) and ccl5 (RANTES)(2.3 times greater than control; p=&lt;0.01) were significantly upregulated in thymex mice compared to controls. In addition, gene expression for the receptors of these chemokines, ccr2 (1.7 times greater than control; p=&lt;0.01) and ccr5 (4.9 times greater than control; p&lt;0.01) were up-regulated in the pWAT of thymex mice. To determine if these chemokines were actively recruiting T cells to the pWAT, we assessed CCR2 and CCR5 expression on CD4+ (Helper) and CD8+ (Cytotoxic) T cells in the pWAT using flow cytometery. Thymex mice had significantly greater proportions of CD4+ CCR2+ (54.4±4.7%, 72.2±4.3%; p=&lt;0.01) and CCR5+ (57.4±5.1%, 75.4±5.0%; p=0.01) cells compared to controls. Similarly, thymex mice had significantly greater proportions of CD8+ CCR2+ (23.1±4.3%, 43.3±5.4%; p=&lt;0.01) and CCR5+(22.7±5.1%, 41.8±7.1%; p=0.02) cells. These results indicate that early life thymectomy induces upregulation of proinflammatory chemokine signaling in the pWAT. These chemokines appear to recruit T cells and potentially other immune cells to the pWAT, contributing to inflammation and potentially metabolic dysfunction. Funding: AHA 940023, NIH R01AG060395, K01AG061271. 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.

Cognate Antigen-Independent Differentiation and Activation of Resident Memory T cells in Chronic Kidney Disease

Resident memory T cells (TRMs) are a memory subset of T cells which are retained locally within barrier and epithelial tissues such as the kidney and have also been noted for their role in perpetuating chronic inflammation. While T cells have gained recognition for their ability to modulate kidney inflammation in various models of kidney injury TRMs are still poorly understood and their contributions to sterile models of chronic kidney disease (CKD) are unknown. We hypothesized that kidney TRMs are activated and modulate kidney inflammation in the setting of CKD. We utilized an aristolochic acid (AA) model of murine CKD to investigate the presence of kidney TRMs via flow cytometry and characterized their transcriptional profile via single cell RNA sequencing (scRNAseq). We also measured kidney cytokine levels with an MSD cytokine plate. Six weeks after AA-induced CKD, kidney CD8+ T cells were significantly increased compared to control mice (29.6 ± 1.6% vs. 4.7 ± 0.6% of CD45+, respectively; p&lt;0.0001), as were CD4+ T cells (34.4 ± 1.4% vs. 8.2 ± 0.7% of CD45+, respectively; p&lt;0.0001). 53.6 ± 2.5% of CD8+ T cells expressed CD103, a TRM marker, in AA mice compared to 4.8 ± 1.2% in control mice, and 36.0 ± 1.7% vs 1.6 ± 0.3% of CD4+ T cells. Kidney protein lysates of AA mice had significantly higher levels of IFNγ than control kidneys (18.9 ± 1.6 vs. 2.9 ± 0.7 pg/mg, respectively; p=0.0003) as well as TNF (98.6 ± 8.9 pg/mg vs. 6.9 ± 0.8 pg/mg, respectively; p=0.0002) and IL-6 (247.6 ± 39.1 pg/mg vs. 75.4 ± 22.5 pg/mg, respectively; p=0.0226). scRNAseq of immune cells revealed T cells bearing TRM markers CD103 and CD49a which lacked expression of tissue egress molecules S1pr1, Sell, Ccr7, and Klf2. scRNAseq analysis of this TRM population also revealed downregulation of Eomes and Tbx21 transcription factors and upregulation of Runx3 transcription factor, all of which correspond with previously described TRM transcriptional profiles. We next evaluated signaling pathways involved with the survival and transcriptional profile of TRMs, namely IL-15 and TGF-β. While Il2rb and Il2rg (encoding IL-15 receptor subunits) were upregulated in TRMs, IL-15 protein lysate levels were not elevated in AA kidneys compared to controls (2,600 ± 145 pg/mg protein vs. 2,281 ± 96 pg/mg protein, respectively; p=0.0831). Tgfbr1 and Tgfbr2 (encoding TGF-β receptor subunits) were also upregulated in TRMs, but TGF-β protein lysate levels were not significantly elevated in AA kidneys compared to controls (2,158 ± 159 pg/mg protein vs. 1,924 ± 202 pg/mg protein, respectively; p=0.3886). Gene ontology pathway analysis showed enrichment of pathways involved in T cell receptor signaling. Because of this we assessed the kidney immune profile of transgenic OT-1 mice, which can only recognize specific ovalbumin residues in the context of MHC class 1 H-2Kb. Six weeks after AA-induced CKD, injured OT-1 mice still had a significant increase in the proportion of CD8+ T cells that were CD103+ compared to vehicle control OT-1 mice (47.3 ± 2.9% vs. 9.6 ± 1.6% of CD45+, respectively; p&lt;0.0001) and CD4+ T cells that were CD103+ (26.7 ± 3.1% vs. 6.4 ± 2.4% of CD45+, respectively; p=0.0040). Here we demonstrate the differentiation and activation of TRMs during AA-induced CKD independent of cognate antigen interaction and highlight their activated profile, as well as an increase in kidney IFNγ, TNF, and IL-6, all proinflammatory cytokines typical of TRMs. Future studies will aim to determine the contributions of TRMs to CKD progression. F32 DK136187, T32 DK007545, R01 DK 118932, U54 DK137301. 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.

