Antigen-presenting Macrophages Research Articles

Macrophages exploit the mannose receptor and JAK-STAT1-MHC-II pathway to drive antigen presentation and the antimycobacterial immune response after BCG vaccination.

Tuberculosis (TB), caused by Mycobacterium tuberculosis ( M. tb), remains one of the leading causes of fatal infectious diseases worldwide. The only licensed vaccine, Mycobacterium bovis Bacillus Calmette-Guérin (BCG), has variable efficacy against TB in adults. Insufficiency of immune cell function diminishes the protective effects of the BCG vaccine. It is critical to clarify the mechanism underlying the antimycobacterial immune response during BCG vaccination. Macrophage mannose receptor (MR) is important for enhancing the uptake and processing of glycoconjugated antigens from pathogens for presentation to T cells, but the roles of macrophage MR in the BCG-induced immune response against M. tb are not yet clear. Here, we discover that macrophage MR deficiency impairs the antimycobacterial immune response in BCG-vaccinated mice. Mechanistically, macrophage MR triggers JAK-STAT1 signaling, which promotes antigen presentation via upregulated MHC-II and induces IL-12 production by macrophages, contributing to CD4 + T cell activation and IFN-γ production. MR deficiency in macrophages reduces the vaccine efficacy of BCG and increases susceptibility to M. tb H37Ra challenge in mice. Our results suggest that MR is critical for macrophage antigen presentation and the antimycobacterial immune response to BCG vaccination and offer valuable guidance for the preventive strategy of BCG immunization.

Potential predictive value of immune-related genes FUCA1 and NCKAP1L for osteosarcoma metastasis

BackgroundOsteosarcoma is a common malignant tumor with a low survival rate after metastasis. Current treatments have not proven to effectively increase patient survival rates. Immunotherapy is a promising new treatment approach, however, immune target therapy has not shown satisfactory results. This study aims to provide new insights and evidence for the use of immunotherapy in osteosarcoma, based on a comprehensive analysis of gene expression data from databases. MethodsGene expression and GSAV analysis were conducted on samples from patients with metastatic and non-metastatic osteosarcoma in the TARGET and GEO databases to identify relevant genes. These genes were further analyzed using GO, KEGG, GSVA, correlation analysis, and immune microenvironment scoring techniques. The tissue location of gene expression was confirmed through single-cell analysis. Validation of gene expression patterns was performed using polymerase chain reaction, western blot, and immunohistochemistry. ResultsThe study identified FUCA1 and NCKAP1L as significantly enriched in non-metastatic osteosarcoma, with higher expression associated with better patient survival rates. Gene function enrichment was primarily related to immune functions, with positive correlations to macrophage phagocytosis, antigen presentation, and macrophage polarization pathways. Analysis of the immune microenvironment revealed a positive correlation between gene expression and immune scores, with increased presence of macrophages, T cells, and B cells in the high expression group. Single-cell analysis and experimental results confirmed the enrichment of FUCA1 and NCKAP1L in macrophages. ConclusionThe identification of FUCA1 and NCKAP1L as potential prognostic biomarkers suggests their potential for improving patient outcomes. Modulation of macrophages may offer a promising strategy for enhancing the immune microenvironment in osteosarcoma.

The tumor-intrinsic role of the m6A reader YTHDF2 in regulating immune evasion.

Tumors evade attacks from the immune system through various mechanisms. Here, we identify a component of tumor immune evasion mediated by YTH domain-containing family protein 2 (YTHDF2), a reader protein that usually destabilizes m6A-modified mRNA. Loss of tumoral YTHDF2 inhibits tumor growth and prolongs survival in immunocompetent tumor models. Mechanistically, tumoral YTHDF2 deficiency promotes the recruitment of macrophages via CX3CL1 and enhances mitochondrial respiration of CD8+ T cells by impairing tumor glycolysis metabolism. Tumoral YTHDF2 deficiency promotes inflammatory macrophage polarization and antigen presentation in the presence of IFN-γ. In addition, IFN-γ induces autophagic degradation of tumoral YTHDF2, thereby sensitizing tumor cells to CD8+ T cell-mediated cytotoxicity. Last, we identified a small molecule compound that preferentially induces YTHDF2 degradation, which shows a potent antitumor effect alone but a better effect when combined with anti-PD-L1 or anti-PD-1 antibodies. Collectively, YTHDF2 appears to be a tumor-intrinsic regulator that orchestrates immune evasion, representing a promising target for enhancing cancer immunotherapy.

HLA-DRB5 promotes immune thrombocytopenia via activating CD8+ T cells.

