PD-L1 Expression In Macrophages Research Articles

BMAL2 promotes eCIRP-induced macrophage endotoxin tolerance.

The disruption of the circadian clock is associated with inflammatory and immunological disorders. BMAL2, a critical circadian protein, forms a dimer with CLOCK, activating transcription. Extracellular cold-inducible RNA-binding protein (eCIRP), released during sepsis, can induce macrophage endotoxin tolerance. We hypothesized that eCIRP induces BMAL2 expression and promotes macrophage endotoxin tolerance through triggering receptor expressed on myeloid cells-1 (TREM-1). C57BL/6 wild-type (WT) male mice were subjected to sepsis by cecal ligation and puncture (CLP). Serum levels of eCIRP 20h post-CLP were assessed by ELISA. Peritoneal macrophages (PerM) were treated with recombinant mouse (rm) CIRP (eCIRP) at various doses for 24h. The cells were then stimulated with LPS for 5h. The levels of TNF-α and IL-6 in the culture supernatants were assessed by ELISA. PerM were treated with eCIRP for 24h, and the expression of PD-L1, IL-10, STAT3, TREM-1 and circadian genes such as BMAL2, CRY1, and PER2 was assessed by qPCR. Effect of TREM-1 on eCIRP-induced PerM endotoxin tolerance and PD-L1, IL-10, and STAT3 expression was determined by qPCR using PerM from TREM-1-/- mice. Circadian gene expression profiles in eCIRP-treated macrophages were determined by PCR array and confirmed by qPCR. Induction of BMAL2 activation in bone marrow-derived macrophages was performed by transfection of BMAL2 CRISPR activation plasmid. The interaction of BMAL2 in the PD-L1 promoter was determined by computational modeling and confirmed by the BIAcore assay. Serum levels of eCIRP were increased in septic mice compared to sham mice. Macrophages pre-treated with eCIRP exhibited reduced TNFα and IL-6 release upon LPS challenge, indicating macrophage endotoxin tolerance. Additionally, eCIRP increased the expression of PD-L1, IL-10, and STAT3, markers of immune tolerance. Interestingly, TREM-1 deficiency reversed eCIRP-induced macrophage endotoxin tolerance and significantly decreased PD-L1, IL-10, and STAT3 expression. PCR array screening of circadian clock genes in peritoneal macrophages treated with eCIRP revealed the elevated expression of BMAL2, CRY1, and PER2. In eCIRP-treated macrophages, TREM-1 deficiency prevented the upregulation of these circadian genes. In macrophages, inducible BMAL2 expression correlated with increased PD-L1 expression. In septic human patients, blood monocytes exhibited increased expression of BMAL2 and PD-L1 in comparison to healthy subjects. Computational modeling and BIAcore assay identified a putative binding region of BMAL2 in the PD-L1 promoter, suggesting BMAL2 positively regulates PD-L1 expression in macrophages. eCIRP upregulates BMAL2 expression via TREM-1, leading to macrophage endotoxin tolerance in sepsis. Targeting eCIRP to maintain circadian rhythm may correct endotoxin tolerance and enhance host resistance to bacterial infection.

Induction of a different immune response in non-titanized compared to titanized polypropylene meshes.

It is well known that pelvic organ prolapse (POP) significantly reduces the quality of life of affected women and in many cases requires corrective surgery. Aim of the study was to compare the immune response against titanized versus non-titanized meshes, especially macrophage polarization and immune checkpoint association. For this, we analyzed 644 POP surgeries, which were performed between 2017 and 2022, in our department. Four of them needed revision surgery caused by erosion. We analyzed the influx of CD68 & CD163 positive macrophages and the expression of immune checkpoint molecules PD-L1 and PD1 in these 4 patients. We identified a large number of CD68 and CD163 positive macrophages and additionally a PD-L1 expression of these cells. Based on the in-vivo results, we isolated monocytes and co-cultivated monocytes with different mesh material covered with or without fibroblasts. We identified a significantly enhanced macrophage activation and PD-L1 expression in macrophages surrounding non-titanized polypropylene mesh material. Encapsulation of the material by fibroblasts was crucial for that. Specifically, CD68-positive macrophages are upregulated (p<0.001), co-expressing PD-L1 (p<0.001) in monocytes co-cultivated with non-titanized polypropylene meshes. Monocytes co-cultivated with titanized polypropylene meshes showed significantly lower expression of CD163 (p=0.027) and PD-L1 (p=0.022). In conclusion, our in vitro data suggest that the titanium coating leads to a decreased polarization of macrophages and to a decreased immune response compared to non-titanized meshes. This could be an indication for the increased incidence of erosion of the non-titanized meshes, which is a severe complication of this procedure and requires revision surgery. STATEMENT OF SIGNIFICANCE: Pelvic organ prolapse is a well-known problem for women and often requires corrective surgery. Polypropylene meshes are often used, which differ in their coating (titanized vs. non-titanized). A severe side effect of these surgeries is mesh erosion, due to onset of inflammation, which requires revision surgery. We examined all erosion cases (4 of 644 patients) with implanted nontitanium-coated meshes by immunohistochemistry and found upregulation of macrophage polarization (as markers CD68 and CD163) and increased expression of the immune checkpoint molecules PD-L1 and PD1. This suggests inflammatory processes and an enhanced immune response. In addition, we set up an in vitro experiment to investigate whether coating plays a role. Here, we demonstrated that the non-titanized meshes elicited a significantly higher immune response in comparison to tetanized meshes, which could lead to the higher erosion rate of the non-titanized meshes. Our results highlight the benefit of titanized meshes, which should lead to a lower revision surgery rate and thus improved patient outcome.

