Recruitment Of Effector Cells Research Articles

CXCR2 perturbation promotes Staphylococcus aureus implant-associated infection.

Introduction. Staphylococcus aureus is the leading cause of acute medical implant infections, representing a significant modern medical concern. The success of S. aureus as a pathogen in these cases resides in its arsenal of virulence factors, resistance to multiple antimicrobials, mechanisms of immune modulation, and ability to rapidly form biofilms associated with implant surfaces. S. aureus device-associated, biofilm-mediated infections are often persistent and notoriously difficult to treat, skewing innate immune responses to promote chronic reoccurring infections. While relatively little is known of the role neutrophils play in response to acute S. aureus biofilm infections, these effector cells must be efficiently recruited to sites of infection via directed chemotaxis. Here we investigate the effects of modulating CXC chemokine receptor 2 (CXCR2) activity, predominantly expressed on neutrophils, during S. aureus implant-associated infection.Hypothesis. We hypothesize that modulation of CXCR2 expression and/or signalling activities during S. aureus infection, and thus neutrophil recruitment, extravasation and antimicrobial activity, will affect infection control and bacterial burdens in a mouse model of implant-associated infection.Aim. This investigation aims to elucidate the impact of altered CXCR2 activity during S. aureus biofilm-mediated infection that may help develop a framework for an effective novel strategy to prevent morbidity and mortality associated with implant infections.Methodology. To examine the role of CXCR2 during S. aureus implant infection, we employed a mouse model of indwelling subcutaneous catheter infection using a community-associated methicillin-resistant S. aureus (MRSA) strain. To assess the role of CXCR2 induction or inhibition during infection, treatment groups received daily intraperitoneal doses of either Lipocalin-2 (Lcn2) or AZD5069, respectively. At the end of the study, catheters and surrounding soft tissues were analysed for bacterial burdens and dissemination, and Cxcr2 transcription within the implant-associated tissues was quantified.Results. Mice treated with Lcn2 developed higher bacterial burdens within the soft tissue surrounding the implant site, which was associated with increased Cxcr2 expression. AZD5069 treatment also resulted in increased implant- and tissues-associated bacterial titres, as well as enhanced Cxcr2 expression.Conclusion. Our results demonstrate that CXCR2 plays an essential role in regulating the severity of S. aureus implant-associated infections. Interestingly, however, perturbation of CXCR2 expression or signalling both resulted in enhanced Cxcr2 transcription and elevated implant-associated bacterial burdens. Thus, CXCR2 appears finely tuned to efficiently recruit effector cells and mediate control of S. aureus biofilm-mediated infection.

Endothelial cells in tumor microenvironment: insights and perspectives.

The tumor microenvironment is a highly complex and dynamic mixture of cell types, including tumor, immune and endothelial cells (ECs), soluble factors (cytokines, chemokines, and growth factors), blood vessels and extracellular matrix. Within this complex network, ECs are not only relevant for controlling blood fluidity and permeability, and orchestrating tumor angiogenesis but also for regulating the antitumor immune response. Lining the luminal side of vessels, ECs check the passage of molecules into the tumor compartment, regulate cellular transmigration, and interact with both circulating pathogens and innate and adaptive immune cells. Thus, they represent a first-line defense system that participates in immune responses. Tumor-associated ECs are involved in T cell priming, activation, and proliferation by acting as semi-professional antigen presenting cells. Thus, targeting ECs may assist in improving antitumor immune cell functions. Moreover, tumor-associated ECs contribute to the development at the tumor site of tertiary lymphoid structures, which have recently been associated with enhanced response to immune checkpoint inhibitors (ICI). When compared to normal ECs, tumor-associated ECs are abnormal in terms of phenotype, genetic expression profile, and functions. They are characterized by high proliferative potential and the ability to activate immunosuppressive mechanisms that support tumor progression and metastatic dissemination. A complete phenotypic and functional characterization of tumor-associated ECs could be helpful to clarify their complex role within the tumor microenvironment and to identify EC specific drug targets to improve cancer therapy. The emerging therapeutic strategies based on the combination of anti-angiogenic treatments with immunotherapy strategies, including ICI, CAR T cells and bispecific antibodies aim to impact both ECs and immune cells to block angiogenesis and at the same time to increase recruitment and activation of effector cells within the tumor.

Enhanced potency of immunotherapy against B-cell precursor acute lymphoblastic leukemia by combination of an Fc-engineered CD19 antibody and CD47 blockade.

CD19-directed immunotherapy has become a cornerstone in the therapy of B-cell precursor acute lymphoblastic leukemia (BCP-ALL). CD19-directed cellular and antibody-based therapeutics have entered therapy of primary and relapsed disease and contributed to improved outcomes in relapsed disease and lower therapy toxicity. However, efficacy remains limited in many cases due to a lack of therapy response, short remission phases, or antigen escape. Here, BCP-ALL cell lines, patient-derived xenograft (PDX) samples, human macrophages, and an in vivo transplantation model in NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mice were used to examine the therapeutic potency of a CD19 antibody Fc-engineered for improved effector cell recruitment (CD19-DE) and antibody-dependent cellular phagocytosis (ADCP), in combination with a novel modified CD47 antibody (Hu5F9-IgG2σ). For the in vivo model, only samples refractory to CD19-DE monotherapy were chosen. Hu5F9-IgG2σ enhanced ADCP by CD19-DE in various BCP-ALL cell line models with varying CD19 surface expression and cytogenetic backgrounds, two of which contained the KMT2A-AFF1 fusion. Also, the antibody combination was efficient in inducing ADCP by human macrophages in pediatric PDX samples with and adult samples with and without KMT2A-rearrangement in vitro. In a randomized phase 2-like PDX trial using seven KMT2A-rearranged BCP-ALL samples in NSG mice, the CD19/CD47 antibody combination proved highly efficient. Our findings support that the efficacy of Fc-engineered CD19 antibodies may be substantially enhanced by a combination with CD47 blockade. This suggests that the combination may be a promising therapy option for BCP-ALL, especially in relapsed patients and/or patients refractory to CD19-directed therapy.