Immune thrombocytopenia (ITP) is an autoimmune disease characterized by a low platelet (PLT) count and a high risk of bleeding, the clinical treatment for which still needs to be upgraded. Based on the critical role of human leukocyte antigen class II heterodimer β5 (HLA-DRB5) in immune system, we herein investigated its effect on ITP. ITP murine models were established by the injection of guinea pig anti-mouse platelet serum (GP-APS), and the PLT of mouse peripheral blood was counted during the modeling. Quantitative real-time reverse transcription polymerase chain reaction, western blot and immunofluorescence assay was performed to quantify expressions of HLA-DRB5, major histocompatibility complex II (MHC-II) and co-stimulatory molecules (CD80, CD86). Flow cytometry was conducted to analyze the percentage of CD8+ T cells. As a result, the PLT count was decreased in mouse peripheral blood. Expressions of HLA-DRB5, MHC-II and co-stimulatory molecules, as well as the percentage of CD8+ T cells were elevated in peripheral blood of ITP mice. HLA-DRB5 knockdown mitigated ITP by increasing peripheral PLT level, downregulating expressions of MHC-II and co-stimulatory molecules and inactivating CD8+ T cells. Collectively, the downregulation of HLA-DRB5 restores the peripheral PLT count in ITP mice by reducing MHC-II-mediated antigen presentation of macrophages to inhibit the activation of CD8+ T cells.

Resident and recruited macrophages differentially contribute to cardiac healing after myocardial ischemia.

Cardiac macrophages are heterogenous in phenotype and functions, which has been associated with differences in their ontogeny. Despite extensive research, our understanding of the precise role of different subsets of macrophages in ischemia/reperfusion (I/R) injury remains incomplete. We here investigated macrophage lineages and ablated tissue macrophages in homeostasis and after I/R injury in a CSF1R-dependent manner. Genomic deletion of a fms-intronic regulatory element (FIRE) in the Csf1r locus resulted in specific absence of resident homeostatic and antigen-presenting macrophages, without affecting the recruitment of monocyte-derived macrophages to the infarcted heart. Specific absence of homeostatic, monocyte-independent macrophages altered the immune cell crosstalk in response to injury and induced proinflammatory neutrophil polarization, resulting in impaired cardiac remodeling without influencing infarct size. In contrast, continuous CSF1R inhibition led to depletion of both resident and recruited macrophage populations. This augmented adverse remodeling after I/R and led to an increased infarct size and deterioration of cardiac function. In summary, resident macrophages orchestrate inflammatory responses improving cardiac remodeling, while recruited macrophages determine infarct size after I/R injury. These findings attribute distinct beneficial effects to different macrophage populations in the context of myocardial infarction.

Integrative analysis of single-cell and bulk transcriptome data reveal the significant role of macrophages in lupus nephritis

ObjectiveWe attempted to identify abnormal immune cell components and signaling pathways in lupus nephritis (LN) and to identify potential therapeutic targets.MethodsDifferentially expressed genes (DEGs) between LN and normal kidney tissues were identified from bulk transcriptome data, and functional annotation was performed. The phenotypic changes in macrophages and aberrant intercellular signaling communications within immune cells were imputed from LN scRNA-seq data using trajectory analysis and verified using immunofluorescence staining. Finally, lentivirus-mediated overexpression of LGALS9, the gene encoding Galectin 9, in THP-1 cells was used to study the functional effect of this gene on monocytic cells.ResultsFrom bulk transcriptome data, a significant activation of interferon (IFN) signaling was observed, and its intensity showed a significantly positive correlation with the abundance of infiltrating macrophages in LN. Analysis of scRNA-seq data revealed 17 immune cell clusters, with macrophages showing the highest enrichment of intercellular signal communication in LN. Trajectory analysis revealed macrophages in LN undergo a phenotypic change from inflammatory patrolling macrophages to phagocytic and then to antigen-presenting macrophages, and secrete various pro-inflammatory factors and complement components. LGALS9 was found significantly upregulated in macrophages in LN, which was confirmed by the immunofluorescence assay. Gene functional study showed that LGALS9 overexpression in THP-1 cells significantly elicited pro-inflammatory activation, releasing multiple immune cell chemoattractants.ConclusionOur results present an important pathophysiological role for macrophages in LN, and our preliminary results demonstrate significant pro-inflammatory effects of LGALS9 gene in LN macrophages.

Current research on the interaction between Helicobacter pylori and macrophages.

Helicobacter pylori (H. pylori) is a gram-negative bacteria with a worldwide infection rate of 50%, known to induce gastritis, ulcers and gastric cancer. The interplay between H. pylori and immune cells within the gastric mucosa is pivotal in the pathogenesis of H. pylori-related disease. Following H. pylori infection, there is an observed increase in gastric mucosal macrophages, which are associated with the progression of gastritis. H. pylori elicits macrophage polarization, releases cytokines, reactive oxygen species (ROS) and nitric oxide (NO) to promote inflammatory response and eliminate H. pylori. Meanwhile, H. pylori has developed mechanisms to evade the host immune response in order to maintain the persistent infection, including interference with macrophage phagocytosis and antigen presentation, as well as induction of macrophage apoptosis. Consequently, the interaction between H. pylori and macrophages can significantly impact the progression, pathogenesis, and resolution of H. pylori infection. Moreover, macrophages are emerging as potential therapeutic targets for H. pylori-associated gastritis. Therefore, elucidating the involvement of macrophages in H. pylori infection may provide novel insights into the pathogenesis, progression, and management of H. pylori-related disease.