High-dose ascorbic acid potentiates immune modulation through STAT1 phosphorylation inhibition and negative regulation of PD-L1 in experimental sepsis.

Sepsis is a complex, multifactorial syndrome characterized by a dysregulated host response to infection, leading to severe organ dysfunction and high mortality rates among critically ill patients. Hypovitaminosis C and vitamin C deficiency are frequently observed in septic patients, prompting interest in the potential therapeutic role of ascorbic acid. Although intravenous administration of ascorbic acid has been investigated in multiple clinical trials for sepsis treatment, the specific immunomodulatory mechanisms underlying its effects remain elusive. This study aimed to investigate the protective effects of high-dose ascorbic acid on experimental sepsis. Results show that intravenous administration of high-dose ascorbic acid (250mg/kg) attenuated sepsis-induced organ dysfunctions in a cecal ligation and puncture (CLP)-induced septic mouse model. Ascorbic acid improved splenic cell apoptosis and increased the number of CD3+ T cells in septic mice induced by CLP. Furthermore, ascorbic acid downregulated PD-L1 expression in livers, reduced PD-1 expression in spleens, and inhibited the phosphorylation of STAT1 at Y701 in multiple organs of CLP-induced septic mice. The in vitro experiments also revealed that 800μM ascorbic acid suppressed STAT1 phosphorylation and inhibited lipopolysaccharide (LPS) and IFN-γ-induced PD-L1 expression in macrophages. These findings suggest that ascorbic acid prevents sepsis-associated organ dysfunction through the p-STAT1/PD-L1 signaling pathway. Our study provides new insights into the potential therapeutic use of ascorbic acid in sepsis.

Tumor-derived small extracellular vesicles promote breast cancer progression by upregulating PD-L1 expression in macrophages

BackgroundThe metastasis of breast cancer (BC) is a complex multi-step pathological process, strictly dependent on the intrinsic characteristics of BC cells and promoted by a predisposing microenvironment. Although immunotherapy has made important progress in metastasis BC, the heterogeneity of PD-L1 in tumor associated macrophages (TAMs) in BC and the underlying mechanisms in the metastasis development of BC are still not completely elucidated. Small extracellular vesicles (sEVs) represent essential interaction mediators between BC cells and TAMs. It is worth noting to explore the underlying mechanisms typical of sEVs and their role in the metastasis development of BC.MethodsThe structure of sEVs was identified by TEM, while the particle size and amounts of sEVs were detected by BCA and NTA analysis. The specific PD-L1 + CD163 + TAM subpopulation in metastasis BC was identified by scRNA-seq data of GEO datasets and verified by IHC and IF. The function of TAMs and sEVs in metastasis BC was explored by RT-qPCR, WB, IF, flow cytometry and in vivo experiment. The expression profiles of plasma sEVs-miRNA in relation to BC metastasis was analyzed using next-generation sequencing. Further detailed mechanisms of sEVs in the metastasis development of BC were explored by bioinformatics analysis, RT-qPCR, WB and luciferase reporter assay.ResultsIn this study, we identified that the immunosuppressive molecule PD-L1 was more abundant in TAMs than in BC cells, and a specific PD-L1 + CD163 + TAM subpopulation was found to be associated with metastasis BC. Additionally, we found that BC cells-derived sEVs can upregulate the PD-L1 expression and induce the M2 polarization, enhancing the metastasis development both in vitro and in vivo. Also, Clinical data showed that sEV-miR-106b-5p and sEV-miR-18a-5p was in relation to BC metastasis development and poor prognosis of BC patients. Further mechanistic experiments revealed that BC-derived sEV-miR-106b-5p and sEV-miR-18a-5p could synergistically promoted the PD-L1 expression in M2 TAMs by modulating the PTEN/AKT and PIAS3/STAT3 pathways, resulting in the enhancement of the BC cells invasion and metastasis.ConclusionsOur study demonstrated that BC-derived sEVs can induce metastasis in BC through miR-106b-5p/PTEN/AKT/PD-L1 and miR-18a-5p/PIAS3/STAT3/PD-L1 pathways in TAMs. Therefore, the inhibition of these specific interactions of signaling pathways would represent a promising target for future therapeutic strategies for treatment of BC.

Expression and role of the immune checkpoint regulator PD-L1 in the tumor-stroma interplay of pancreatic ductal adenocarcinoma.