A novel complement-fixing IgM antibody targeting GPC1 as a useful immunotherapeutic strategy for the treatment of pancreatic ductal adenocarcinoma

BackgroundPancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive cancers with a very low survival rate at 5 years. The use of chemotherapeutic agents results in only modest prolongation of survival and is generally associated with the occurrence of toxicity effects. Antibody-based immunotherapy has been proposed for the treatment of PDAC, but its efficacy has so far proved limited. The proteoglycan glypican-1 (GPC1) may be a useful immunotherapeutic target because it is highly expressed on the surface of PDAC cells, whereas it is not expressed or is expressed at very low levels in benign neoplastic lesions, chronic pancreatitis, and normal adult tissues. Here, we developed and characterized a specific mouse IgM antibody (AT101) targeting GPC1.MethodsWe developed a mouse monoclonal antibody of the IgM class directed against an epitope of GPC1 in close proximity to the cell membrane. For this purpose, a 46 amino acid long peptide of the C-terminal region was used to immunize mice by an in-vivo electroporation protocol followed by serum titer and hybridoma formation.ResultsThe ability of AT101 to bind the GPC1 protein was demonstrated by ELISA, and by flow cytometry and immunofluorescence analysis in the GPC1-expressing "PDAC-like" BXPC3 cell line. In-vivo experiments in the BXPC3 xenograft model showed that AT101 was able to bind GPC1 on the cell surface and accumulate in the BXPC3 tumor masses. Ex-vivo analyses of BXPC3 tumor masses showed that AT101 was able to recruit immunological effectors (complement system components, NK cells, macrophages) to the tumor site and damage PDAC tumor tissue. In-vivo treatment with AT101 reduced tumor growth and prolonged survival of mice with BXPC3 tumor (p < 0.0001).ConclusionsThese results indicate that AT101, an IgM specific for an epitope of GPC1 close to PDAC cell surface, is a promising immunotherapeutic agent for GPC1-expressing PDAC, being able to selectively activate the complement system and recruit effector cells in the tumor microenvironment, thus allowing to reduce tumor mass growth and improve survival in treated mice.

Proteomic Profiling of Different Antiphospholipid Antibody-Positive Phenotypes: Results from Antiphospholipid Syndrome Alliance for Clinical Trials and International Networking (APS ACTION) Registry

Background. Antiphospholipid syndrome (APS) is an autoimmune disease with thrombotic and obstetric complications arising via a model of immunothrombosis. Patients may present with a spectrum of phenotypes, including thrombotic (tAPS), obstetric (oAPS), or catastrophic/microvascular APS (C/MAPS), while others may have antiphospholipid antibodies (aPL) without disease manifestations. The mechanisms underlying the development of these diverse phenotypes remain uncertain. Proteomic profiling was used in other thrombotic and microvascular disorders to highlight potential mechanisms of disease pathogenesis and may have a role in understanding the pathophysiology of APS. We performed multiplex plasma proteomic profiling in aPL-positive patients with different clinical phenotypes to gain a greater understanding of potential immunothrombotic mechanisms in the pathogenesis of APS. Methods. We utilized samples from APS ACTION Registry. The inclusion criteria were positive aPL per Updated Sapporo Classification Criteria tested within one year prior to the enrollment. Multiplex proteomic profiling measuring approximately 7,000 unique proteins (SomaLogic; Boulder, CO, USA) was performed on 40 primary aPL-positive patient plasma samples (10 each of tAPS with/without oAPS, oAPS only, C/MAPS, and positive aPL without APS classification), and 10 of healthy controls. Differentially abundant proteins among all phenotypic groups and in pairwise comparisons were determined by applying ANOVA and t-tests to log-normalized data, respectively, with p-values &amp;lt;0.05 considered statistically significant (Qlucore Omics Explorer, Lund, Sweden). Tests were adjusted for false discovery (q&amp;lt;0.1) to minimize the likelihood of false positives. Pathway enrichment analysis was performed using Metascape (https://metascape.org) and Ingenuity IPA (Qiagen, Venlo, Netherlands) platforms. Results. The median age of patients was 48 years; 30% were men, 70% had triple aPL-positivity, and no one had a concurrent diagnosis of lupus. A set of concordant and differentially abundant proteins clustered patients with 4 APS clinical phenotypes and controls (Figure A) with a high statistical significance (p&amp;lt;0.0007) and a high false discovery confidence (q&amp;lt;0.05). Proteins in the identified set belonged to several highly enriched pathways such as neutrophil degranulation (p&amp;lt;10 -10), humoral immune response (p&amp;lt;10 -9), coagulation (p&amp;lt;10 -6), and alternative complement (p&amp;lt;10 -5) [data not shown]. Pathway enrichment analysis of protein sets identified in pairwise comparisons of APS phenotypes (aPL vs C/MAPS, aPL vs tAPS, tAPS vs C/MAPS) revealed involvement of several common pathways associated with inflammation, collagen and fibroblast signaling, cellular and cytoskeletal activation, humoral immune response, myeloid and effector cell differentiation and recruitment, and immunothrombosis such as Pathogen-Induced Cytokine Storm, Neutrophil Extracellular Trap (NET), and collagen-induced platelet-activating GP6 signaling (Figure B). For several pathways, the measure of activation related to increased or predicted to be increased proteins annotated within a particular pathway positively correlated with the clinical “distance” between APS subtypes, from the most clinically related C/MAPS vs tAPS to the most distant aPL vs C/MAPS suggesting an “evolution” from one phenotype to a more severe in terms of activation of specific pathways. Specifically notable is the higher activation of the NET signaling pathway in the aPL than in C/MAPS phenotype suggesting that the presence of aPL antibodies activates this immunothrombotic pathway. Conclusions. Plasma proteome of APS subtypes is characterized by alteration in several cellular processes, particularly receptor signaling, signal transduction, regulation of cellular differentiation, neutrophil, complement, coagulation and cytokine activation notable in all individuals with aPL-positivity. Pathways activated in the non-thrombotic (aPL) phenotype and escalating activation of several pathways from non-thrombotic to the most thrombotic (C/MAPS) phenotype provide important data for the understanding of APS pathogenesis and informing the model of APS-related immunothrombosis.