Abstract 725: Listeria-inspired phagosome escape drives STING responses to CD47 blockade

Abstract Purpose: Through phagocytosis, antigen-presenting cells (APCs) engulf and neutralize malignant cells. ​However, engulfed tumor cells are destroyed in phagolysosomes leaving few peptides to enter cytosol, where cross-presentation of antigens and immune activation occurs. Listeria (L.) monocytogenes escape phagolysosomes by secreting a pore-forming protein Listeriolysin O (LLO). Our breakthrough centers on harnessing LLO to create LLO-CD47, a novel protein-antibody conjugate that fuses the myeloid checkpoint anti-CD47 to LLO. By promoting tumor cell-phagocytosis and the release of tumor peptides and DNA from phagolysosomes, we hypothesize that LLO-CD47 enhances the immune functionalities of APCs. Procedures: Anti-CD47 was conjugated to LLO using a water-soluble SPDP crosslinker and purified by affinity chromatography. Transmission electron microscopy (TEM) was used to visualize the integrity of macrophage phagolysosomes following treatment. C57BL/6 mouse bone marrow-derived macrophages (BMDMs) were used to study M2-to-Ml polarization, tumor cell phagocytosis, STING activation, and antigen presentation. Mice bearing orthotopically implanted spontaneously metastatic murine triple-negative 4T1 tumors and E0771 tumors received intratumoral (IT) or intraperitoneal (IP) injection. CD8+ T cells or tumor-associated macrophages (TAMs) were depleted from tumor-bearing mice using an anti-CD8 antibody or anti-CSF-1R antibody prior to treatment. Results: 1. LLO-CD47 skews BMDMs from M2-to-M1 inflammatory phenotypes and enhances the phagocytosis of E0771 tumor cells. 2. BMDMs visualized by TEM show breaches in phagosome membranes following LLO-CD47, but not anti-CD47, treatment. 3. LLO-CD47 increases levels of phosphorylated STING, IFN, and TNFa in BMDMs in vitro and TAMs in vivo, relative to anti-CD47. 4. IT delivery of LLO-CD47 inhibits the growth and metastasis of orthotopically implanted murine breast tumors relative to anti-CD47. Harvested tumors show increased M1 polarized macrophages and CD8+ T cells, which express high levels of PD-1. 5. Co-administration of IP anti-PD-1 and LLO-CD47 delays the progression of breast tumor metastasis and prolongs animal survival relative to animals treated with anti-PD-1 and anti-CD47. 6. The elimination of CD8+ T cells or TAMs abrogates the antitumor effect of LLO-CD47. 7. IP administration of LLO-CD47 showed acceptable toxicity with stable body weight gain and no long-term hematological, renal, or hepatic toxicities. Conclusion: Conjugation of LLO to anti-CD47 enhances macrophage STING activation, antigen presentation, and tumor cell phagocytosis. Our work represents the first antibody-drug conjugate specifically designed to enhance immune activation against cancer.​ Citation Format: Benjamin R. Schrank, Yifan Wang, Abin Antony, Wen Jiang. Listeria-inspired phagosome escape drives STING responses to CD47 blockade [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 725.

Factors associated with genetic markers for rotator cuff disease in patients with atraumatic rotator cuff tears.

For atraumatic rotator cuff tears, genetics contributes to symptomatic tear risk and may influence rotator cuff healing after surgical repair. But little is known about how genetic factors influence rotator cuff tear patient characteristics at presentation. We collected saliva samples for genotyping from atraumatic rotator cuff tear patients. We examined nine single nucleotide polymorphisms (SNPs) associated with cuff tears in prior literature. We estimated associations of SNP dosage with (1) age at tear diagnosis, (2) bilateral atraumatic tear prevalence, and (3) tear size. Linear regression was used to estimate associations with diagnosis age adjusted for sex and principal components. Logistic regression and ordinal logistic regression were used to estimate associations with bilateral tear prevalence and tear size category, respectively, adjusting for age, sex, and principal components. Of 344 eligible patients, 336 provided sufficient samples for genotyping. Median age at tear diagnosis was 61, 22% (N = 74) had bilateral atraumatic tears, and 9% (N = 29) had massive tears. SNP rs13107325 in the SLC39A8 gene and rs11850957 in the STXBP6 gene were associated with younger diagnosis age even after accounting for multiple comparisons (rs13107325: -4 years, 95% CI = -6.5, -1.4; rs11850957: -2.7 years, 95% CI = -4.3, -1.1). No other significant associations were observed with diagnosis age, tear size, or bilateral tear prevalence. SLC39A8 encodes a Mn transporter. STXBP6 may play a role in inflammatory responses by altering phagocytosis and antigen presentation of monocytes and macrophages. Further research is needed to determine if genetic markers can be used alongside patient characteristics to aid in identifying optimal surgical repair candidates.