The role of CX3CR1+ FALC-associated macrophages in health and inflammation

Abstract The omentum is a visceral adipose tissue that contains fat-associated lymphoid clusters (FALC) to aid in peritoneal immunity. These clusters consist of large aggregates of lymphocytes, dendritic cells, macrophages, and innate lymphoid cells that participate in the clearance of pathogens. Tissue-resident macrophages (TRM) are immune cells found in every tissue and can be defined by their origins, consisting of subsets maintained either by embryonic precursors or by bone-marrow progenitors. A recent study found embryonically derived TRM express at least one marker amongst Tim4, LYVE1, FOLR2 while bone marrow derived macrophages maintain CCR2 expression. Previous publications have shown the existence of LYVE1+ macrophages surrounding each cluster, here we study an unexamined subset of FALC-associated macrophages that are imbedded within the lymphoid structures. Using confocal imaging, flow cytometry analysis with reporter/ conditional knock-out mice, we have characterized this TRM subset as CX3CR1+ Tim4- LYVE1lo/- and CCR2 dependent. In addition, we examined the interaction between CX3CR1+ macrophages and CCL19+ fibroblastic reticular cells by generating CCL19-driven Csf1 depleted mice and found CX3CR1+ macrophages were partially depleted. Lastly, we looked at the role of tissue-resident macrophages in the omentum in early ovarian tumor burden and found omental/peritoneal TRM macrophage depletion by Wt1-driven Csf1 depletion led to increased tumor burden.

Transcriptomic analysis reveals the critical role of chemokine signaling in the anti-atherosclerosis effect of Xuefu Zhuyu decoction

