CCR5 Expression Research Articles

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.

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 1841: TAK-500 is a clinical stage immune-cell directed antibody drug conjugate (iADC) inducing STING activation in CCR2-expressing intratumor myeloid cells and favorable immunomodulation

Abstract Introduction: Myeloid cells are present in the tumor microenvironment (TME) of most human solid cancers, playing an essential role to influence local immunomodulatory functions. STING signaling in intratumor myeloid cells can enhance interferon (IFN) production leading to enhanced local innate and adaptive immunity, and synergism with other anti-tumor mechanisms. Here, we used a novel iADC, TAK-500, to selectively activate STING in CCR2-expressing cells. This approach enables systemic delivery and favors intratumor accumulation of a STING agonist to potentially achieve anti-tumor activity. Methods: TAK-500 and mTAK-500, a murine surrogate, were employed to study the immunomodulatory role and anti-tumor effects in preclinical models. CCR2 protein expression was characterized in the TME of >1,000 primary human tumors including non-small cell lung cancer (NSCLC, 2 cohorts, N= 411), colorectal carcinomas (CRC, 2 cohorts, N=350) and pancreatic ductal adenocarcinomas (PDAC, 1 cohort, N=228) represented in tissue microarrays using multiplexed quantitative immunofluorescence (mQIF). Results: TAK-500 triggered dose-dependent monocyte activation in vitro. mTAK-500 induced robust activation of innate and adaptive immune responses both in vitro and in vivo. In syngeneic mouse models with CCR2 expressing intratumoral myeloid cells, mTAK-500 treatment caused accumulation and activation of CD8+ effector T-cells in the TME resulting in prominent anti-tumor activity and enhanced survival. The baseline levels of CCR2-expressing mMDSCs in mouse models positively associated with anti-tumor response to mTAK-500. mQIF analysis of human tumors confirmed high expression of CCR2 in myeloid cells, lower expression in tumor infiltrating lymphocytes (TILs) and absence in tumor epithelial cells. Using an mQIF panel for simultaneous measurement of DAPI, CK, CCR2, CD11b and CD68, we identified CCR2 protein expression in 94% of NSCLCs, 87% CRCs and 89% PDACs. The levels of CCR2 protein were significantly higher in NSCLC than in CRC and PDAC. In NSCLC, high CCR2 protein in myeloid cells was associated with the presence of activating EGFR/KRAS mutations and increased CD8+ TILs and local PD-L1 expression. In CRC, higher levels of CCR2 in CD68+ macrophages were seen in tumors harboring microsatellite instability than in microsatellite stable carcinomas. Conclusion: The iADC TAK-500 and mTAK-500 induces CCR2-dependent immune cell activation and anti-tumor effect. CCR2 is highly expressed in intratumor myeloid cells from NSCLC. High CCR2 is associated with enhanced local adaptive immune responses and specific clinical/molecular tumor subsets. TAK-500 is currently being evaluated clinically as a single agent and in combination with pembrolizumab in adults with select locally advanced or metastatic solid tumors (NCT05070247). Citation Format: Kurt A. Schalper, Atsushi Matsuda, Michelle Ganno-Sherwood, Angel E. Maldonado-Lopez, Emily Rosentrater, Angelo Porciuncula, Dong Mei Zhang, Camilla L. Christensen, Samantha A. Merrigan, Tiquella Hatten, Hong Myung Lee, Min Young Lee, Linlin Dong, Jian Huang, Natasha Iartchouk, Jianing Wang, He Xu, Tomoki Yoneyama, Konstantin I. Piatkov, Carole E. Harbison, Alex Parent, Neil Lineberry, Adnan O. Abu-Yousif, Vicky Appleman. TAK-500 is a clinical stage immune-cell directed antibody drug conjugate (iADC) inducing STING activation in CCR2-expressing intratumor myeloid cells and favorable immunomodulation [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 1841.