Creeping fat exhibits distinct Inflammation-specific adipogenic preadipocytes in Crohn's disease.

Creeping fat (CrF) is an extraintestinal manifestation observed in patients with Crohn's disease (CD). It is characterized by the accumulation of mesenteric adipose tissue (MAT) that wraps around the intestinal wall. Although the role of CrF in CD is still debated, multiple studies have highlighted a correlation between CrF and inflammation, as well as fibrostenosais of the intestine, which contributes to the worsening of CD symptoms. However, the mechanism underlying the potential role of CrF in the development of Crohn's fibrosis remains an enigma. This study aimed to analyze CrF comprehensively using single-cell RNA sequencing analysis. The data was compared with transcriptomic data from adipose tissue in other disease conditions, such as ulcerative colitis, lymphedema, and obesity. Our analysis classified two lineages of preadipocyte (PAC) clusters responsible for adipogenesis and fibrosis in CrF. Committed PACs in CrF showed increased cytokine expression in response to bacterial stimuli, potentially worsening inflammation in patients with CD. We also observed an increase in fibrotic activity in PAC clusters in CrF. Co-analyzing the data from patients with lymphedema, we found that pro-fibrotic PACs featured upregulated pentraxin-3 expression, suggesting a potential target for the treatment of fibrosis in CrF. Furthermore, PACs in CrF exhibited a distinct increase in cell-to-cell communication via cytokines related to inflammation and fibrosis, such as CCL, LIGHT, PDGF, MIF, and SEMA3. Interestingly, these interactions also increased in PACs of the lymphedema, whereas the increased MIF signal of PACs was found to be a distinct characteristic of CrF. In immune cell clusters in CrF, we observed high immune activity of pro-inflammatory macrophages, antigen-presenting macrophages, B cells, and IgG+ plasma cells. Finally, we have demonstrated elevated IgG+ plasma cell infiltration and increased pentraxin-3 protein levels in the fibrotic regions of CrF in CD patients when compared to MAT from both UC patients and healthy individuals. These findings provide new insights into the transcriptomic features related to the inflammation of cells in CrF and suggest potential targets for attenuating fibrosis in CD.

Humanized Monoclonal Antibody IMM47, Targeting CD24, Exhibits Exceptional Anti-Tumor Efficacy By Blocking the CD24/Siglec-10 Interaction and Can be Used As Monotherapy or in Combination with Anti-PD1 Antibodies for Cancer Immunotherapy