Immune checkpoint inhibitors (ICI), e.g., targeting programmed cell death protein 1-ligand 1 (PD-L1) or its receptor PD-1, have markedly improved the therapy of many cancers but so far failed in pancreatic ductal adenocarcinoma (PDAC). Macrophages represent one of the most abundant immune cell populations within the tumor microenvironment (TME) of PDAC being able to either support or restrain tumor progression depending on their phenotype. To better understand treatment failure of PD-L1/PD-1 inhibitors in PDAC, this study examined PD-L1 expression in the context of a dynamic TME in PDAC with a particular focus on the impact of macrophages. Formalin-fixed and paraffin embedded tissue samples of primary PDAC tissues and corresponding liver metastases were used for immunohistochemical analyses. Serial sections were stained with antibodies detecting Pan-Cytokeratin, CD68, CD163, CD8, and PD-L1.To investigate whether the PD-1/PD-L1 axis and macrophages contribute to immune escape of PDAC cells, a stroma enriched 3D spheroid coculture model was established in vitro, using different PDAC cell lines and macrophages subtypes as well as CD8+ T cells. Functional and flow cytometry analyses were conducted to characterize cell populations. Immunohistochemical analyses revealed that PD-L1 is mainly expressed by stroma cells, including macrophages and not PDAC cells in primary PDAC tissues and corresponding liver metastases. Notably, high local abundance of macrophages and strong PD-L1 staining were commonly found at invasion fronts of tumoral lesions between CD8+ T cells and tumor cells. In order to investigate whether PD-L1 expressing macrophages impact the response of PDAC cells to treatment with PD-L1/PD-1 inhibitors, we developed a spheroid model comprising two different PDAC cell lines and different ratios of in vitro differentiated primary M1- or M2-like polarized macrophages. In line with our in situ findings, high PD-L1 expression was observed in macrophages rather than PDAC cells, which was further increased by the presence of PDAC cells. The effector phenotype of co-cultured CD8+ T cells exemplified by expression of activation markers and release of effector molecules was rather enhanced by PDAC macrophage spheroids, particularly with M1-like macrophages compared to mono-culture spheroids. However, this was not associated with enhanced PDAC cell death. ICI treatment with either Durvalumab or Pembrolizumab alone or in combination with Gemcitabine hardly affected the effector phenotype of CD8+ T cells along with PDAC cell death. Thus, despite strong PD-L1 expression in macrophages, ICI treatment did not result in an enhanced activation and cytotoxic phenotype of CD8+ T cells. Overall, our study revealed novel insights into the interplay of PDAC cells and macrophages in the presence of ICI.

CAA-derived IL-6 induced M2 macrophage polarization by activating STAT3

BackgroundTumor-associated macrophages (TAMs) are the most abundant types of immune cells in the tumor microenvironment (TME) of breast cancer (BC). TAMs usually exhibit an M2 phenotype and promote tumor progression by facilitating immunosuppression. This study aimed to investigate the effect of CAA-derived IL-6 on macrophage polarization in promoting BC progression.MethodsHuman BC samples and adipocytes co-cultured with 4T1 BC cells were employed to explore the properties of CAAs. The co-implantation of adipocytes and 4T1 cells in mouse tumor-bearing model and tail vein pulmonary metastasis model were constructed to investigate the impact of CAAs on BC malignant progression in vivo. The functional assays, qRT-PCR, western blotting assay and ELISA assay were employed to explore the effect of CAA-derived IL-6 on macrophage polarization and programmed cell death protein ligand 1 (PD-L1) expression.ResultsCAAs were located at the invasive front of BC and possessed a de-differentiated fibroblast phenotype. CAAs facilitated the malignant behaviors of 4T1 cells in vitro, and promoted 4T1 tumor growth and pulmonary metastasis in vivo. The IHC staining of both human BC specimens and xenograft and the in vitro experiment indicated that CAAs could enhance infiltration of M2 macrophages in the TME of 4T1 BC. Furthermore, CAA-educated macrophages could enhance malignant behaviors of 4T1 cells in vitro. More importantly, CAAs could secret abundant IL-6 and thus induce M2 macrophage polarization by activating STAT3. In addition, CAAs could upregulate PD-L1 expression in macrophages.ConclusionsOur study revealed that CAAs and CAA-educated macrophages enhanced the malignant behaviors of BC. Specifically, CAA-derived IL-6 induced migration and M2 polarization of macrophages via activation STAT3 and promoted macrophage PD-L1 expression, thereby leading to BC progression.

Gastric cancer-derived exosomes facilitate pulmonary metastasis by activating ERK-mediated immunosuppressive macrophage polarization.

Gastric cancer (GC) with pulmonary metastasis is one of the deadliest diseases in the world; however, the underlying pathological mechanisms and potential therapeutic targets remain to be elucidated. As exosomes play indispensable roles in the formation of premetastatic niches (PMN) and cancer metastasis. Therefore, investigating the underlying mechanisms of exosome-mediatedpulmonary metastasis of GC may shed new light on identifying novel therapeutic targets for GC treatment.GC-derived exosomes were isolated from the conditioned medium of mouse forestomach carcinoma(MFC) cell line. The effects of MFC-derived exosomeson pulmonary macrophage polarization were analyzed byreverse- transcription polymerase chain reactionand flow cytometry. Expression of PD-L1 and other proteins was evaluated by Western blot. Exosomal microRNAs (miRNAs) were analyzed by microarray.GC-derived exosomes (GC-exo) accumulated in high numbers in the lungs and were ingested by macrophages. The extracellular-signal-regulated kinase (ERK) signaling pathway was activated by GC-exo, inducing macrophage immunosuppressive-phenotype differentiation and increased PD-L1 expression. miRNA-sequencing identified 130 enriched miRNAs in GC-exo. Among the enriched miRNAs, miR-92a-3p plays a major role in activating ERK signaling via inhibition of PTEN expression. In addition, inhibiting ERK signaling with PD98059 significantly reduced the expression of PD-L1 in macrophages and, therefore, reversed the immunosuppressive PMN and inhibited the colonization of GC cells in the lungs. This study identified a novel mechanism of GC-exo mediated PD-L1 expression in lung macrophages that facilitates lung PMN formation and GC pulmonary metastasis, which also provided a potential therapeutic target for GC with pulmonary metastasis treatment.