Ethnopharmacological relevanceThe process of atherosclerosis (AS) is complicated. Transcriptomics technology can assist in discovering the underlying mechanisms and exploring the key targets of Traditional Chinese Medicine (TCM) against atherosclerosis. AimThis study aimed to investigate targets and signaling pathways significantly related to AS and the potential intervention targets of Xuefu Zhuyu decoction by transcriptomics. Materials and methodsAS models were established by subjecting ApoE-/-mice to an 8-week high-fat diet. Structural changes and plaque formation in the aortic root were observed using hematoxylin-eosin staining (HE staining), while Oil Red O staining was employed to visualize lipid deposition within the aortic root plaque. Movat staining and immunohistochemical staining were conducted to examine the components present in the aortic root plaque. Macrophage content within the plaque was observed through immunofluorescence. Additionally, mRNA sequencing was performed on aortic tissues to identify differentially expressed genes. Enrichment analysis was performed using GO and KEGG analysis. Visualization of the protein-protein interaction (PPI) network was achieved using Cytoscape 3.7.1 and STRING. Western blotting (WB) was employed to assess the protein expression of major differentially expressed genes in the aortic tissue. The drug freeze-dried powder of Xuefu Zhuyu decoction was prepared and the RAW264.7 cells were induced by lipopolysaccharide (LPS) to build an in vitro model. Real-time quantitative PCR was employed to measure the mRNA expression of major differential genes. ResultsAfter ApoE-/- mice were fed with an 8-week high-fat diet, observable changes included the thinning of the aortic root wall, the accumulation of foam cells within the plaque, and the formation of cholesterol crystals in the model group. Treatment with Xuefu Zhuyu (XFZY) decoction for 12 weeks significantly reduced the lipid deposition and the number of macrophages (P < 0.05) and significantly increased the collagen content within the plaque (P < 0.01). Enrichment analysis revealed a high enrichment of the Cytokine-cytokine receptor interaction pathway and Chemokine signaling pathway. Noteworthy genes involved in this response included Ccl12, Ccl22, Cx3cr1, Ccr7, Ccr2, Tnfrsf25, and Gdf5. Xuefu Zhuyu decoction significantly downregulated the expression of CX3CL1 and CX3CR1 (P < 0.05) and upregulated the expression of GDF5 (P < 0.01). Compared with control group, in cell models, the mRNA expressions of Ccl12, Ccl22, and Ccr2 were significantly upregulated (P < 0.05 or P < 0.01). Xuefu Zhuyu decoction significantly downregulated the expression of Ccl12, Ccl22, Cx3cr1, Ccr7 and Ccr2 (P < 0.05 or P < 0.01). ConclusionXuefu Zhuyu decoction demonstrates effective regulation of plaque components, retarding plaque progression and preserving plaque stability by modulating lipid metabolism and inflammatory responses. Subsequent transcriptome analysis identified the Cytokine-cytokine receptor interaction and Chemokine signaling pathway as potential key pathways for the therapeutic effects of Xuefu Zhuyu decoction. This insight not only provides crucial avenues for further exploration into the mechanisms underlying Xuefu Zhuyu decoction but also offers valuable perspectives and hypotheses for enhancing disease prevention and treatment strategies.

A TriAdj-Adjuvanted Chlamydia trachomatis CPAF Protein Vaccine Is Highly Immunogenic in Pigs.

Chlamydia trachomatis (Ct) infections are the most common sexually transmitted infection (STI). Despite effective antibiotics for Ct, undetected infections or delayed treatment can lead to infertility, ectopic pregnancies, and chronic pelvic pain. Besides humans, chlamydia poses similar health challenges in animals such as C. suis (Cs) in pigs. Based on the similarities between humans and pigs, as well as their chlamydia species, we use pigs as a large biomedical animal model for chlamydia research. In this study, we used the pig model to develop a vaccine candidate against Ct. The vaccine candidate consists of TriAdj-adjuvanted chlamydial-protease-like activity factor (CPAF) protein. We tested two weekly administration options-twice intranasal (IN) followed by twice intramuscular (IM) and twice IM followed by twice IN. We assessed the humoral immune response in both serum using CPAF-specific IgG (including antibody avidity determination) and also in cervical and rectal swabs using CPAF-specific IgG and IgA ELISAs. The systemic T-cell response was analyzed following in vitro CPAF restimulation via IFN-γ and IL-17 ELISpots, as well as intracellular cytokine staining flow cytometry. Our data demonstrate that while the IN/IM vaccination mainly led to non-significant systemic immune responses, the vaccine candidate is highly immunogenic if administered IM/IN. This vaccination strategy induced high serum anti-CPAF IgG levels with strong avidity, as well as high IgA and IgG levels in vaginal and rectal swabs and in uterine horn flushes. In addition, this vaccination strategy prompted a pronounced cellular immune response. Besides inducing IL-17 production, the vaccine candidate induced a strong IFN-γ response with CD4 T cells. In IM/IN-vaccinated pigs, these cells also significantly downregulated their CCR7 expression, a sign of differentiation into peripheral-tissue-homing effector/memory cells. Conclusively, this study demonstrates the strong immunogenicity of the IM/IN-administered TriAdj-adjuvanted Ct CPAF vaccine candidate. Future studies will test the vaccine efficacy of this promising Ct vaccine candidate. In addition, this project demonstrates the suitability of the Cs pre-exposed outbred pig model for Ct vaccine development. Thereby, we aim to open the bottleneck of large animal models to facilitate the progression of Ct vaccine candidates into clinical trials.