Novel neuroendocrine role of γ-aminobutyric acid and gastrin-releasing peptide in the host response to influenza infection

Gastrin-releasing peptide (GRP), an evolutionarily conserved neuropeptide, significantly contributes to influenza-induced lethality and inflammation in rodent models. Because GRP is produced by pulmonary neuroendocrine cells (PNECs) in response to γ-aminobutyric acid (GABA), we hypothesized that influenza infection promotes GABA release from PNECs that activate GABAB receptors on PNECs to secrete GRP. Oxidative stress was increased in the lungs of influenza A/PR/8/34 (PR8)-infected mice, as well as serum glutamate decarboxylase 1, the enzyme that converts L-glutamic acid into GABA. The therapeutic administration of saclofen, a GABAB receptor antagonist, protected PR8-infected mice, reduced lung proinflammatory gene expression of C-C chemokine receptor type 2 (Ccr2), cluster of differentiation 68 (Cd68), and Toll like receptor 4 (Tlr4) and decreased the levels of GRP and high-mobility group box 1 (HMGB1) in sera. Conversely, baclofen, a GABAB receptor agonist, significantly increased the lethality and inflammatory responses. The GRP antagonist, NSC77427, as well as the GABAB antagonist, saclofen, blunted the PR8-induced monocyte infiltration into the lung. Together, these data provide the first report of neuroregulatory control of influenza-induced disease.

Expression of Homing Receptors in IgM+IgD+CD27+ B Cells and Their Frequencies in Appendectomized and/or Tonsillectomized Individuals

ABSTRACT Background In humans, blood circulating IgM+IgD+CD27+ B cells are considered analogous to those described in the marginal zone of the spleen and are involved in important immunological processes. The homing receptors they express, and the organs involved in their development (for example, intestinal organs in rabbits) are only partially known. We recently reported that this population is heterogeneous and composed of at least two subsets: one expressing high levels of IgM – IgMhi B cells – and another low levels – IgMlo B cells. Objectives To evaluate the expression of homing receptors on IgD+CD27+ IgMhi and IgMlo B cells and quantify their frequencies in blood of control and appendectomized and/or tonsillectomized volunteers. Materials and Methods Using spectral flow cytometry, the simultaneous expression of 12 previously reported markers that differentiate IgMhi B cells and IgMlo B cells and of α4β7, CCR9, CD22 and CCR10 were evaluated in blood circulating B cells of control and appendectomized and/or tonsillectomized volunteers. Results The existence of phenotypically defined IgMlo and IgMhi B cell subsets was confirmed. They differentially expressed intestinal homing receptors, and the expression of α4β7 and CCR9 seems to determine new IgM subpopulations. IgMlo and IgMhi B cells were detected at lower frequencies in the appendectomized and/or tonsillectomized volunteers relative to controls. Conclusions Human blood circulating IgD+CD27+ IgMlo and IgMhi B cell subsets differentially express homing receptors, and it is necessary to investigate if mucosal organs are important in their development.

Varicella Zoster Virus infects mucosal associated Invariant T cells.

Mucosal Associated Invariant T (MAIT) cells are innate-like T cells that respond to conserved pathogen-derived vitamin B metabolites presented by the MHC class I related-1 molecule (MR1) antigen presentation pathway. Whilst viruses do not synthesize these metabolites, we have reported that varicella zoster virus (VZV) profoundly suppresses MR1 expression, implicating this virus in manipulation of the MR1:MAIT cell axis. During primary infection, the lymphotropism of VZV is likely to be instrumental in hematogenous dissemination of virus to gain access to cutaneous sites where it clinically manifests as varicella (chickenpox). However, MAIT cells, which are found in the blood and at mucosal and other organ sites, have yet to be examined in the context of VZV infection. The goal of this study was to examine any direct impact of VZV on MAIT cells. Using flow cytometry, we interrogated whether primary blood derived MAIT cells are permissive to infection by VZV whilst further analysing differential levels of infection between various MAIT cell subpopulations. Changes in cell surface extravasation, skin homing, activation and proliferation markers after VZV infection of MAIT cells was also assessed via flow cytometry. Finally the capacity of MAIT cells to transfer infectious virus was tested through an infectious center assay and imaged via fluorescence microscopy. We identify primary blood-derived MAIT cells as being permissive to VZV infection. A consequence of VZV infection of MAIT cells was their capacity to transfer infectious virus to other permissive cells, consistent with MAIT cells supporting productive infection. When subgrouping MAIT cells by their co- expression of a variety cell surface markers, there was a higher proportion of VZV infected MAIT cells co-expressing CD4+ and CD4+/CD8+ MAIT cells compared to the more phenotypically dominant CD8+ MAIT cells, whereas infection was not associated with differences in co-expression of CD56 (MAIT cell subset with enhanced responsiveness to innate cytokine stimulation), CD27 (co-stimulatory) or PD-1 (immune checkpoint). Infected MAIT cells retained high expression of CCR2, CCR5, CCR6, CLA and CCR4, indicating a potentially intact capacity for transendothelial migration, extravasation and trafficking to skin sites. Infected MAIT cells also displayed increased expression of CD69 (early activation) and CD71 (proliferation) markers. These data identify MAIT cells as being permissive to VZV infection and identify impacts of such infection on co- expressed functional markers.