Targeted xCT-mediated Ferroptosis and Protumoral Polarization of Macrophages Is Effective against HCC and Enhances the Efficacy of the Anti-PD-1/L1 Response.

Tumor-associated macrophages (TAMs) play an essential role in tumor progression, metastasis, and antitumor immunity. Ferroptosis has attracted extensive attention for its lethal effect on tumor cells, but the role of ferroptosis in TAMs and its impact on tumor progression have not been clearly defined. Using transgenic mouse models, this study determines that xCT-specific knockout in macrophages is sufficient to limit tumorigenicity and metastasis in the mouse HCC models, achieved by reducing TAM recruitment and infiltration, inhibiting M2-type polarization, and activating and enhancing ferroptosis activity within TAMs. The SOCS3-STAT6-PPAR-γ signaling may be a crucial pathway in macrophage phenotypic shifting, and activation of intracellular ferroptosis is associated with GPX4/RRM2 signaling regulation. Furthermore, that xCT-mediated macrophage ferroptosis significantly increases PD-L1 expression in macrophages and improves the antitumor efficacy of anti-PD-L1 therapy is unveiled. The constructed Man@pSiNPs-erastin specifically targets macrophage ferroptosis and protumoral polarization and combining this treatment with anti-PD-L1 exerts substantial antitumor efficacy. xCT expression in tumor tissues, especially in CD68+ macrophages, can serve as a reliable factor to predict the prognosis of HCC patients. These findings provide further insight into targeting ferroptosis activation in TAMs and regulating TAM infiltration and functional expression to achieve precise tumor prevention and improve therapeutic efficacy.

Ataxia telangiectasia mutated kinaseinhibition promotes irradiation-induced PD-L1 expression in tumour-associated macrophages through IFN-I/JAK signalling pathway.

Tumour-associated macrophages (TAMs) are one of the primary sources of PD-L1 expression in the tumour microenvironment (TME). Ionizing radiation (IR) promotes PD-L1 expression in tumour cells. However, the effect of IR on macrophage PD-L1 expression and the underlying mechanisms remain unclear. ATM kinase, as the key kinase for initiating DNA damage repair (DDR) process, is associated with innate immune STING axis activation. Here, we explored the molecular mechanism implicated in macrophage PD-L1 expression regulated by IR as well as the role of ATM kinase in this process. IR-regulated PD-L1 expression in macrophages and associated signalling pathways were explored by in vitro studies using murine and human macrophage cell lines. A colorectal xenograft murine model was employed to demonstrate the impact of targeting ATM and PD-L1 expression in TAMs following IR on growth of tumour in vivo. IR up-regulated PD-L1 expression in macrophages, which was further augmented by ATM kinase inhibition. ATM inhibition increased IR-induced DNA damage, which activated STING/interferon regulatory factor 3 (IRF3) signalling pathway and up-regulated type I interferon (IFN-I) expression in macrophages. IFN-I bound to the IFN α receptor 1 on macrophages, activated the downstream JAK1 and STAT1/3 signalling and eventually led to PD-L1 up-expression. ATM inhibition augmented IR-induced PD-L1 expression in macrophages and CD8+ T cell infiltration, and promoted anti-tumour efficacy in vivo. These results suggested that ATM inhibition promoted IR-induced PD-L1 expression through the activation of innate immunity in TAMs, which provided a novel approach to enhance the anti-tumour efficacy of RT.

Human Umbilical Cord Mesenchymal Stem Cells Promote Macrophage PD-L1 Expression and Attenuate Acute Lung Injury in Mice.