Agarose spot migration assay to measure the chemoattractant potential of extracellular vesicles: applications in regenerative medicine and cancer metastasis

BackgroundThe recruitment of effector cells is one of the novel functions described for extracellular vesicles (EVs) that needs further study. For instance, cell recruitment by mesenchymal stromal cell derived-EVs (MSC-EVs) is one of the features by which MSC-EVs may induce regeneration and ameliorate tissue injury. On the other hand, increasing evidence suggests that cancer EVs play an important role in the preparation of the pre-metastatic niche (PMN) by recruiting their primary tumour cells. Understanding and measuring the potential of MSC-EVs or cancer-EVs to induce cell migration and recruitment is essential for cell-free therapeutic approaches and/or for a better knowledge of cancer metastasis, respectively. In this context, classical in vitro migration assays do not completely mimic the potential situation by which EVs exert their chemotactic capacity.ResultsWe adapted an agarose spot migration assay as an in vitro system to evaluate the cell recruitment capacity of locally delivered or localized EVs. Cell migration was tracked for 12 h or 48 h, respectively. Thereafter, endpoint migration images and time-lapse videos were analysed to quantify several parameters aiming to determine the migration of cells to either MSC-EV or pro-metastatic EV. The number of cells contained inside the agarose spots, the migration distance, the area occupied by cells, the directionality of the cell movement, and the Euclidean distance were measured. This multi-parametric evaluation revealed the potential of different MSC-EV preparations to recruit endothelial cells and to detect an enhanced recruitment capacity of highly metastatic PC3-derived EVs (PC3-EVs) compared to low-metastatic LNCaP-EVs in a tumour cell-specific manner.ConclusionsOverall, this agarose spot migration assay may offer a diversity of measurements and migration settings not provided by classical migration assays and reveal its potential use in the EV field in two different contexts with recruitment in common: regeneration and cancer metastasis.

Dendritic cells regulate innate cell trafficking and lymph node organization during inflammation

Abstract Organization of immune cells within lymph nodes (LNs) is critical for effective host defense. During vaccination or infection, layers of innate cell activity in the outer periphery of the LN provide immediate protection, while antigen-presenting dendritic cells (DCs) migrate into the T cell zone (TZ), located deeper in the LN, to initiate later-stage adaptive responses. Using mouse models of vaccination and infection, we now find that DC activation and repositioning into the TZ is tightly associated with localized recruitment of multiple innate effector cells, including neutrophils, monocytes, and NK cells, which also rapidly infiltrate the TZ. As activated DCs migrated towards the TZ, they passed by the high endothelial venules (HEVs), where they expressed multiple inflammatory chemokines and high levels of the integrin ligand, ICAM-1, generating a chemokine- and adhesion molecule-rich matrix ripe for innate cell migration. Depletion of DCs, or conditional ablation of ICAM-1, significantly impaired innate cell trafficking into LNs, and cells which did enter remained largely trapped near the HEVs and were less able to infiltrate the TZ. While essential for innate defense, neutrophil influx also led to large-scale disruption of the TZ and impaired early T cell activation. However, over time this was controlled by DCs and DC-recruited monocytes, which efferocytosed the infiltrating neutrophils and cleared the TZ to allow productive adaptive responses to occur. Thus, in addition to their classical roles in antigen presentation, DCs orchestrate innate cell recruitment and activation across the entire LN, to promote both effective innate defense as well as rapid restoration of LN architecture for the generation of adaptive immunity. Supported by grant from NIH (1F31AI161316-01).