Background: To evaluate the anti-tumor mechanism of IMM47 by blocking the interaction of CD24/Siglec-10 through macrophage antigen presentation. Methods: IMM47 was constructed and produced using an in-house-developed CHO-K1 cell expression system. BLI, ELSIA, and flow cytometry methods were used to measure the EC50 of IMM47 binding, ADCC, ADCP, ADCT and CDC activities. hSiglec-10 Tg C57BL/6 mice congenitally transplanted with MC38-hCD24 were used to analyze the in vivo efficacy. IMM47 monotherapy or in combination with SIRP-Fc fusion protein (IMM01) or anti-PD-1 antibodies were used to test IMM47's anti-tumor efficacy in SCID mice with Jeko-1 or MCF-7 tumor cell xenotransplantation and hPD-1 Tg C57BL/6 mice with MC38-hCD24/hPD-L1 tumor cell homologous transplantation models. Results: The BLI assay results showed that the affinity between IMM47 and CD24 was 0.876 nM. ELISA results showed that the EC50s of IMM47 binding to CD24 were 0.2847 nM. The EC50 of IMM47 binding to REH, Hela, MCF-7, and HCC1954 cells were 14.09 nM, 7.14 nM, 7.54 nM and 54.89 nM, respectively. IMM47 can specifically bind to CD24-positive cells but not to CD24-negative cells. IMM47 binding to CD24 is independent of the glycosylation modification of the extracellular domain. IMM47 can only bind to CD24 in humans and chimpanzees but not in other species, including mice, rats, pigs, dogs, and cynomolgus. IMM47 does not bind to human erythrocytes but strongly binds to granulocytes. Mechanism of action study results showed that IMM47 blocks the binding of CD24 to Siglec-10, with ADCC EC50 values of 0.030 nM, 0.162 nM, 0.017 nM, and 0.202 nM on REH, Hela, MCF-7, and HCC1954, respectively. IMM47 has strong ADCT activity with an EC50 of 0.333 nM against REH. IMM47 has an ADCP EC50 of 0.067 nM and 0.099 nM on REH and MCF-7, respectively. The phagocytic activity of IMM47 was significantly stronger than that of IMM01, a SIRPα-Fc fusion protein. The CDC activity results showed that the EC50 of IMM47 on REH cells was 27.29 nM. With hSiglec-10 Tg C57BL/6 mice congenitally transplanted with MC38-hCD24 cells, in vivo efficacy assays showed that IMM47 has strong anti-tumor activity, with higher spleen M1 and M2 immune cells. After the IMM47 treatment, when tumor cells were reinoculated into mice, they did not form tumors, suggesting a memory immune response had been established. After the IMM47 treatment, tumors in 5 of 6 mice were completely cured at a dose of 3 mg/kg, with a dose-dependent relationship being observed. Pharmacodynamic in vivo study in the Jeko-1 tumor cell heterologous transplantation SCID mouse model, IMM47 exhibits strong anti-tumor activity and a dose-dependent relationship. The combination of IMM47 and IMM01 has significantly better efficacy than that of any single drug in the model of MCF-7 xenograft SCID mice. The combination of IMM47 and the PD-1 antibody Tislelizumab in the MC38-hCD24/hPD-L1 tumor cells in the homologously transplanted PD-1 transgenic C57BL/6 mouse model showed the best efficacy, and tumors in all mice were eliminated. The efficacy of IMM47 combined with either the PD-1 antibody Opdivo or Keytruda is better than that of monotherapy, and tumors in 5/6 and 6/6 mice were eliminated when combined with Opdivo and Keytruda, respectively. Four weeks after drug withdrawal, the re-inoculated cells could not form tumors and were eventually eliminated, while the re-inoculated cells in the control group grew normally and formed tumors. Conclusion: Our research showed that the humanized anti-CD24 mAb IMM47 had excellent anti-tumor effect. The extracellular domain's N-glycosylation alteration has no effect on IMM47's ability to bind to CD24. The in vitro assays revealed that IMM47 exhibits significant ADCC, ADCP, ADCT, and CDC activities. IMM47 displays strong anti-tumor activity in transgenic mouse models, and a memory immune response is generated after treatment. Additionally, an in vivo pharmacodynamics assay of IMM47 in combination with different PD-1 antibodies revealed that IMM47 exhibits synergistic therapeutic efficacy when combined with Tislelizumab, Opdivo, and Keytruda. This suggests that IMM47 has significant potential for use in the clinical setting for cancer immunotherapy, either alone or in combination with other immune checkpoint inhibitors. Finally, IMM47 avoids the adverse effects displayed by some anti-CD47 antibodies because it does not bind to human red blood cells and has a high safety profile.

Inhalation of Citrus Reticulata essential oil alleviates airway inflammation and emphysema in COPD rats through regulation of macrophages

Ethnopharmacological relevanceChronic obstructive pulmonary disease (COPD) is a chronic inflammatory respiratory disease. Citrus Reticulata peel, the dried ripe peel of Citrus Reticulata species, has been found to have anti-inflammatory and cough attenuation effects. However, the therapeutic effects and its precise underlying mechanisms of atomizing inhalation using Citrus Reticulata essential oil (CREO) have not yet been fully elucidated. Aim of the studyThe aim of this study was to assess the therapeutic effects of Citrus Reticulata essential oil and its associated anti-inflammatory mechanisms in COPD rat model. MethodsA total of 80 SD rats were randomized into four groups: control group (Con), COPD model group (COPD), COPD + ipratropium bromide (IB), and COPD + citrus reticulata essential oil (CREO). To induce COPD in rats, cigarette smoke (CS) exposure was used, while CREO and IB groups were administered through atomizing inhalation. The clinical signs, pathological lesions of the lung, percentages of antigen-presenting lung macrophages (CD11b/c+/CD86+ cells) and CD8+ T cells, and the content and mRNA expression of cytokines of the lung were analyzed. ResultsThe findings revealed that atomizing inhalation of Citrus reticulata essential oil had therapeutic effects on COPD rats. The treatment resulted in improvement in the body weight and mental status of COPD rats, reduced pathological injury of the lung, and increased proportion of CD11b/c+/CD86+ cells in lung macrophages, while also decreasing the number of CD8+ T cells. In addition, the Citrus Reticulata essential oil reduced the contents of IL-18, IL-17A, IL-12p70, and GM-CSF, downregulated the relative mRNA expression of IFN-γ, IL-4, and MMP-12, and upregulated the mRNA expression of IL-10. ConclusionsCitrus reticulata essential oil can alleviate histological injury of the lung and regulate macrophages and CD8+ T cells in COPD rats. The study suggests that citrus reticulata essential oil could be a potential therapeutic agent for COPD.