Acute lung injury (ALI)/acute respiratory distress syndrome (ARDS) remains a serious clinical problem but has no approved pharmacotherapy. Mesenchymal stem cells (MSCs) represent an attractive therapeutic tool for tissue damage and inflammation owing to their unique immunomodulatory properties. The present study aims to explore the therapeutic effect and underlying mechanisms of human umbilical cord MSCs (UC-MSCs) in ALI mice. In this study, we identify a novel mechanism for human umbilical cord-derived MSCs (UC-MSCs)-mediated immunomodulation through PGE2-dependent reprogramming of host macrophages to promote their PD-L1 expression. Our study suggests that UC-MSCs or primed- UC-MSCs offer new therapeutic approaches for lung inflammatory diseases. Lipopolysaccharide (LPS)-induced ALI mice were injected with 5×105 UC-MSCs via the tail vein after 4 hours of LPS exposure. After 24 hours of UC-MSC administration, the total protein concentration and cell number in the bronchoalveolar lavage fluid (BALF) and cytokine levels in the lung tissue were measured. Lung pathological changes and macrophage infiltration after UCMSC treatment were analyzed. Moreover, in vitro co-culture experiments were performed to analyze cytokine levels of RAW264.7 cells and Jurkat T cells. UC-MSC treatment significantly improved LPS-induced ALI, as indicated by decreased total protein exudation concentration and cell number in BALF and reduced pathological damage in ALI mice. UC-MSCs could inhibit pro-inflammatory cytokine levels (IL-1β, TNF-α, MCP-1, IL-2, and IFN-γ), while enhancing anti-inflammatory cytokine IL-10 expression, as well as reducing macrophage infiltration into the injured lung tissue. Importantly, UC-MSC administration increased programmed cell death protein ligand 1 (PD-L1) expression in the lung macrophages. Mechanistically, UC-MSCs upregulated cyclooxygenase-2 (COX2) expression and prostaglandin E2 (PGE2) secretion in response to LPS stimulation. UC-MSCs reduced the inflammatory cytokine levels in murine macrophage Raw264.7 through the COX2/PGE2 axis. Furthermore, UC-MSC- derived PGE2 enhanced PD-L1 expression in RAW264.7 cells, which in turn promoted programmed cell death protein 1 (PD-1) expression and reduced IL-2 and IFN-γ production in Jurkat T cells. Our results suggest that UC-MSCs attenuate ALI via PGE2-dependent reprogramming of macrophages to promote their PD-L1 expression.

Hemangiosarcoma cells induce M2 polarization and PD-L1 expression in macrophages

Hemangiosarcoma (HSA) is a malignant tumor derived from endothelial cells. Tumor-associated macrophages are one of the major components of tumor microenvironment and crucial for cancer development. The presence and function of macrophages in HSA have not been studied because there is no syngeneic model for HSA. In this study, we evaluated two mouse HSA cell lines and one immortalized mouse endothelial cell line for their usefulness as syngeneic models for canine HSA. Our results showed that the ISOS-1 cell line developed tumors with similar morphology to canine HSA. ISOS-1 cells highly expressed KDM2B and had similar KDM2B target expression patterns with canine HSA. Moreover, we determined that in both ISOS-1 and canine HSA tumors, macrophages were present as a major constituent of the tumor microenvironment. These macrophages were positive for CD204, an M2 macrophage marker, and express PD-L1, an immune checkpoint molecule. Canine HSA with macrophages expressing PD-L1 had a smaller number of T-cells in tumor tissues than tumors with PD-L1 negative macrophages. ISOS-1-conditioned medium could induce M2 polarization and PD-L1 expression in RAW264.7 mouse macrophage cell line and mouse peritoneal macrophages. These results show that ISOS-1 can be used as a syngenic model for canine HSA and suggest that macrophages play an important role in immune evasion in HSA. Using the syngeneic mouse model for canine HSA, we can further study the role of immune cells in the pathology of HSA.

Endoplasmic reticulum stress promotes the release of exosomal PD-L1 from head and neck cancer cells and facilitates M2 macrophage polarization

BackgroundEndoplasmic reticulum (ER) stress has been found to foster the escape of cancer cells from immune surveillance and upregulate PD-L1 expression. However, the underlying mechanisms are unknown.MethodsWhile analyzing the protein levels using immunofluorescence and Western blotting, the RNA levels were measured using qRT-PCR. Ten injection of exosomes into six-week-old nude mice was made through the tail vein once every other day in total.ResultsThe expression of certain ER stress markers such as PERK (PKR-like endoplasmic reticulum kinase), ATF6 (activating transcription factor 6), and GRP78 (glucose-regulated protein 78), was found to be upregulated in the oral squamous cell carcinoma (OSCC) tissues and related to poor overall survival. There is a positive relationship between the extent of ER stress-related proteins and a cluster of PD-L1 expression and macrophage infiltration among the OSCC tissues. Further, incubation with exosomes derived from ER-stressed HN4 cells (Exo-ER) was found to upregulate PD-L1 extents in macrophages in vitro and in vivo, and macrophage polarization toward the M2 subtype was promoted by upregulating PD-L1.ConclusionsER stress causes OSCC cells to secrete exosomal PD-L1 and upregulates PD-L1 expression in macrophages to drive M2 macrophage polarization. The delineation of a new exosome-modulated mechanism was made for OSCC–macrophage crosstalk driving tumor development and to be examined for its therapeutic use.Graphical abstractExosomal PD-L1 secreted by ER-stressed OSCC cells promoted M2 macrophage polarization. BvoEQJGxSejnxUc-5MZRg5Video .

Ferric Ammonium Citrate Upregulates PD-L1 Expression through Generation of Reactive Oxygen Species.

Iron plays an important role in macrophage polarization by altering metabolic and redox status. However, the impact of iron on the immune status of macrophages is still controversial. In this study, we report that ferric ammonium citrate (FAC) upregulates PD-L1 expression in macrophages. FAC not only altered the phenotype of macrophages but also led to enriching immune-modulatory T cell subsets. Since iron is known to be a constituent of coenzymes facilitating metabolic processes in mitochondria, we examined the metabolic status of FAC-overloaded macrophages by measuring the oxygen consumption rate (OCR) and the represented coenzyme, aconitase. In addition to enhancement of metabolic processes, FAC accelerated the Fenton reaction in macrophages, which also contributed to the facilitation of oxygen consumption. We reasoned that the enhancement of the OCR leads to the production of reactive oxygen species (ROS), which are directly linked to PD-L1 induction. Using ferrostatin, rotenone, and N-acetyl-L-cysteine, we confirmed that metabolic and redox regulation is responsible for FAC-mediated PD-L1 expression. Furthermore, we suggested that FAC-induced ROS production may explain FAC-mediated pro- and anti-inflammatory responses in macrophages. These findings may extend our understanding of regulating iron concentration during immune checkpoint therapy in cancer patients.