Interaction of mediators and effector cells in cashew nut-induced anaphylaxis

BackgroundThe underlying mechanisms of an immediate food-induced allergic reaction involve mast cell degranulation and recruitment of other effector cells, such as lymphocytes, eosinophils, and basophils. How the interaction of various mediators and cells results in anaphylaxis is not fully understood. ObjectiveTo evaluate changes in platelet-activating factor (PAF), platelet-activating factor acetylhydrolase (PAF-AH), tryptase, eosinophils, basophils, and eosinophil cationic protein (ECP) in cashew nut-induced anaphylaxis. MethodsOpen cashew nut challenges were performed on 106 children (aged 1-16 years), sensitized to cashew nut, with earlier allergic reaction to cashew nut or no known exposure. PAF, PAF-AH, tryptase, ECP, eosinophils, and basophils were measured at 4 time points. ResultsOf 72 challenges with positive results, 34 were defined as anaphylactic. Eosinophil count decreased progressively during an anaphylactic reaction at all 4 time points (P < .005*) compared with baseline. Although significant PAF elevation was observed 1 hour from moderate-to-severe reaction (P = .04*), PAF seemed to peak especially in anaphylaxis but did not achieve statistical significance. PAF peak ratio (peak PAF/baseline PAF) was significantly greater in anaphylactic reactions compared with the no-anaphylaxis group (P = .008*). Maximal percentage change in eosinophils revealed negative correlation to severity score and PAF peak ratio (Spearman's rho −0.424 and −0.516, respectively). Basophils decreased significantly in moderate-to-severe reactions and in anaphylaxis (P < .05*) compared with baseline. Delta-tryptase (peak tryptase minus baseline) did not differ significantly between anaphylaxis and the no-anaphylaxis subgroups (P = .05). ConclusionPAF is a specific anaphylaxis biomarker. Marked decline of eosinophils during anaphylaxis may be related to robust secretion of PAF reflecting migration of eosinophils to target tissues.

Effect of stimulator of interferon genes (STING) signaling on radiation-induced chemokine expression in human osteosarcoma cells.

Cancer cell-intrinsic mechanisms affecting radiation immunomodulation could be exploited to optimize systemic effects of localized radiation. Radiation-induced DNA damage is sensed by cyclic GMP-AMP synthase (cGAS), which ultimately activates stimulator of interferon (IFN) genes (STING). Resultant expression of soluble mediators such as CCL5 and CXCL10 can facilitate recruitment of dendritic cells and immune effector cells into the tumor. The primary objectives of this study were to determine the baseline expression levels of cGAS and STING in OSA cells and evaluate the dependence of OSA cells on STING signaling for eliciting radiation-induced expression of CCL5 and CXCL10. cGAS and STING expression, and CCL5/CXCL10 expression in control cells, STING-agonist treated cells, and cells treated with 5 Gy ionizing radiation were assessed utilizing RTqPCR, Western blot, and ELISA. U2OS and SAOS-2 OSA cells were deficient in STING relative to human osteoblasts (hObs), while SAOS-2-LM6 and MG63 OSA cells expressed equivalent amounts of STING compared to hObs. A dependence on baseline or induced STING expression was observed for STING-agonist, and radiation-induced, expression of CCL5 and CXCL10. This finding was confirmed by performing siRNA knockdown of STING in MG63 cells. These results show that STING signaling is necessary for radiation-induced expression of CCL5 and CXCL10 in OSA cells. Additional studies are necessary to determine whether STING expression in OSA cells in vivo alters immune cell infiltrates after radiation exposure. These data may also have implications for other potentially STING-dependent characteristics such as resistance to oncolytic virus cytotoxicity.

Mast cells selectively produce inflammatory mediators and impact the early response to Chlamydia reproductive tract infection.

Chlamydia trachomatis (C. trachomatis) is a Gram-negative obligate intracellular bacterium that causes reproductive tract complications in women, including ectopic pregnancies and tubal factor infertility. We hypothesized that mast cells, which are common at mucosal barriers, may contribute to responses to Chlamydia infection and aimed to define human mast cell responses to C. trachomatis. Human cord blood-derived mast cells (CBMCs) were exposed to C. trachomatis to assess bacterial uptake, mast cell degranulation, gene expression, and production of inflammatory mediators. The role of formyl peptide receptors and Toll-like receptor 2 (TLR2) were investigated using pharmacological inhibitors and soluble TLR2. Mast cell-deficient mice and littermate controls were used to examine the in vivo role of mast cells in influencing the immune response to Chlamydia infection in the female reproductive tract. C. trachomatis bacteria were taken up by human mast cells but did not replicate efficiently inside CBMCs. C. trachomatis-activated mast cells did not degranulate but maintained viability and exhibited cellular activation with homotypic aggregation and upregulation of ICAM-1. However, they significantly enhanced the gene expression of IL1B, CCL3, NFKB1, CXCL8, and IL6. Inflammatory mediators were produced, including TNF, IL-1β, IL-1RA, IL-6, GM-CSF, IL-23, CCL3, CCL5, and CXCL8. Endocytic blockade resulted in reduced gene expression of IL6, IL1B, and CCL3, suggesting C. trachomatis induced mast cell activation in both extracellular and intracellular locations. The IL-6 response to C. trachomatis was reduced when CBMCs were treated with C. trachomatis coated with soluble TLR2. Mast cells derived from TLR2-deficient mice also demonstrated a reduced IL-6 response to C. muridarum. Five days following C. muridarum infection, mast cell-deficient mice showed attenuated CXCL2 production and significantly reduced numbers of neutrophils, eosinophils, and B cells in the reproductive tract when compared with mast cell-containing littermates. Taken together, these data demonstrate that mast cells are reactive to Chlamydia spp. through multiple mechanisms that include TLR2-dependent pathways. Mast cells also play an important role in shaping in vivo immune responses in Chlamydia reproductive tract infection through both effector cell recruitment and modification of the chemokine microenvironment.