IFNγ-induction of TH1-like regulatory T cells controls antiviral responses.

Regulatory T (Treg) cells are an immunosuppressive population that are required to maintain peripheral tolerance and prevent tissue damage from immunopathology, via anti-inflammatory cytokines, inhibitor receptors and metabolic disruption. Here we show that Treg cells acquire an effector-like state, yet remain stable and functional, when exposed to interferon gamma (IFNγ) during infection with lymphocytic choriomeningitis and influenza A virus. Treg cell-restricted deletion of the IFNγ receptor (encoded by Ifngr1), but not the interleukin 12 (IL12) receptor (encoded by Il12rb2), prevented TH1-like polarization (decreased expression of T-bet, CXC motif chemokine receptor 3 and IFNγ) and promoted TH2-like polarization (increased expression of GATA-3, CCR4 and IL4). TH1-like Treg cells limited CD8+ T cell effector function, proliferation and memory formation during acute and chronic infection. These findings provide fundamental insights into how Treg cells sense inflammatory cues from the environment (such as IFNγ) during viral infection to provide guidance to the effector immune response. This regulatory circuit prevents prolonged immunoinflammatory responses and shapes the quality and quantity of the memory T cell response.

The CCR6/CCL20 axis expands RORγt+ Tregs to protect from glomerulonephritis

Previous studies have identified a unique Treg population, which expresses the Th17 characteristic transcription factor RORγt. These RORγt+ Tregs possess enhanced immunosuppressive capacity, which endows them with great therapeutic potential. However, as a caveat, they are also capable of secreting pro-inflammatory IL-17A. Since the sum function of RORγt+ Tregs in glomerulonephritis (GN) remains unknown, we studied the effects of their absence. Purified CD4+ T cell populations, containing or lacking RORγt+ Tregs, were transferred into immunocompromised RAG1 knockout mice and the nephrotoxic nephritis model of GN was induced. Absence of RORγt+ Tregs significantly aggravated kidney injury, demonstrating overall kidney-protective properties. Analyses of immune responses showed that RORγt+ Tregs were broadly immunosuppressive with no preference for a particular type of T cell response. Further characterization revealed a distinct functional and transcriptional profile, including enhanced production of IL-10. Expression of the chemokine receptor CCR6 marked a particularly potent subset, whose absence significantly worsened GN. As an underlying mechanism, we found that chemokine CCL20 acting through receptor CCR6 signaling mediated expansion and activation of RORγt+ Tregs. Finally, we also detected an increase of CCR6+ Tregs in kidney biopsies, as well as enhanced secretion of chemokine CCL20 in 21 patients with anti-neutrophil cytoplasmic antibody associated GN compared to that of 31 healthy living donors, indicating clinical relevance. Thus, our data characterize RORγt+ Tregs as anti-inflammatory mediators of GN and identify them as promising target for Treg directed therapies.