Critically-ill COVID-19 susceptibility gene CCR3 shows natural selection in sub-Saharan Africans

The prevalence of COVID-19 critical illness varies across ethnicities, with recent studies suggesting that genetic factors may contribute to this variation. The aim of this study was to investigate natural selection signals of genes associated with critically-ill COVID-19 in sub-Saharan Africans. Severe COVID-19 SNPs were obtained from the HGI website. Selection signals were assessed in 661 sub-Sahara Africans from 1000 Genomes Project using integrated haplotype score (iHS), cross-population extended haplotype homozygosity (XP-EHH), and fixation index (Fst). Allele frequency trajectory analysis of ancient DNA samples were used to validate the existing of selection in sub-Sahara Africans. We also used Mendelian randomization to decipher the correlation between natural selection and critically-ill COVID-19. We identified that CCR3 exhibited significant natural selection signals in sub-Sahara Africans. Within the CCR3 gene, rs17217831-A showed both high iHS (Standardized iHS = 2) and high XP-EHH (Standardized XP-EHH = 2.5) in sub-Sahara Africans. Allele frequency trajectory of CCR3 rs17217831-A revealed natural selection occurring in the recent 1,500 years. Natural selection resulted in increased CCR3 expression in sub-Sahara Africans. Mendelian Randomization provided evidence that increased blood CCR3 expression and eosinophil counts lowered the risk of critically ill COVID-19. Our findings suggest that sub-Saharan Africans are resistant to critically ill COVID-19 due to natural selection and identify CCR3 as a potential novel therapeutic target.

Palmatine Attenuated Lipopolysaccharide-Induced Acute Lung Injury by Inhibiting M1 Phenotype Macrophage Polarization via NAMPT/TLR2/CCR1 Signaling.

The present work was conducted to research the potential mechanism of palmatine (PAL) on lipopolysaccharide (LPS)-caused acute lung injury (ALI). Network pharmacology and bioinformatic analyses were carried out. Mice were intragastrically treated with PAL and intratracheally stimulated with LPS. LPS-induced RAW264.7 cells were employed for the in vitro model. The MPO activity, W/D ratio, neutrophils, total cell number in BALF, and histopathological alteration were examined. The levels of TNF-α, IL-1β, IL-6, IL-18, IL-4, and IL-10 in serum, BALF, and supernatant were examined by ELISA. The mRNA expressions of iNOS, CD68, Arg1, Ym1, and CD206 and protein expressions of NAMPT, TLR2, CCR1, and NLRP3 inflammasome were detected by PCR, WB, and immunofluorescence. The NAMPT inhibitor FK866, TLR2 inhibitor C29, CCR1 inhibitor BX471, NAMPT-overexpression (OE) plasmid, and TLR2-OE plasmid were used for mechanism research. As a result, PAL relieved the symptoms of ALI. PAL inhibited M1 phenotype indices and promoted M2 phenotype indices in both LPS-induced mice and RAW264.7 cells. PAL also inhibited the expressions of NAMPT, TLR2, CCR1, and NLRP3 inflammasome. The treatments with FK866, NAMPT-OE plasmid, C29, TLR2-OE plasmid, and BX471 proved that PAL exerted its effect via NAMPT/TLR2/CCR1. Molecular docking suggested that PAL might combine with NAMPT. In conclusion, PAL ameliorated LPS-induced ALI by inhibiting M1 phenotype macrophage polarization via NAMPT/TLR2/CCR1 signaling.

Gut-derived trimethylamine N-oxide promotes CCR2-mediated macrophage infiltration in acute kidney injury.

Inflammation is crucial in the development of acute kidney injury (AKI) and subsequent chronic kidney disease (CKD) following renal ischaemia-reperfusion (IR) injury. Gut microbiota metabolites trigger inflammation and affect IR-induced renal damage. Yet the driving factors and mechanisms are unclear. Trimethylamine N-oxide (TMAO), a gut-derived choline metabolite, is a strong pro-inflammatory factor that increases in patients with AKI and CKD. We hypothesized that TMAO can promote renal injury caused by IR. Mice subjected to unilateral renal IR to induce AKI and CKD were fed a high-choline diet to observe the effects of TMAO on kidney inflammation, fibrosis and macrophage dynamics. A choline-rich diet altered the gut microbiota and elevated TMAO levels, exacerbating IR-induced AKI and subsequent CKD. Single-cell analysis identified a distinct subset of CCR2+ macrophages derived from monocytes as key responders to TMAO, intensifying immune cell interactions and worsening renal injury. TMAO promoted sustained CCR2 expression after IR, increasing macrophage infiltration. CCR2 deletion and antagonist RS-102895 improved TMAO-induced inflammation and fibrosis and alleviated renal injury induced by IR. Our study provides valuable insights into the link between TMAO and IR-induced renal inflammation and fibrosis, emphasizing the critical role of TMAO-mediated macrophage infiltration via CCR2 as a key therapeutic target in the acute and chronic phases after IR.