Abstract 4894: The anti-ROR1 ADC STRO-003 demonstrates immune-modulating properties that may enhance checkpoint blockade

Abstract There is growing evidence that tumor-targeted cytotoxins can also enhance anti-tumor immunity by inducing immunogenic cell death (ICD) in tumor cells and promoting recruitment of immune effector cells. We sought to investigate the immune stimulating potential of STRO-003, an antibody drug conjugate (ADC) composed of an anti-Receptor Orphan Receptor Kinase 1 (ROR1) antibody conjugated to a topoisomerase I targeted exatecan warhead via a cleavable linker. ROR1 is an oncofetal transmembrane receptor with restricted expression in normal tissues. Its overexpression in several cancer indications has been described, including in ovarian, non-small cell lung carcinoma (NSCLC), triple negative breast cancer (TNBC), and hematological malignancies, thus making it an ideal ADC target. We have previously demonstrated potent in vivo activity of STRO-003 in a panel of ROR1-expressing lung cancer patient-derived xenograft (PDX) models. Here we show that the exatecan warhead, SC3386, and STRO-003 ADC induced ICD in vitro as evidenced by presentation of cell-surface calreticulin and release of HMGB1. Consistent with this finding, STRO-003-treated cells elicited monocyte activation in a PBMC co-culture assay. To determine if these immunogenic properties could improve therapeutic efficacy, we evaluated STRO-003 in combination with an immune checkpoint inhibitor in a mouse syngeneic model expressing ROR1. We have demonstrated that STRO-003 in combination with checkpoint inhibition significantly enhances efficacy in a syngeneic mouse model and supports durable anti-tumor immunity. Follow-up vaccination studies were performed to further explore the significance of STRO-003-induced ICD and protective immunity in vivo. These results demonstrate that tumor cells pre-treated with STRO-003 or SC3386 undergo potent immunogenic cell damage which can, in turn, mount protective immunity in vivo. Citation Format: Alice Yam, Helena Kiefel, Andrew McGeehan, Robert Yuan, Sihong Zhou, Dayson Moreira, Indrani Dutta, Xiaofan Li, Cuong Tran, Gang Yin, Kristin Bedard, Trevor Hallam. The anti-ROR1 ADC STRO-003 demonstrates immune-modulating properties that may enhance checkpoint blockade. [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 4894.

Abstract 5139: Chemerin suppresses prostate tumor growth by modulating the recruitment of NK and CD8+ T-cells to tumor microenvironment

Abstract Infiltration of immune cells into the tumor microenvironment (TME) can regulate growth and survival of malignant cells, thereby affecting tumorigenesis and tumor progression. While immune checkpoint inhibitors and agonism of costimulatory molecules can act to stimulate leukocytes, the lack of adequate recruitment of effector cells into TME often results in suboptimal immune responses. Chemerin (retinoic acid receptor responder 2, RARRES2) is an endogenous chemoattractant that is widely expressed by many tissues and recruits innate leukocytes through its receptor, CMKLR1 which is expressed on NK cells, some dendritic cells, and macrophages. Previous studies show that RARRES2 is downregulated across multiple tumor types including prostate cancer. Here, using a syngeneic transgenic adenocarcinoma mouse prostate (TRAMP-C1) model, we show that forced overexpression of RARRES2 by tumor cells results in significant tumor suppression and recruitment of NK and CD8+ T-cells within the TME. Male C57/BL/6 mice were inoculated with either RARRES2-overexpressing, vector control (VC) or mixed (50:50; VC: RARRES2) TRAMP-C1 cells. Chemerin overexpression in TRAMP-C1 cells did not alter their phenotypic behavior in vitro, but significantly suppressed tumor growth in vivo in mice. Furthermore, we observed a significantly higher influx of NK (CD3-CD19-NK1.1+) and CD8+ T-cells (CD3+CD8+) in tumor-infiltrating leukocytes (TILs) from mice with mixed tumors compared with VC tumors. Depletion of NK cells, CD4+, or CD8+ T cells showed that NK cells and CD8+ T-cells, but not CD4+ T-cells, were required for chemerin-dependent suppression of TRAMP-C1 tumor growth. We found significant increases in splenic NK and CD8 T cells, and a concomitant significant decrease in splenic G-MDSC in mice with chemerin-expressing tumors compared to controls, suggesting chemerin expression within the TME has an effect on systemic immune responses. Treatment of control tumors with anti-PD1 checkpoint inhibition did not result in significant tumor suppression, while anti-PD1 treatment of chemerin-expressing tumors significantly reduced tumor growth, doubling the complete response rate in these unresponsive tumors. Thus, for the first time we have demonstrated that increasing chemerin expression within the prostate TME can suppress growth by augmenting recruitment of NK and CD8+ T-cells and has the potential to be combined with checkpoint inhibitors in order to improve response rates in non-responsive tumors. Overall, these data suggest that a tumor-targeted chemerin therapeutic may have potential to treat human prostate cancers. Citation Format: Muhammad A. Saeed, Kevin Kim, Russell K. Pachynski. Chemerin suppresses prostate tumor growth by modulating the recruitment of NK and CD8+ T-cells to tumor microenvironment. [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 5139.