CCL5/CCR5-mediated peripheral inflammation exacerbates blood‒brain barrier disruption after intracerebral hemorrhage in mice

BackgroundOwing to metabolic disequilibrium and immune suppression, intracerebral hemorrhage (ICH) patients are prone to infections; according to a recent global analysis of stroke cases, approximately 10 million new-onset ICH patients had experienced concurrent infection. However, the intrinsic mechanisms underlying the effects of infection related peripheral inflammation after ICH remain unclear.MethodsLipopolysaccharide (LPS) was intraperitoneally injected into ICH model mice to induce peripheral inflammation. Neurobehavioral deficits, blood‒brain barrier (BBB) disruption, and the expression of CCR5, JAK2, STAT3, and MMP9 were evaluated after treatment with recombinant CCL5 (rCCL5) (a CCR5 ligand), maraviroc (MVC) (an FDA-approved selective CCR5 antagonist), or JAK2 CRISPR plasmids.ResultsOur study revealed that severe peripheral inflammation increased CCL5/CCR5 axis activation in multiple inflammatory cell types, including microglia, astrocytes, and monocytes, and aggravated BBB disruption and neurobehavioral dysfunction after ICH, possibly in part through the JAK2/STAT3 signaling pathway.ConclusionsCCR5 might be a potential target for the clinical treatment of infection-induced exacerbation of BBB disruption following ICH.

Implications of LAG3 and CTLA4 immune checkpoints beyond PD-1/PD-L1 as a potential target in determining the prognosis of uveal melanoma patients

BackgroundResponse rate of PD-1/PD-L1 immunotherapeutic blockade agents in uveal melanoma (UM) is poor. Lymphocyte activation gene 3 (LAG3) and cytotoxic T-lymphocyte-associated protein 4 (CTLA4) are the two promising immune checkpoint...

Enhanced TLR5-denpendent migration and activation of antigen-loaded airway dendritic cells by flagellin.

Toll-like receptor 5 (TLR5) agonist flagellin is an effective mucosal adjuvant via intranasal (i.n.) administration. Previous studies demonstrated that the mucosal adjuvanticity of flagellin depends on TLR5 signaling of airway epithelial cells. Since dendritic cells (DCs) play a central role in antigen sensitization and the initiation of primary immune responses, we wondered how DCs were modulated by the intranasally administrated flagellin. In this study, a mouse model of i.n. immunization with ovalbumin (OVA), a model antigen, in the presence or absence of flagellin was utilized. We found that nasal administration of flagellin enhanced the coadministered antigen specific antibody responses, and T cell clonal expansion in a TLR5-dependent manner. However, neither the entering of flagellin to nasal lamina propria, nor the uptake of coadministered antigen by nasal resident DCs was associated with TLR5 signaling. In contrary, migration of antigen-loaded DCs from the nasal cavity to the cervical lymph nodes (CLN) and activation of DC in the CLN were both enhanced through TLR5 signaling. Furthermore, for the DCs, flagellin enhanced the expression of CCR7, which was pivotal for DCs in the priming site migrating to draining lymph nodes. Interestingly, the migration, activation and chemokine receptor expression levels of antigen-loaded DC were all significantly higher than that of bystander DCs. In conclusion, intranasally administrated flagellin enhanced TLR5-dependent antigen loaded DCs' migration and activation, but not antigen uptake.

CCL5 is a potential bridge between type 1 and type 2 inflammation in asthma

Type 1 (T1) inflammation (marked by IFN-γ expression) is now consistently identified in subsets of asthma cohorts, but how it contributes to disease remains unclear. We sought to understand the role of CCL5 in asthmatic T1 inflammation and how it interacts with both T1 and type 2 (T2) inflammation. CCL5, CXCL9, and CXCL10 messenger RNA expression from sputum bulk RNA sequencing, as well as clinical and inflammatory data were obtained from the Severe Asthma Research Program III (SARP III). CCL5 and IFNG expression from bronchoalveolar lavage cell bulk RNA sequencing was obtained from the Immune Mechanisms in Severe Asthma (IMSA) cohort and expression related to previously identified immune cell profiles. The role of CCL5 in tissue-resident memory T-cell (TRM) reactivation was evaluated in a T1high murine severe asthma model. Sputum CCL5 expression strongly correlated with T1 chemokines (P< .001 for CXCL9 and CXCL10), consistent with a role in T1 inflammation. CCL5high participants had greater fractional exhaled nitric oxide (P= .009), blood eosinophils (P<.001), and sputum eosinophils (P= .001) in addition to sputum neutrophils (P= .001). Increased CCL5 bronchoalveolar lavage expression was unique to a previously described T1high/T2variable/lymphocytic patient group in the IMSA cohort, with IFNG trending with worsening lung obstruction only in this group (P= .083). In a murine model, high expression of the CCL5 receptor CCR5 was observed in TRMs and was consistent with a T1 signature. Arole for CCL5 in TRM activation was supported by the ability of the CCR5 inhibitor maraviroc to blunt reactivation. CCL5 appears to contribute to TRM-related T1 neutrophilic inflammation in asthma while paradoxically also correlating with T2 inflammation and with sputum eosinophilia.