Remodeling the immune microenvironment for gastric cancer therapy through antagonism of prostaglandin E2 receptor 4

Gastric cancer is highly prevalent among digestive tract tumors. Due to the intricate nature of the gastric cancer immune microenvironment, there is currently no effective treatment available for advanced gastric cancer. However, there is promising potential for immunotherapy targeting the prostaglandin E2 receptor subtype 4 (EP4) in gastric cancer. In our previous study, we identified a novel small molecule EP4 receptor antagonist called YY001. Treatment with YY001 alone demonstrated a significant reduction in gastric cancer growth and inhibited tumor metastasis to the lungs in a mouse model. Furthermore, administration of YY001 stimulated a robust immune response within the tumor microenvironment, characterized by increased infiltration of antigen-presenting cells, T cells, and M1 macrophages. Additionally, our research revealed that YY001 exhibited remarkable synergistic effects when combined with the PD-1 antibody and the clinically targeted drug apatinib, rather than fluorouracil. These findings suggest that YY001 holds great promise as a potential therapeutic strategy for gastric cancer, whether used as a standalone treatment or in combination with other drugs.

ASO-mediated knockdown or kinase inhibition of G2019S-Lrrk2 modulates lysosomal tubule-associated antigen presentation in macrophages

Genetic variation around the LRRK2 gene affects risk for both familial and sporadic Parkinson's disease (PD). LRRK2 levels have become an appealing target for potential PD therapeutics with LRRK2 antisense oligonucleotides (ASOs) now moving toward clinical trials. However, LRRK2 has been suggested to play a fundamental role in peripheral immunity, and it is currently unknown if targeting increased LRRK2 levels in peripheral immune cells will be beneficial or deleterious. Here it was observed that G2019S macrophages exhibited increased stimulation-dependent lysosomal tubule formation (LTF) and MHC-II trafficking from the perinuclear lysosome to the plasma membrane in an mTOR-dependent manner with concomitant increases in pro-inflammatory cytokine release. Both ASO-mediated knockdown of mutant Lrrk2 and LRRK2 kinase inhibition ameliorated this phenotype and decreased these immune responses in control cells. Given the critical role of antigen presentation, lysosomal function, and cytokine release in macrophages, it is likely LRRK2-targeting therapies with systemic activity may have therapeutic value with regard to mutant LRRK2, but deleterious effects on the peripheral immune system, such as altered pathogen control in these cells, should be considered when reducing levels of non-mutant LRRK2.

IMM47, a humanized monoclonal antibody that targets CD24, exhibits exceptional anti-tumor efficacy by blocking the CD24/Siglec-10 interaction and can be used as monotherapy or in combination with anti-PD1 antibodies for cancer immunotherapy.

This study evaluates the anti-tumor mechanism of IMM47, a humanized anti-CD24 mAb. Biolayer interferometry, ELISA and flow cytometry methods were used to measure the IMM47 binding, affinity, ADCC, ADCP, ADCT and CDC activities. In vivo therapeutical efficacy was measured in transplanted mouse models. IMM47 significantly binds granulocytes but not human erythrocytes and blocks CD24's ability to bind to Siglec-10. IMM47 has strong ADCC, ADCT and ADCP activity against REH cells. IMM47's in vivo pharmacodynamics showed that IMM47 has strong anti-tumor effects in human siglec-10 transgenic mouse models with a memory immune response. IMM47 also has powerful synergistic therapeutic efficacy when combined with Tislelizumab, Opdivo and Keytruda, by blocking CD24/Siglec-10 interaction through macrophage antigen presentation with strong ADCC, ADCP, ADCT and CDC activities and with a safe profile. IMM47 binding to CD24 is independent of N-glycosylation modification of the extracellular domain.

Identification, expression pattern and functional characterization of IFN-γ involved in activating JAK-STAT pathway in Sebastes schlegeli.