Effects of High-Fat and Low-Carbohydrate Diet Combined with Radiation Therapy on Tumor Microenvironment in LLC Tumor-Bearing Mice

<h3>Purpose/Objective(s)</h3> Metabolic reprogramming of tumor immune cells can regulate immune function. To add a sentence linking tumor metabolism with radioimmunity. In this study, the high heterogeneity and reprogramming of tumor cell metabolism were utilized to explore the effects of high fat and low carbohydrate diet combined with radiotherapy on tumor immune microenvironment of tumor mice bearing LLC from the perspective of tumor microenvironment. <h3>Materials/Methods</h3> Lewis lung cancer (LLC) model was established in female C57BL/6J mice aged 6 to 8 weeks and around 18g, and they were given a high fat and low carbohydrate diet (45F, fat 45.00% of energy) combined with radiotherapy. The body weight, tumor growth, blood glucose and blood ketone (βHB) values, survival time, median survival time and treatment safety of tumor-bearing mice were observed. Immunohistochemistry, multiple immunofluorescence and flow cytometry were used to detect the expression of tumor-associated microangiogenesis molecules (CD34), lymphatic microvessels (LYVE-1), collagen fibers and immune cell phenotypes (CD3, CD4, CD8, Treg and PD1). <h3>Results</h3> High fat and low carbohydrate diet combined with radiotherapy (45F+RT) group had the best tumor control, and the tumor volume was significantly smaller than the control group (p < 0.001), and tumor weight in the combined treatment group (45F+RT) was significantly lower than that in the normal diet (PT, fat 12.11% of energy) and 45F group (p < 0.001); The blood glucose in 45F group was lower than that in PT group, but there was no statistical difference (p < 0.05); It was found that the βHB value in the high-fat and low-carbohydrate diet group was higher than that in the normal diet group. There were statistically significant differences in the second and third weeks (1.00±0.20mmol/L vs 0.63±0.06mmol/L, 0.90±0.17mmol/L vs 0.70±0.10mmol/L, p < 0.05). The median survival time of mice in each group was 38 days in the PT group, 55 days in the PT+RT group and 41 days in the 45F group, but not in the 45F+RT group. Immunohistochemistry and Multiple immunofluorescences showed that CD8 expression was up-regulated and PD1, CD34, LYVE-1 and collagen fiber expression were down-regulated in 45F+RT group. Flow cytometry showed that the proportion of CD8+T cells in 45F+RT group was higher than that in PT group and PT+RT group (p < 0.05) and higher than 45F group, but the difference was not statistically significant (p < 0.05); The proportion of CD4+T cells in PT+RT and 45F+RT groups was higher than that in PT and 45F groups (p < 0.05); The proportion of Treg in T cells in 45F+RT group was lower than that in PT+RT group, but the difference was not statistically significant (p < 0.05). <h3>Conclusion</h3> High fat and low carbohydrate diet combined with radiotherapy can enhance the recruitment and function of immune effector cells in tumor microenvironment and inhibit tumor microangiogenesis, thereby inhibiting tumor growth.

Dupilumab: Advances in the off-label usage of IL4/IL13 antagonist in dermatoses.

Type 2 immune response refers toa complicated series of immune responses characterized by Th2 polarization and Th2 cytokines secretion. The IgE secretion, airway hypersensitivity, and effector cell recruitment (eosinophils, mast cells, basophils) in skin lesion and peripheral blood stream could be upregulated during the activation of type 2 immune response. Th1/Th2 ratio, also referred as Th1/Th2 balance, represent the T lymphocytes immune pattern to a certain degree: Th1-dominated responses are often involved in intracellular infections (e.g., mycobacterium tuberculosis) and autoimmune diseases (e.g., Graves' disease) while Th2-dominated responses are involved in allergic conditions (e.g., atopic dermatitis, eczema), IgE mediated diseases (e.g., urticaria), and fibrotic dermatoses (e.g., keloids). Dupilumab, as one of the most widely applied Th2 cytokine inhibitors, could block the bioactivity of IL-14/IL-13 via competitively binding to the common IL-4Rα subunit shared by IL-4 and IL-13 receptors. In addition to the direct inhibition of type 2 response, dupilumab is also effective in autoimmune and some infectious skin diseases through indirect regulation of type 1 immune response. The pathological mechanism of Th2 responses and advanced clinical application of dupilumab in skin diseases will be summarized and discussed in the review.

751P Preclinical evaluation of SG-3-06686, a positive allosteric modulator of CXCR3

The CXCR3 chemokine system plays a critical role in generating productive anti-tumor immune response at multiple steps. CXCR3 upregulation serves as an important driver for the recruitment of anti-tumorigenic effector cells including CD8+ T cells, NK cells, cDC1s, and Th1 cells from the blood into the tumor microenvironment (TME). Within tumor proximal lymph nodes, efficient CXCR3 signaling contributes to proper CD4 and CD8 T cell positioning to promote the differentiation and expansion of tumor antigen specific T cells.