Abstract CT174: A phase 1 study of JBH492, an anti C-C chemokine receptor 7 antibody-drug conjugate (anti-CCR7 ADC), assessing safety and efficacy in lymphoid malignancies

Abstract Background: CCR7 is overexpressed in lymphoid malignancies. JBH492 is a first-in-class CCR7-targeting ADC, comprising a humanized, Fc-silenced anti-CCR7 IgG1 antibody conjugated to maytansinoid microtubule inhibitor DM4 via a cleavable linker. Preclinical studies support avidity-driven differential binding in CCR7-high tumor cells compared to CCR7-low healthy immune cells, and in vivo animal studies demonstrated robust and durable efficacy in multiple clinically relevant lymphoma models (Dang et al. 2022). Methods: This is an ongoing first-in-human, open-label, phase I/Ib, multi-center study in patients (pts) with relapsed/refractory (r/r) chronic lymphocytic leukemia (CLL) and non-Hodgkin’s lymphoma (NHL) (NCT04240704), which completed enrollment for dose-escalation phase. Five dose levels of JBH492 monotherapy ranging from 0.4 to 3.6 mg/kg (intravenous administration every 3 weeks [q3W]) were tested with 3 - 7 pts enrolled per dose level. Preclinical data from xenografts predicted tumor stasis at 1.6 mg/kg q3W. The objective of dose escalation was to assess safety, tolerability, pharmacokinetics (PK), immunogenicity, and preliminary efficacy of JBH492 and to determine maximum tolerated and recommended doses. Pts with r/r CLL and NHL, who failed standard of care therapy (at least 2 prior regimens) and who were intolerant/ineligible to approved therapies, were included; CCR7 expression confirmation was not required prior enrollment. Results: Total 25 pts with r/r stage II-IV NHL (n = 24, [adult T cell leukemia-lymphoma, 3; cutaneous T cell lymphoma, 4; diffuse large B cell lymphoma, 7; follicular lymphoma, 1; Mantle cell lymphoma, 1; marginal zone lymphoma, 2; peripheral T cell lymphoma, 3; others, 3]) and CLL (n = 1) with median age 69.0 y (43 - 85) were enrolled. Mean duration of exposure was 13.5 wks. One pt had dose-limiting toxicity — grade (G) 3 thrombocytopenia lasting for 13 d. Overall, 23 (92%) pts had at least 1 adverse event (AE) (G ≥ 3, n = 11). Treatment-related AEs (TRAEs) were reported in 15 (60%) pts (G ≥ 3, n = 3). Of 6 (24%) pts who had serious AEs (SAEs) (G ≥ 3, n = 3), 2 (8%) had TRSAE. The most frequent AEs (≥ 20%) included anemia (32%), thrombocytopenia (28%), neutropenia and dry eye (20% each). Two deaths due to underlying disease were reported. PK for total ADC was roughly dose proportional. Four NHL pts (2 T-NHL and 2 B-NHL) had partial response (PR) and 2 had stable disease, with PRs observed at doses between 2.4 and 3.6 mg/kg q3W. Post data-cut-off, 1 pt with T-NHL achieved CR following 4 cycles of treatment. No correlation between CCR7 expression and response could be drawn (CCR7 expression was observed in all samples from 14 pts). Conclusions: The data from this phase I study suggest that targeting CCR7 with JBH492 is a promising therapeutic strategy for lymphoid malignancies with manageable hematological and non-hematological safety profile. [K.I. and S.L. contributed equally to this study.] Citation Format: Pau Abrisqueta, Reinhard Marks, Irit Avivi, Martin Wermke, Francesca Lim, Tae Min Kim, Alba Cabirta Touzon, Kristiina Karihtala, Shinichi Makita, Lillian Werner, Chiaki Tanaka, Anhthu Dang, Anwesha Chaudhury, Seth Rice, Rowshan Chowdhury, Deborah Knee, Belén Gomezcarrillo, Elissa Furutani, Koji Izutsu, Sirpa Leppä. A phase 1 study of JBH492, an anti C-C chemokine receptor 7 antibody-drug conjugate (anti-CCR7 ADC), assessing safety and efficacy in lymphoid malignancies [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 CT174.