IFN-γ (interferon gamma) is a critical cytokine in the immune system involved both directly and indirectly in antiviral activity, stimulation of bactericidal activity, antigen presentation and activation of macrophages via the Janus kinase/signal transducer and activator of transcription (JAK-STAT) pathway. The IFN-γ function is best described in cell defense against intracellular pathogens in mammals, but IFN-γ cytokine-induced metabolic change and its role in anti-infection remain unknown in teleost fish. In this study, a novel IFN-γ (SsIFN-γ) was identified from black rockfish (Sebastes schlegeli) by rapid amplification of cDNA ends (RACE). The open reading frame (ORF) of SsIFN-γ encoded a putative protein of 215 amino acids and shares 60.2%-93.5% overall sequence identities with other teleost IFN-γ. SsIFN-γ was distributed ubiquitously in all the detected tissues and immune cells, which was highly expressed in the spleen, gills, head kidney by quantitative real-time PCR. The mRNA expression of SsIFN-γ was significantly upregulated in the spleen, head kidney, head kidney (HK) macrophages and peripheral blood lymphocytes (PBLs) during pathogen infection. Meanwhile, the recombinant protein (rSsIFN-γ) exhibited an immunomodulatory function to enhance respiratory burst activity and nitric oxide response of HK macrophages. Furthermore, rSsIFN-γ could effectively upregulate the expression of macrophage proinflammatory cytokine, the expression of JAK-STAT signaling pathway related genes and interferon-related downstream genes in the head kidney and spleen. Luciferase assays showed ISRE and GAS activity were obviously enhanced after rSsIFN-γ treatment. These results indicated that SsIFN-γ possessed apparent immunoregulatory properties and played a role in fighting pathogen infection which will be helpful to further understanding of the immunologic mechanism of teleosts IFN-γ in innate immunity.

Sialidase facilitates Porphyromonas gingivalis immune evasion by reducing M1 polarization, antigen presentation, and phagocytosis of infected macrophages.

Porphyromonas gingivalis (P. gingivalis), a major pathogen of periodontitis, can evade host immune defenses. Previously, we found that P. gingivalis W83 sialidase gene mutant strain (ΔPG0352) was more easily cleared by macrophages. The aims of this study were to investigate the effects of sialidase in P. gingivalis on the polarization, antigen presentation, and phagocytosis of infected macrophages and to clarify the mechanism of P. gingivalis immune evasion. Human monocytes U937 were differentiated to macrophages and infected with P. gingivalis W83, ΔPG0352, comΔPG0352, and Escherichia coli (E. coli). The phagocytosis of macrophages was observed by transmission electron microscopy and flow cytometry. ELISA or Griess reaction were used to examine the levels of interleukin-12 (IL-12), inducible nitric oxide synthase (iNOS) and interleukin-10 (IL-10), and the expressions of CD68, CD80 and CD206 were determined by flow cytometry. The expression of major histocompatibility complex-II (MHC-II) was detected by immunofluorescence. A rat periodontitis model was established to determine the M1 and M2 polarization of macrophages. Compare with P. gingivalis W83, ΔPG0352 increased the levels of IL-12, iNOS, CD80, and MHC-II and inhibited the levels of IL-10 and CD206. Macrophages phagocytosed 75.4% of ΔPG0352 and 59.5% of P. gingivalis W83. In the rat periodontitis model, the levels of M1 and M2 macrophages in P. gingivalis W83 group were both higher than those in ΔPG0352 group, while the ratio of M1/M2 was higher in the ΔPG0352 group. Alveolar bone absorption was lower in ΔPG0352 group. Sialidase facilitates P. gingivalis immune evasion by reducing M1 polarization, antigen presentation, and phagocytosis of infected macrophages.

PD-L1 Expression on Circulating Antigen Presenting Macrophages in Blood Predicts PFS & OS in an Array of Metastatic Cancer Types Treated with PD-L1/PD-1 Immunotherapies

Abstract Cancer Associated Macrophage Like cells (CAMLs) are antigen presenting circulating stromal cells found in the blood of cancer patients (pts). Recently, PD-L1 expression on CAMLs was described in breast (BC), lung (LC) and renal cell carcinomas (RCC), appearing to parallel the inflammatory PD-L1 state of the tumor microenvironment, and acting as a possible predictive biomarker for PD-L1/PD-1 immunotherapeutics (IMTs). We monitored CAML PD-L1 from 48 pts in an array of solid tumors, prior PD-L1/PD-1 IMTs induction, to evaluate CAML’s ability to predict response by progression free survival (PFS) and overall survival (OS). Three single blind multi-year prospective studies were run to test PD-L1 CAML expression against PFS & OS, prior to induction of therapy in metastatic (m) LC (n=24), mBC (n=16) or mRCC (n=8). We recruited pts with pathologically confirmed progressing disease after at least one failed prior line of therapy. Blood samples (7.5 mL) were taken prior to start of new therapeutic regimes, i.e. pembrolizumab (n=36) or nivolumab (n=12). Whole blood was filtered by CellSieve microfilters to collect CAMLs, stain for PD-L1, and then scored as a binary high or low, to evaluate PFS & OS hazard ratios (HRs) by censored univariate & multivariate analysis at 24 months. CAMLs were found in 98% of all tested samples. Overall, pts with high CAML PD-L1 expression treated with IMT had significantly better PFS (HR=2.9, 95%CI=1.4–6.1, p=0.0067) and better OS (HR=2.7, 95%CI=1.2–6.1, p=0.0317) vs patients with low CAML PD-L1. These data suggests that PD-L1 expression in circulating CAMLs appears to predict pts with increased benefit to PD-L1/PD-1 IMTs in an array of tumor subtypes, though larger studies are needed to validate these findings. Private Funding by Creatv MicroTech, Inc. and supported grants by NCI-NIH (R43CA206840), and the Defense Advanced Research Projects Agency (DARPA)(W911NF-14-C-0098).