C-C chemokine receptor 5 and acute graft-versus-host disease.

BackgroundThe C–C chemokine receptor 5 (CCR5) is mainly expressed in a variety of immune cells. It interacts with multiple chemokine ligands that mediate the trafficking and recruitment of effector cells toward sites of inflammation. CCR5 not only plays a critical role in cell growth, activation, differentiation, adhesion, and migration but also participates in the development of acute graft‐versus‐host disease (GVHD) after allogeneic hematopoietic cell transplantation.MethodsThis is a literature review article. The research design method is an evidence‐based rapid review. The present discourse aim is first to scrutinize and assess the available literature on CCR5 and acute GVHD. Standard literature and database searches were implemented, gathered relevant material, and extracted information was then assessed.ResultsCCR5 is a marker of GVHD effector cells, and CCR5 expression is elevated when acute GVHD occurs. CCR5 blockade with maraviroc in clinical trials results in a low incidence of acute GVHD. The immune mechanism includes that CCR5 blockade inhibits donor T cell migration and recruitment toward target organs, reduces the absolute numbers of donor T cells, is capable of slightly suppressing dendritic cell maturation, and reduces the percentage of Th1 and Th17 subsets. CCR5 blockade also inhibits internalization and activation of chemokines, inhibits proliferation and chemotaxis of T cells, and decreases the production of TNF‐α and IFN‐γ. In addition, there may be a form of crosstalk between CCR5 and CCR2. Inconsistently, infusion of CCR5−/− Tregs into lethally irradiated mice significantly increased the infiltration of CD4+ and CD8+ T cells into the liver, resulting in earlier and more severe GVHD.ConclusionThis review indicates that CCR5 plays an important role in pathogenesis and development of acute GVHD. Elucidating its role in different immune cells will aid the development of targeted therapeutic treatments.

Plasticity of monocytes and macrophages in cirrhosis of the liver.

Cirrhosis of the liver is a systemic condition with raising prevalence worldwide. Patients with cirrhosis are highly susceptible to develop bacterial infections leading to acute decompensation and acute-on-chronic liver failure both associated with a high morbidity and mortality and sparse therapeutic options other than transplantation. Mononuclear phagocytes play a central role in innate immune responses and represent a first line of defence against pathogens. Their function includes phagocytosis, killing of bacteria, antigen presentation, cytokine production as well as recruitment and activation of immune effector cells. Liver injury and development of cirrhosis induces activation of liver resident Kupffer cells and recruitment of monocytes to the liver. Damage- and pathogen-associated molecular patterns promote systemic inflammation which involves multiple compartments besides the liver, such as the circulation, gut, peritoneal cavity and others. The function of circulating monocytes and tissue macrophages is severely impaired and worsens along with cirrhosis progression. The underlying mechanisms are complex and incompletely understood. Recent 'omics' technologies help to transform our understanding of cellular diversity and function in health and disease. In this review we point out the current state of knowledge on phenotypical and functional changes of monocytes and macrophages during cirrhosis evolution in different compartments and their role in disease progression. We also discuss the value of potential prognostic markers for cirrhosis-associated immuneparesis, and future immunotherapeutic strategies that may reduce the need for transplantation and death.

Organ-specific host differential gene expression analysis in systemic candidiasis: A systems biology approach

Patients admitted to the hospital with coronavirus disease (COVID-19) are at risk for acquiring mycotic infections in particular Candidemia. Candida albicans (C. albicans) constitutes an important component of the human mycobiome and the most common cause of invasive fungal infections. Invasive yeast infections are gaining interest among the scientific community as a consequence of complications associated with severe COVID-19 infections. Early identification and surveillance for Candida infections is critical for decreasing the COVID-19 mortality. Our current study attempted to understand the molecular-level interactions between the human genes in different organs during systematic candidiasis. Our research findings have shed light on the molecular events that occur during Candidiasis in organs such as the kidney, liver, and spleen. The differentially expressed genes (up and down-regulated) in each organ will aid in designing organ-specific therapeutic protocols for systemic candidiasis. We observed organ-specific immune responses such as the development of the acute phase response in the liver; TGF-pathway and genes involved in lymphocyte activation, and leukocyte proliferation in the kidney. We have also observed that in the kidney, filament production, up-regulation of iron acquisition mechanisms, and metabolic adaptability are aided by the late initiation of innate defense mechanisms, which is likely related to the low number of resident immune cells and the sluggish recruitment of new effector cells. Our findings point to major pathways that play essential roles in specific organs during systemic candidiasis. The hub genes discovered in the study can be used to develop novel drugs for clinical management of Candidiasis.