LncRNA 8244-ssc-miR-320-CCR7 Regulates IFN-β during SVA Infecting PK-15 Cells.

Seneca Valley virus (SVV), a member of the Picornaviridae family, is an oncolytic RNA virus that can cause idiopathic vesicular disease and increase mortality in newborn piglets. Although research on the pathogenic characteristics, epidemiology, pathogenic mechanism, and clinical diagnosis of SVA has increased due to its emergence and prevalence, the interaction between SVA and its host lncRNA has not been fully studied. This study used qualcomm sequencing to analyze differentially expressed lncRNAs and found that during SVA infection, lncRNA 8244 was significantly down-regulated in both PK-15 cells and piglets. Further analysis through quantitative real-time PCR and dual luciferase experiments demonstrated that lncRNA8244 could compete with ssc-miR-320 to regulate the expression of CCR7. The lncRNA824-ssc-miR-320-CCR7 axis activated the TLR-mediated signaling pathway, which recognized viral molecules and induced the expression of IFN-β. These findings provide new insight into the interaction between lncRNA and SVA infection, which could lead to a better understanding of SVA pathogenesis and contribute to the prevention and control of SVA disease.

CCR5∆32 and SDF1 3'A: Gene Variants, Expression and Influence on Biological Markers for the Clinical Progression to AIDS among HIV-1 Virus Controllers in a Mixed Population of the Amazon Region of Brazil.

CCR5Δ32 and SDF1-3'A polymorphisms were investigated in a cohort of viremia controllers, without the use of therapy, along with their influence on CD4+ T lymphocytes (TLs), CD8+ TLs, and plasma viral load (VL). The samples were analyzed from 32 HIV-1-infected individuals classified as viremia controllers 1 and 2 and viremia non-controllers, from both sexes, mostly heterosexuals, paired with 300 individuals from a control group. CCR5∆32 polymorphism was identified by PCR amplification of a fragment of 189 bp for the wild-type allele and 157 bp for the allele with the ∆32 deletion. SDF1-3'A polymorphism was identified by PCR, followed by enzymatic digestion (restriction fragment length polymorphism) with the Msp I enzyme. The relative quantification of gene expression was performed by real-time PCR. The distribution of allele and genotype frequencies did not show significant differences between the groups. The gene expression of CCR5 and SDF1 was not different between the profiles of AIDS progression. There was no significant correlation between the progression markers (CD4+ TL/CD8+ TL and VL) and the CCR5∆32 polymorphism carrier status. The 3'A allele variant was associated with a marked loss of CD4+ TLs and a higher plasma VL. Neither CCR5∆32 nor SDF1-3'A was associated with viremia control or the controlling phenotype.

JAK inhibition ameliorates bone destruction by simultaneously targeting mature osteoclasts and their precursors