Understanding the impact of chemotherapy on the immune landscape of high-grade serous ovarian cancer

ObjectivesWe quantitatively characterized the change in temporospatial expression of repressive and stimulatory checkpoints across immune cell populations in the tumor microenvironment in a cohort of high grade serous ovarian carcinomas (HGSOC) using matched samples before and after neoadjuvant platinum-based chemotherapy. MethodsUsing retrospectively collected matched tissue samples from 9 patients, cell populations were assessed using multiplex immunofluorescence using the Vectra Multispectral Imaging System (Perkin Elmer). We used multiple panels to assess: tumor (AE1/AE3), T cells (CD3, CD8, FOXP3), macrophages (CD68) as well as immune checkpoints (C3aR, PD-1, PD-L1, LAG3, IDO, ICOS, GITR). IHC staining was performed for folate receptor status. Changes in immune cell populations as well as intensities of associated repressive and stimulatory proteins were assessed pre- to post-treatment. ResultsWe observed a consistently high pre-treatment stromal macrophage population which is reduced post-chemotherapy with post-treatment enrichment in macrophage PD-L1 expression. While inhibitory checkpoint expression on T cells was heterogeneous post-chemotherapy, we observed a change in the ThICOS+:Treg ratio which resulted in ThICOS+ cells outnumbering Treg cells post-treatment. Spatial analysis revealed the proximity of Treg cells to ThICOS+ T cells decreased post-treatment. We also observed upward shifts in Teff:Treg T cell ratios with retention of immune checkpoints PD-1, LAG3 and GITR. ConclusionsIn this unique dataset of pre and post matched chemotherapy treated HGSOC patients, we observed changes in immune cell subsets expressing repressive or stimulatory proteins resulting in immune compositions more favorable to checkpoint modulations, suggesting novel therapeutic strategies in the recurrent setting.

Human Umbilical Cord Mesenchymal Stem Cells Promote Macrophage PD-L1 Expression and Attenuate Acute Lung Injury in Mice

Human Umbilical Cord Mesenchymal Stem Cells Promote Macrophage PD-L1 Expression and Attenuate Acute Lung Injury in Mice

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

A Sampling of Highlights from the Literature: Article Recommendations from Our Deputy and Senior Editors.