Abstract 6349: Targeting the CXCR3 pathway with a novel peptide drug candidate mobilizes the immune system to enhance anti-tumor immunity

Abstract Immune-checkpoint inhibitor (ICI) therapy releases the molecular “brakes” on the immune system thereby promoting robust anti-tumor immune responses. However, many patients do not respond to ICI therapy due to development of primary and secondary resistance, and this population represents a large unmet medical need. The critical role of C-X-C motif chemokine receptor 3 (CXCR3) signaling in eliciting an effective response to anti-PD-1 therapy has been recently demonstrated. The CXCR3 chemokine system is instrumental in effector cell recruitment to the tumor and augments intratumoral CD8+ T cell proliferation and function, which are key mechanisms driving anti-tumor immunity and responses to ICI therapy. We used our proprietary peptide discovery platform to identify a unique microbiome-derived peptide, SG-3-00802, from bacterial strains associated with response to anti-PD1 inhibitors in patients with melanoma. We subsequently determined that CXCR3 is the target receptor for SG-3-00802 and demonstrated that SG-3-00802 enhanced the activity of CXCR3 in the presence of its endogenous ligands CXCL9/CXCL10/CXCL11. Optimization of SG-3-00802 pharmacological properties led to the selection of a novel drug development candidate SG-3-00802DC with improved potency and PK properties. Mechanistically, it increases CXCR3 activation by CXCL11 by greater than 10-fold from a nM to a pM range. As a positive allosteric modulator, SG-3-00802DC can potentially alter the conformation of the primary orthosteric binding site of CXCR3 and enhance the binding affinity of CXCL11, causing increased CXCR3 signaling activity, which is known to drive TIL infiltration that improves overall survival in mouse tumor models and cancer patients. Supporting this concept, we demonstrated that SG-3-00802DC showed anti-tumor activity in pre-clinical mouse tumor models, alone and in combination with anti-PD-1, improved overall survival and increased recruitment of CXCR3+ effector cells into the tumor microenvironment. Numerous strategies are currently undergoing clinical evaluation to improve long-lasting disease control in broader patient populations by combining ICIs with approved and novel therapeutic agents and procedures. SG-3-00802DC, with its well validated and unique mechanism of action to safely target the CXCR3-driven anti-tumor immune response offers a novel orthogonal approach complementary not only to immunotherapies, but also as a combination strategy with chemotherapy or radiotherapy. Citation Format: Dhwani Haria, Jina Lee, Justy Guagua, Archana Nagaraja, Kyle Roskamp, Bum-Yeol Hwang, Divya Ravichandar, Michi Willcoxon, Todd DeSantis, Karim Dabbagh, Helena Kiefel. Targeting the CXCR3 pathway with a novel peptide drug candidate mobilizes the immune system to enhance anti-tumor immunity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6349.

Effects of high-fat and low-carbohydrate diet combined with radiation therapy on tumor microenvironment in LLC tumor-bearing mice.

e20543 Background: Metabolic reprogramming of tumor immune cells can regulate immune function. To add a sentence linking tumor metabolism with radioimmunity. In this study, the high heterogeneity and reprogramming of tumor cell metabolism were utilized to explore the effects of high fat and low carbohydrate diet combined with radiotherapy on tumor immune microenvironment of tumor mice bearing LLC from the perspective of tumor microenvironment. Methods: Lewis lung cancer (LLC) model was established in female C57BL/6J mice aged 6 to 8 weeks and around 18g, and they were given a high fat and low carbohydrate diet (45F, fat 45.00% of energy) combined with radiotherapy. The body weight, tumor growth, blood glucose and blood ketone (βHB) values, survival time, median survival time and treatment safety of tumor-bearing mice were observed. Immunohistochemistry, multiple immunofluorescence and flow cytometry were used to detect the expression of tumor-associated microangiogenesis molecules (CD34), lymphatic microvessels (LYVE-1), collagen fibers and immune cell phenotypes (CD3, CD4, CD8, Treg and PD1). Results: High fat and low carbohydrate diet combined with radiotherapy (45F+RT) group had the best tumor control, and the tumor volume was significantly smaller than the control group (p &lt; 0.001), and tumor weight in the combined treatment group (45F+RT) was significantly lower than that in the normal diet (PT, fat 12.11% of energy) and 45F group (p &lt; 0.001); The blood glucose in 45F group was lower than that in PT group, but there was no statistical difference (p &lt; 0.05); It was found that the βHB value in the high-fat and low-carbohydrate diet group was higher than that in the normal diet group. There were statistically significant differences in the second and third weeks (1.00±0.20mmol/L vs 0.63±0.06mmol/L, 0.90±0.17mmol/L vs 0.70±0.10mmol/L, p &lt; 0.05). The median survival time of mice in each group was 38 days in the PT group, 55 days in the PT+RT group and 41 days in the 45F group, but not in the 45F+RT group. Immunohistochemistry and Multiple immunofluorescence showed that CD8 expression was up-regulated and PD1, CD34, LYVE-1 and collagen fiber expression were down-regulated in 45F+RT group. Flow cytometry showed that the proportion of CD8+T cells in 45F+RT group was higher than that in PT group and PT+RT group (p &lt; 0.05) and higher than 45F group, but the difference was not statistically significant (p &lt; 0.05); The proportion of CD4+T cells in PT+RT and 45F+RT groups was higher than that in PT and 45F groups (p &lt; 0.05); The proportion of Treg in T cells in 45F+RT group was lower than that in PT+RT group, but the difference was not statistically significant (p &lt; 0.05). Conclusions: High fat and low carbohydrate diet combined with radiotherapy can enhance the recruitment and function of immune effector cells in tumor microenvironment and inhibit tumor microangiogenesis, thereby inhibiting tumor growth.