BackgroundRheumatoid arthritis (RA) is characterized by chronic inflammation and resultant cartilage/bone destruction because of aberrantly activated osteoclasts. Recently, novel treatments with several Janus kinase (JAK) inhibitors have been shown to successfully ameliorate arthritis-related inflammation and bone erosion, although their mechanisms of action for limiting bone destruction remain unclear. Here, we examined the effects of a JAK inhibitor on mature osteoclasts and their precursors by intravital multiphoton imaging.MethodsInflammatory bone destruction was induced by local injection of lipopolysaccharides into transgenic mice carrying reporters for mature osteoclasts or their precursors. Mice were treated with the JAK inhibitor, ABT-317, which selectively inhibits the activation of JAK1, and then subjected to intravital imaging with multiphoton microscopy. We also used RNA sequencing (RNA-Seq) analysis to investigate the molecular mechanism underlying the effects of the JAK inhibitor on osteoclasts.ResultsThe JAK inhibitor, ABT-317, suppressed bone resorption by blocking the function of mature osteoclasts and by targeting the migratory behaviors of osteoclast precursors to the bone surface. Further exhaustive RNA-Seq analysis demonstrated that Ccr1 expression on osteoclast precursors was suppressed in the JAK inhibitor-treated mice; the CCR1 antagonist, J-113863, altered the migratory behaviors of osteoclast precursors, which led to the inhibition of bone destruction under inflammatory conditions.ConclusionsThis is the first study to determine the pharmacological actions by which a JAK inhibitor blocks bone destruction under inflammatory conditions; this inhibition is beneficial because of its dual effects on both mature osteoclasts and immature osteoclast precursors.

Abstract P2-26-15: CCR7 expression in Inflammatory breast cancer

Abstract Background: Inflammatory breast cancer is an aggressive breast cancer characterized by florid congestion of lymphovascular spaces by tumor emboli. CCR7 is an immune cell receptor that mediates traffic of immune cells into lymphatics that can be expressed on tumor cells. An RNA-seq screen of tumor promoting mammary glands in mice identified CCR7 as an upregulated signal in mammary glands that promoted IBC-like skin invasion. We examined the expression of CCR7 in IBC cell lines and patients to determine the prevalence of this receptor in IBC. Methods: Protein lysates from IBC and non-IBC cell lines were subjected to immunoblotting using anti CCR7 (R&amp;D systems). An IBC tissue microarray from post-chemotherapy mastectomy specimens of 39 patients, each with three replicates was subjected to immunohistochemical staining for CCR7 (Invitrogen, catalog number MA5-31992) performed using a Leica Bond RX autostainer with an incubation time of 60 minutes at 1:15,000 after 20 minutes of heat-induced antigen retrieval at pH 6.0. In 15 cases there were no tumor cells observed in the cores. Staining was scored by an expert breast pathologist for intensity and percent tumor stained. Staining patterns were scored. Descriptive statistics were examined for representation by receptor subtype. Results: CCR7 staining was strong in all lines examined including MCF7, SUM149, SUM190, MDA-IBC, and SUM159. Among 24 IBC patient cores with tumor in the tissue, 23 (96%) expressed CCR7 in tumor, 15 with complete membranous staining and 9 with incomplete membranous staining. In one case with LVSI the emboli were CCR7 positive. Among the 24 positive cases, 22 were 3+ intensity while two were 2+. Nine CCR7+ cases were estrogen receptor (ER) ER+, 8 ER-, and 6 unknown. HER2 status is pending. Conclusions: CCR7 expression is present across tumor subtypes in IBC cell lines and in both ER+ and ER- patient tumors. Given developing novel pharmacologic targeting of CCR7, this target warrants further investigation in IBC and other breast cancers. Citation Format: Wintana Balema, Savitri Krishnamurthy, Alison Lawrence, Megan Rodriguez, Richard Larson, Natalie Fowlkes, Naoto T. Ueno, Wendy Woodward. CCR7 expression in Inflammatory breast cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P2-26-15.

Identification of CCR2 as a hub in septic myocardial injury and cardioprotection of silibinin.

Myocardial injury is a serious complication of sepsis associated with high morbidity and mortality. Our previous work has confirmed that silibinin (SIL) alleviates septic myocardial injury, but the specific molecular mechanism has not been fully elucidated. This study aimed to identify its potential targets through network pharmacology combined with experimental verification. Firstly, a total of 29 overlapping genes between sepsis and SIL targets were obtained from RNA-seq analysis and the known databases. Subsequently, KEGG and GO analysis showed that these genes were enriched in immune response and cytokine-cytokine receptor interaction pathways. Notably, CCR2 was identified as an important candidate hub by protein-protein interaction analysis and molecular docking approach. In vivo experiments showed that SIL treatment significantly improved survival rate and cardiac function in septic mice, accompanied by decreased CCR2 expression. Moreover, in vitro experiments obtained the similar results. Especially, CCR2 siRNA attenuated inflammation response. In conclusion, this study systematically elucidated the key target of SIL in the treatment of septic myocardial injury. These findings provide valuable insights into the targets of sepsis and offer new avenues for exploring drug effect systematically.