TAM receptor tyrosine kinase MERTK as a therapeutic target in the tumor immune microenvironment in a murine Krasmutant lung cancer model

Abstract TAM receptor tyrosine kinase MERTK is involved in apoptotic cell clearance to maintain tissue homeostasis. During efferocytosis, MERTK activation also promotes an immunosuppressive phenotype, which can be hijacked by tumor cells to escape immune attacking. Here, we determined the impact of MERTK inhibition on immune cell subsets in the tumor microenvironment in mice with subcutaneous murine CMT167 (Kras-G12V) lung cancer grafts. Although MERTK expression was not detected on tumor associated dendritic cells (DCs), there was a significant increase in the incidence of DCs in tumors from Mertk knockout (KO) mice compared to wild type mice by day 5 after inoculation of tumor cells. Characterization of tumor-associated macrophages by flow cytometry revealed three distinct populations expressing different levels of MHCII (CD11b +MHCII high, CD11b +MHCII +and CD11b highMHCII neg). Tumors from Mertk KO mice had significantly greater numbers of MHCII highmacrophages by day 10 after tumor cell inoculation and reduced numbers of MHCII negmacrophages by day 19. Together, the increased incidence of dendritic cells and the shift toward a more antigen-presenting macrophage phenotype are expected to promote anti-tumor immunity in Mertk KO mice. Indeed, CMT167 tumor growth was significantly reduced in Mertk KO mice and this effect was abrogated in Mertk KO scid mice, which lack functional B and T cells. Treatment with MRX-2843, a MERTK-selective inhibitor currently in Phase 1 clinical trials, also reduced tumor growth in mice. Together, these data demonstrate a critical role for MERTK as a mediator of tumor progression in the tumor immune microenvironment and implicate MRX-2843 as an effective strategy to exploit this finding in the clinic. The research was supported by the National Cancer Institute of the National Institutes of Health (Emory Lung Cancer Spore, Award Number P50CA217691). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

Abstract LB195: Immunotherapies that repolarize macrophages and CD4 T cells enhance the effect of chemotherapy in high-grade serous ovarian cancer

Abstract We asked if single-cell RNA sequencing (scRNA-seq) analysis of tumor immune cells in high-grade serous ovarian cancer (HGSOC) would reveal ways to enhance effects of neo-adjuvant chemotherapy (NACT). Analysis of 64,097 immune cells from seven HGSOC omental metastases showed that positive effects of NACT on immune responses were counterbalanced by induction of immunosuppressive mechanisms, especially related to antigen presentation and Treg activity. We identified stabilin-1 (Clever-1) on macrophages and FOXP3 in Tregs as targets and validated actions of their inhibitors in vitro. ScRNA-seq analysis of 69,781 cells from chemotherapy-treated syngeneic mouse HGSOC tumors showed significant agreement with the patient results. Combinations of chemotherapy with anti-stabilin1 antibody and/or FOXP3 inhibitor (AZD8701), FOXP3i, significantly increased median survival of mice with established peritoneal disease. Long-term survivors (300 days+) were resistant to tumor rechallenge. Analysis of bulk RNA sequencing data from treated murine tumors confirmed our hypothesis and suggested potential mechanisms to explain the anti-tumor response seen in the murine models. Tumors treated with chemotherapy plus anti-stabilin1 antibody showed significant enrichment of anti-tumor macrophage signature and antigen presentation pathway compared to chemotherapy alone. Tumors treated with chemotherapy plus FOXP3i in combination with anti-stabilin1 antibody showed significant upregulation of antigen presentation, T cell activation and cytotoxicity and T helper 1 differentiation pathways. There was significant reduction in FOXP3 positive cells and significant increase of Tbet positive cells in FOXP3i treated murine tumors suggesting repolarization of naive T cells in the tumor microenvironment towards a more anti-tumor T helper phenotype. As there are early phase clinical trials of FOXP3 inhibitor and anti-stabilin1 (Clever-1) antibody in patients with advanced cancer, these results suggest ways of improving response to chemotherapy not only in HGSOC but in other cancers that are treated by NACT. Citation Format: Samar Elorbany, Chiara Berlato, Larissa Carnevalli, Eleni Maniati, Jun Wang, Simon Barry, Ranjit Manchanda, Julia Kzhyshkowska, Frances Balkwill. Immunotherapies that repolarize macrophages and CD4 T cells enhance the effect of chemotherapy in high-grade serous ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB195.