CAR T cells can be engineered to overcome antigen loss (from Fig. 2C of Landoni et al., Cancer Immunol Res 2021)One factor limiting chimeric antigen receptor (CAR) T-cell therapy efficacy against solid tumors is CAR antigen loss. Johnson et al. show that engineering CAR T cells to express the RNA RN7SL1 increases their antitumor efficacy in multiple models. RN7SL1 enhances CAR T-cell expansion and effector-memory differentiation, and also is transferred in extracellular vesicles to other immune cells that then promote activation of endogenous T cells that can reject solid tumors with CAR antigen loss. By testing various combinations of CAR constructs, Hirabayashi et al. find that T cells engineered to be dual targeting—with each CAR signaling through a different costimulatory endodomain, only one of which includes a CD3ζ-chain—provide the most effective antitumor effects and prevent antigen loss.Johnson LR, …, Minn AJ. Cell 2021 Sep 16;184:4981–95.e14.Hirabayashi K, …, Dotti G. Nat Cancer 2021 Sep 23;2:904–18.Local delivery of cytokine-ending mRNA could improve immunotherapy efficacy (by Gwillhickers via Wikimedia Commons)How to effectively deliver immunotherapy locally to tumors remains a challenge. Hotz et al. test the intratumoral delivery of a mixture of mRNAs encoding four cytokines/cytokine fusion proteins (IL12 single chain, IFNα4, GM-CSF, and IL15 sushi) in multiple tumor models. They find the treatment effectively activates antitumor immunity and induces immune memory; therapeutic effects are observed for primary, distant, and metastatic tumors. Combining the mRNA mixture with immune checkpoint blockade further enhances therapeutic benefit. The data suggest that this treatment strategy has the potential to improve current immunotherapy regimens, and it is now being tested in clinical trials.Hotz C, …, Wiederschain D. Sci Transl Med 2021 Sep 8;13:eabc7804.Pancreatic adenocarcinoma frequently evades the immune system (by Min Yu via NIH Flickr)Pancreatic adenocarcinoma (PDAC) is largely refractory to immunotherapy. Freed-Pastor et al. have developed organoid models of the subset of PDACs they predict to have high-affinity neoantigens and find that some of these organoids evade the immune system after transplantation into immune-competent mice. Immune-evasive organoids and human PDACs are infiltrated with dysfunctional neoantigen-specific CD8+ T cells and have high CD155 expression. Blockade of CD155/TIGIT signaling, together with anti–PD-1 blockade and CD40 agonism, limited immune evasion and CD40-agonist resistance in the mice, identifying a potential combination therapeutic strategy for PDAC.Freed-Pastor WA, …, Jacks T. Cancer Cell 2021 Oct 11;39:1342–60.e14.A CRISPR screen links tumor suppressor genes and the adaptive immune system (by Ernesto del Aguila III via NIH Flickr)Genetic screens for drivers of tumorigenesis are often conducted using systems that do not account for the complexity of the tumor microenvironment, including pressure exerted by the immune system. By performing transcriptome-wide CRISPR screens in mouse tumor models using animals with and without functional adaptive immunity, Martin et al. find an enrichment for loss of tumor suppressor genes as a requirement for tumor growth in the presence of an adaptive immune system. Loss of one tumor suppressor gene, GNA13, increases recruitment of tumor-associated macrophages by enhancing CCL2 secretion. These data highlight potential new functions for tumor suppressor genes that could be harnessed for immunotherapy.Martin TD, …, Elledge SJ. Science 2021 Sep 17;373:1327–35.Engineering IL-2 to be a partial agonist can enhance adoptive cell therapy (by Emw from Wikimedia Commons)IL-2 is often used in the generation of T cells for adoptive cell therapy because it promotes T-cell expansion. However, it also induces terminal differentiation and expression of coinhibitory molecules, limiting therapeutic efficacy. Mo et al. identify an IL-2 partial agonist, H9T, that promotes CD8+ T–cell expansion but not terminal differentiation; rather, it drives transcriptional, epigenetic, and metabolic profiles that restrict T-cell exhaustion and maintain a stem cell–like state. T-cell receptor transgenic T cells and CAR T cells expanded with H9T show enhanced antitumor activity in mice compared with cells expanded in IL-2, highlighting the potential for engineering cytokines to increase adoptive cell therapy efficacy.Mo F, …, Leonard WJ. Nature 2021 Sep 23;597:544–8.A feedback loop facilitates tumor metastasis (by AleXXw via Wikimedia Commons)Tumor cells use various mechanisms to disseminate from the primary tumor to a metastatic site. Morrissey et al. show that tumor-derived exosomes drive polarization of macrophages in the premetastatic niche to an immune-suppressive phenotype, thereby promoting tumor metastasis. Macrophage interaction with tumor-derived exosomes leads to upregulation of PD-L1 and metabolic alterations, which act to further increase macrophage PD-L1 expression. The data highlight a feedback loop that potentiates immune suppression and identifies tumor-derived exosomes, macrophages, and metabolism-related factors as key players in the process.Morrissey SM, …, Yan J. Cell Metab 2021 Oct 5;33:2040–58.e10.

Apatinib triggers autophagic and apoptotic cell death via VEGFR2/STAT3/PD-L1 and ROS/Nrf2/p62 signaling in lung cancer

BackgroundRecently, a variety of clinical trials have shown that apatinib, a small-molecule anti-angiogenic drug, exerts promising inhibitory effects on multiple solid tumors, including non-small cell lung cancer (NSCLC). However, the underlying molecular mechanism of apatinib on NSCLC remains unclear.MethodsMTT, EdU, AO/EB staining, TUNEL staining, flow cytometry, colony formation assays were performed to investigate the effects of apatinib on cell proliferation, cell cycle distribution, apoptosis and cancer stem like properties. Wound healing and transwell assays were conducted to explore the role of apatinib on migration and invasion. The regulation of apatinib on VEGFR2/STAT3/PD-L1 and ROS/Nrf2/p62 signaling were detected. Furthermore, we collected conditioned medium (CM) from A549 and H1299 cells to stimulate phorbol myristate acetate (PMA)-activated THP-1 cells, and examined the effect of apatinib on PD-L1 expression in macrophages. The Jurkat T cells and NSCLC cells co-culture model was used to assess the effect of apatinib on T cells activation. Subcutaneous tumor formation models were established to evaluate the effects of apatinib in vivo. Histochemical, immunohistochemical staining and ELISA assay were used to examine the levels of signaling molecules in tumors.ResultsWe showed that apatinib inhibited cell proliferation and promoted apoptosis in NSCLC cells in vitro. Apatinib induced cell cycle arrest at G1 phase and suppressed the expression of Cyclin D1 and CDK4. Moreover, apatinib upregulated Cleaved Caspase 3, Cleaved Caspase 9 and Bax, and downregulated Bcl-2 in NSCLC cells. The colony formation ability and the number of CD133 positive cells were significantly decreased by apatinib, suggesting that apatinib inhibited the malignant and stem-like features of NSCLC cells. Mechanistically, apatinib inhibited PD-L1 and c-Myc expression by targeting VEGFR2/STAT3 signaling. Apatinib also inhibited PD-L1 expression in THP-1 derived macrophages stimulated by CM from NSCLC cells. Furthermore, apatinib pretreatment increased CD69 expression and IFN-γ secretion in stimulated Jurkat T cells co-cultured with NSCLC cells. Apatinib also promoted ROS production and inhibited Nrf2 and p62 expression, leading to the autophagic and apoptotic cell death in NSCLC. Moreover, apatinib significantly inhibited tumor growth in vivo.ConclusionOur data indicated that apatinib induced autophagy and apoptosis in NSCLC via regulating VEGFR2/STAT3/PD-L1 and ROS/Nrf2/p62 signaling.