Phenotypic and functional characterization of pharmacologically expanded Vγ9Vδ2 T cells in pigtail macaques

While gaining interest as treatment for cancer and infectious disease, the clinical efficacy of Vγ9Vδ2T cell-based immunotherapeutics has to date been limited. An improved understanding of γδ Tcell heterogeneity across lymphoid and non-lymphoid tissues, before and after pharmacological expansion, is required. Here, we describe the phenotype and tissue distribution of Vγ9Vδ2T cells at steady state and following invivo pharmacological expansion in pigtail macaques. Intravenous phosphoantigen administration with subcutaneous rhIL-2 drove robust expansion of Vγ9Vδ2T cells in blood and pulmonary mucosa, while expansion was confined to the pulmonary mucosa following intratracheal antigen administration. Peripheral blood Vγ9Vδ2T cell expansion was polyclonal, and associated with a significant loss of CCR6 expression due to IL-2-mediated receptor downregulation. Overall, we show the tissue distribution and phenotype of invivo pharmacologically expanded Vγ9Vδ2T cells can be altered based on the antigen administration route, with implications for tissue trafficking and the clinical efficacy of Vγ9Vδ2T cell immunotherapeutics.

Phenotypic diversity of human adipose tissue-resident NK cells in obesity.

Natural killer (NK) cells have emerged as key mediators of obesity-related adipose tissue inflammation. However, the phenotype of NK cell subsets residing in human adipose tissue are poorly defined, preventing a detailed understanding of their role in metabolic disorders. In this study, we applied multicolor flow cytometry to characterize CD56bright and CD56dim NK cells in blood and adipose tissue depots in individuals with obesity and identified surface proteins enriched on adipose tissue-resident CD56bright NK cells. Particularly, we found that adipose tissue harbored clusters of tissue-resident CD56bright NK cells signatured by the expression of CD26, CCR5 and CD63, possibly reflecting an adaptation to the microenvironment. Together, our findings provide broad insights into the identity of NK cells in blood and adipose tissue in relation to obesity.

Analysis of intervertebral disc CCR6 and IL-6 gene levels with short-term postoperative low back pain after spinal fusion in lumbar degenerative disease.

Previous studies have reported that specific pro-inflammatory cytokines or chemokines are more highly expressed in painful than in nonpainful intervertebral discs (IVDs). However, few studies have investigated their correlation with postsurgical outcomes or the relationship between postoperative pain and inflammatory cytokines in IVDs. Thus, the present study examined the correlation among the gene expression levels of pro-inflammatory cytokines and chemokines in IVD tissues removed during surgery and low back pain (LBP), leg pain (LP), and leg numbness (LN) at one year after spinal fusion surgery in patients with a lumbar degenerative disease (LDD). Chemokine and cytokine gene expression levels were measured in IVD samples from 48 patients with LDD. The associations between chemokine and cytokine gene expression levels and pain intensity (numeric rating scale [NRS]) were also analyzed. A correlation analysis was performed between gene expression in each IVD and preoperative and postoperative pain intensity. In the preoperative analysis, CCR6 was associated with NRSLBP (r = -0.291, P = 0.045). Postoperative pain analysis revealed correlations between postoperative NRSLBP and CCR6 (r = -0.328, P = 0.023) and between postoperative NRSLBP and IL-6 (r = -0.382, P = 0.007). Furthermore, patients with high postoperative LBP intensity (NRSLBP ≥ 7) also had high LBP intensity (NRSLBP ≥ 6) before surgery, and a correlation was observed (r = 0.418, P = 0.003). None of the gene mRNAs correlated with NRSLP or NRSLN, respectively. CCR6 and IL-6 gene expression in the IVD was associated with postoperative LBP intensity and may indicate a need for postoperative pain management.