Role of CCR1/5/7 in hepatocellular carcinoma: a study on prognostic evaluation, molecular subtyping, and association with immune infiltration.

This study aims to assess the prognostic value of the C-C motif chemokine receptor (CCR) gene family in hepatocellular carcinoma (HCC) and its relationship with immune infiltration and molecular subtypes of HCC. The evaluation of the GSE14520 dataset and TCGA database confirmed the prognostic significance of CCR. Building upon the correlation between CCR1, CCR5, and CCR7 and favorable prognosis, we further validated the prognostic importance of CCR1, CCR5, and CCR7 in ICGC database and an independent cohort from Guangxi autonomous region. Then, we constructed a risk prognosis model. Additionally, we observed significant positive correlations between CCR1, CCR5, and CCR7 and the infiltration of B cells, T cells, and macrophages in HCC. Subsequently, we conducted CCK assays, Transwell assays, and colony formation assays to evaluate the molecular biological functions of CCR1, CCR5, and CCR7. These experiments further confirmed that upregulation of CCR1, CCR5, and CCR7 can individually inhibit the proliferation, migration, and stemness of HCC cells. By analyzing the relationship between expression levels and tumor mutation frequency, we discovered that patients with high CCR1 expression were more likely to be classified as non-proliferative HCC. Similar conclusions were observed for CCR5 and CCR7. The association of CCR1, CCR5, and CCR7 with the molecular subtypes of HCC suggests that they may serve as intermediary molecules linking immune status and molecular subtypes in HCC. In summary, CCR1, CCR5, and CCR7 have the potential to serve as prognostic biomarkers for HCC and regulate HCC progression by influencing immune cell infiltration.

Abstract 2731: CCR5-001, a novel drug Fc-conjugate (DFC) targeting CCR5, demonstrates potent efficacy in a colorectal cancer mouse model

Abstract Background The approval of immune checkpoint inhibitors (ICI) such as PD-(L)1 inhibitors has revolutionized cancer therapy and established a role for immune effector cells, most notably CD8+ T cells, in tumor control and destruction. While anti-PD-(L)1 therapy has demonstrated durable responses, only a small subset of patients benefit and respond to therapy. CCR5 is expressed on multiple immune cells (e.g. myeloid-derived suppressor cells) and CCR5-dependent migration of these cells contribute to an immune-suppressive tumor microenvironment (TME) thereby limiting response to ICI therapy. In addition, expression of CCR5 on cancer cells has been linked to increased metastasis and resistance to ICI therapy. Upregulation of CCL5/CCR5 has been associated with poor outcomes in multiple solid tumor indications, including colorectal cancer (CRC). Therefore, targeting CCR5 has the potential to increase response rates to ICI therapy and improve outcomes. Herein, we describe a first-in-class CCR5 targeting DFC, CCR5-001, a multivalent conjugate of a potent, small molecule CCR5 antagonist conjugated to an immune-silent proprietary human IgG1 Fc. This CCR5 targeting DFC has the potential to improve responses to ICI therapy by preventing pro-tumorigenic reprogramming of the TME induced by CCL5/CCR5 expressing tumor and myeloid cells. Methods Binding of CCR5-001 to hCCR5 was measured by surface plasmon resonance (SPR) and to CCR5+ PathHunter cells (DiscoverX) by flow cytometry. Functional activity was determined in CCR5+ PathHunter β-arrestin assay with CCL3 to induce CCR5 signaling according to manufacturer’s instructions. Efficacy was determined in an immune-competent syngeneic mouse model using the CRC cell line, MC38. CCR5-001 was dosed at 20 mg/kg twice per week for two weeks. Tumor volumes were recorded and statistical analysis was conducted by t-test (Mann-Whitney) or two-way ANOVA in Prism. Results CCR5-001 displayed a KD of 4.2 nM to immobilized hCCR5 by SPR and IC50 of 0.63-0.98 nM for binding to engineered CCR5+ PathHunter cells. This high affinity binding translated to potent functional inhibition of CCL3-induced CCR5 signaling with an IC50 of 12.7 nM in CCR5+ cell-based assay. CCR5-001 demonstrated potent, statistically significant (p = 0.0052) tumor growth inhibition of 76% by day 13 in a syngeneic mouse model. Conclusions CCR5-001 demonstrated potent CCR5 binding and functional inhibition of CCR5 signaling in cell-based assays. The in vitro activity translated to robust efficacy as a monotherapy in a syngeneic mouse model. Based on these results and other emerging data, CCR5-001 represents an exciting new avenue for targeting CCR5 with DFCs in solid cancers where the pathology is driven by the CCR5/CCL5 axis. Citation Format: Simon Döhrmann, James Levin, Nicholas Dedeic, Amanda Almaguer, Doug Zuill, Elizabeth Abelovski, Jeffrey B. Locke, Joanne Fortier, Maria Hernandez, Qiping Zhao, Karin Amundson, Madison Moniz, Hongyuan Chen, Dhanya Panickar, Thanh Lam, Grayson Hough, Thomas P. Brady, Allen Borchardt, Jason N. Cole, Leslie W. Tari. CCR5-001, a novel drug Fc-conjugate (DFC) targeting CCR5, demonstrates potent efficacy in a colorectal cancer mouse model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2731.