Abstract 2758: Multiplex immunofluorescence (mIF) reveals differences in tumor immune microenvironment between molecularly-defined subsets of small cell lung cancer

Abstract Introduction: Despite the recent approval of immune checkpoints inhibitors (ICI) as a treatment option in the extensive-stage small cell lung cancer (SCLC) setting, survival has not significantly changed in the last decades. Recent scientific efforts have led to the identification of 4 major subtypes defined by expression of three transcription factors: ASCL1 (SCLC-A), NEUROD1 (SCLC-N), POU2F3 (SCLC-P) with the fourth subtype characterized by increased expression of immune genes (Inflamed subtype - SCLC-I). Transcriptomic results, along with recent immunotherapy trials, suggest that modulation of tumor immune microenvironment (TIME) could potential be critical to achieving clinical responses in a subset of patients, hence a comprehensive study of the TIME in SCLC is imperative. Here we report the feasibility of a multiplex immunofluorescence (mIF) methodology to analyze the TIME in SCLC. Methods: FFPE sections from surgically resected SCLC (N=4, one representative case across all SCLC subtypes) were identified from the ICON Project at UT MD Anderson Cancer Center. We used mIF to identify and quantify immune markers grouped into two 6-antibody panels: Panel 1: cytokeratin (CK, AE1/AE3), CD3, CD8, PD-1, PD-L1 and CD68; Panel 2: CK, CD20, granzyme B, FOXP3, CD45RO, and CD57. Finally, genomic (WES), transcriptomic (RNA sequencing) and proteomic (RPPA) data from these cases were integrated with the mIF data. Results: SCLC molecular subtypes (SCLC-A, N, P, I) were classified using transcriptomic and proteomic data. Analysis of TIME unveils a higher immune cell infiltration within SCLC-I subtype compared with the other cases representing, immune “cold” SCLC subtypes. SCLC-I subtype showed a 2-13 folder higher (range) immune cell density than SCLC-A, N and P subtypes (measured as a median of cell density). Specifically, T cells (CD3+) (695 and 242 cells/mm2, for SCLC-I and the median for the other subtypes respectively), T cytotoxic cells (CD3+ CD8+) (206 and 105), activated T cells (CD3+ CD8+ granzyme+) (20 and 2), antigen experienced ‘like' T cells (CD3+ PD-1+) (17 and 0), memory T cells (CD3+ CD45RO+) (328 and 91) and macrophages (CD68+) (773 and 57). PD-L1 expression in malignant cells did not show significant differences within the 4 SCLC subtypes. However, PD-L1 expression in macrophages was significantly higher in the SCLC-I subtype, suggesting an increase of IFN-gamma in the TIME. Conclusions: TIME study show the use of mIF in SCLC tumors to be feasible, and could potentially provide key information towards the identification of SCLC patients that could benefit from ICI. For the first time we complemented transcriptomic data from SCLC tumors with mIF analysis unveiling the complex interplay of the host immune response and malignant cells. Our preliminary results warrant further studies to explore the role of TIME in immunotherapeutic response in SCLC. Citation Format: Pedro Rocha, C. Allison Stewart, Edwin Parra, Luisa M. Solis, Carl M. Gay, Robert J. Cardnell, Naohiro Uraoka, Alejandro Francisco-Cruz, Hitoshi Dejima, Yuanxin Xi, Lixia Diao, Jing Wang, Marcelo V. Negrao, Jianjun Zhang, Ignacio Wistuba, Don L. Gibbons, Lauren A. Byers. Multiplex immunofluorescence (mIF) reveals differences in tumor immune microenvironment between molecularly-defined subsets of small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2758.

Anti-PD-L1 Therapy Does Not Improve Survival in a Murine Model of Lethal Staphylococcus aureus Pneumonia.

Staphylococcus aureus (SA) bacterial pneumonia is a common cause of sepsis in intensive care units. Immune checkpoint inhibitors (CPIs) that target programmed cell death protein 1 (PD-1) and its ligand (PD-L1) have been proposed for the treatment of sepsis. However, in our systematic review of sepsis preclinical models, none of the models examined CPIs in pneumonia. Mice were inoculated intratracheally with vehicle control, low dose (LD)- or high dose (HD)-SA. Immune cell recruitment and checkpoint molecule expression were examined at 4, 24, and 48 hours after infection. Infected animals, treated with control or anti-PD-L1 antibodies, were assessed for survival, bacterial burden, lung immunophenotypes, and mediator production. LD-SA and HD-SA produced lethality of 15% and 70%, respectively, by 168 hours. At 24 hours, LD-infected animals exhibited increased lung monocyte PD-L1 expression (P = .0002) but lower bacterial counts (P = .0002) compared with HD animals. By 48 hours, either infection induced lung neutrophil and macrophage PD-L1 expression (P < .0001). Anti-PD-L1 treatment at the time of infection and at 24 hours following infection with low to high doses of SA reduced PD-L1 detection but did not affect survival or bacterial clearance. Anti-PD-L1 therapy did not alter survival in this pneumonia model. Preclinical studies of additional common pathogens and septic foci are needed.