Abstract 3606: Tumor-specific CD8+ Tc9 cells activate host CD4+ T cells to control antigen-lost tumors

Abstract Cancer immunotherapies relying on targeted destruction of cancer cells by potent antitumor T cells have achieved unprecedented success in recent years. As a main form of cancer immunotherapies, adoptive T cell therapy (ACT) has shown a durable response in certain cancer patients. However, this response is often short-lived and tumor relapse occurs due to the outgrowth of antigen-lost-variant (ALV) tumors and poor antitumor immune response. Here, We reported that adoptively transfer of murine tumor-specific CD8+ Tc9 but not Tc1/CTL cells achieved long-term control of tumor growth in vivo. Here, we demonstrated that murine tumor-specific Tc9 cells not only killed antigen-expressing primary tumors but also controlled the outgrowth of antigen-lost relapsed tumors by recruiting and activating host effector CD4+ T cells that recognized relapsed tumors. Tc9 cells secreted IL-24 and recruited CCR7-expressing conventional type-2 dendritic cells (cDC2) into tumor-draining lymph nodes to prime host CD4+ T cell response against relapsed tumors. Depleting host CD4+ T cells or deficiency in CCR7 expression impaired Tc9 cell ability to control the outgrowth of relapsed tumor. We also observed that intratumoral IL24 expression was positively correlated with gene signatures of cDC2 and CD4+ T cells in human cancers and expression of IL24 and cDC2 and CD4+ T cell gene signatures were associated with patient’s overall survival. Thus, this study uncovers a novel mechanism underlying activation of tumor-specific CD4+ T cells in vivo. Citation Format: Liuling Xiao, Qing Yi. Tumor-specific CD8+ Tc9 cells activate host CD4+ T cells to control antigen-lost tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3606.

JAK/STAT signaling pathway affects CCR5 expression in human CD4+ T cells.

CCR5 serves as R5-tropic HIV co-receptor. Knocking out CCR5 in HIV patients, which has occurred <10 times, is believed important for cure. JAK/STAT inhibitors tofacitinib and ruxolitinib inhibit CCR5 expression in HIV+ viremic patients. We investigated the association of JAK/STAT signaling pathway with CCR5/CCR2 expression in human primary CD4+ T cells and confirmed its importance. Six of nine JAK/STAT inhibitors that reduced CCR5/CCR2 expression were identified. Inhibitor-treated CD4+ T cells were relatively resistant, specifically to R5-tropic HIV infection. Furthermore, single JAK2, STAT3, STAT5A, and STAT5B knockout and different combinations of JAK/STAT knockout significantly reduced CCR2/CCR5 expression of both RNA and protein levels, indicating that CCR5/CCR2 expression was positively regulated by JAK-STAT pathway in CD4+ T cells. Serum and glucocorticoid-regulated kinase 1 (SGK1) knockout affected CCR2/CCR5 gene expression, suggesting that SGK1 is involved in CCR2/CCR5 regulation. If cell surface CCR5 levels can be specifically and markedly down-regulated without adverse effects, that may have a major impact on the HIV cure agenda.