Structural basis of G protein-Coupled receptor CMKLR1 activation and signaling induced by a chemerin-derived agonist.

Chemokine-like receptor 1 (CMKLR1), also known as chemerin receptor 23 (ChemR23) or chemerin receptor 1, is a chemoattractant G protein-coupled receptor (GPCR) that responds to the adipokine chemerin and is highly expressed in innate immune cells, including macrophages and neutrophils. The signaling pathways of CMKLR1 can lead to both pro- and anti-inflammatory effects depending on the ligands and physiological contexts. To understand the molecular mechanisms of CMKLR1 signaling, we determined a high-resolution cryo-electron microscopy (cryo-EM) structure of the CMKLR1-Gi signaling complex with chemerin9, a nanopeptide agonist derived from chemerin, which induced complex phenotypic changes of macrophages in our assays. The cryo-EM structure, together with molecular dynamics simulations and mutagenesis studies, revealed the molecular basis of CMKLR1 signaling by elucidating the interactions at the ligand-binding pocket and the agonist-induced conformational changes. Our results are expected to facilitate the development of small molecule CMKLR1 agonists that mimic the action of chemerin9 to promote the resolution of inflammation.

SRC2 controls CD4+ T cell activation via stimulating c-Myc-mediated upregulation of amino acid transporter Slc7a5.

T cell activation stimulates substantially increased protein synthesis activity to accumulate sufficient biomass for cell proliferation. The protein synthesis is fueled by the amino acids transported from the environment. Steroid nuclear receptor coactivator 2 (SRC2) is a member of a family of transcription coactivators. Here, we show that SRC2 recruited by c-Myc enhances CD4+ T cell activation to stimulate immune responses via upregulation of amino acid transporter Slc7a5. Mice deficient of SRC2 in T cells (SRC2fl/fl/CD4Cre) are resistant to the induction of experimental autoimmune encephalomyelitis (EAE) and susceptible to Citrobacter rodentium (C. rodentium) infection. Adoptive transfer of naive CD4+ T cells from SRC2fl/fl/CD4Cre mice fails to elicit EAE and colitis in Rag1/ recipients. Further, CD4+ T cells from SRC2fl/fl/CD4Cre mice display defective T cell proliferation, cytokine production, and differentiation both in vitro and in vivo. Mechanically, SRC2 functions as a coactivator to work together with c-Myc to stimulate the expression of amino acid transporter Slc7a5 required for T cell activation. Slc7a5 fails to be up-regulated in CD4+ T cells from SRC2fl/fl/CD4Cre mice, and forced expression of Slc7a5 rescues proliferation, cytokine production, and the ability of SRC2fl/fl/CD4Cre CD4+ T cells to induce EAE. Therefore, SRC2 is essential for CD4+ T cell activation and, thus, a potential drug target for controlling CD4+ T cell-mediated autoimmunity.

Store-independent activation of Orai1 by SPCA2 in mammary tumors.

Ca(2+) is an essential and ubiquitous second messenger. Changes in cytosolic Ca(2+) trigger events critical for tumorigenesis, such as cellular motility, proliferation, and apoptosis. We show that an isoform of Secretory Pathway Ca(2+)-ATPase, SPCA2, is upregulated in breast cancer-derived cells and human breast tumors, and suppression of SPCA2 attenuates basal Ca(2+) levels and tumorigenicity. Contrary to its conventional role in Golgi Ca(2+) sequestration, expression of SPCA2 increased Ca(2+) influx by a mechanism dependent on the store-operated Ca(2+) channel Orai1. Unexpectedly, SPCA2-Orai1 signaling was independent of ER Ca(2+) stores or STIM1 and STIM2 sensors and uncoupled from Ca(2+)-ATPase activity of SPCA2. Binding of the SPCA2 amino terminus to Orai1 enabled access of its carboxyl terminus to Orai1 and activation of Ca(2+) influx. Our findings reveal a signaling pathway in which the Orai1-SPCA2 complex elicits constitutive store-independent Ca(2+) signaling that promotes tumorigenesis.

Targeting macrophages for enhancing CD47 blockade-elicited lymphoma clearance and overcoming tumor-induced immunosuppression.

Tumor-associated macrophages (TAMs) are often the most abundant immune cells in the tumor microenvironment (TME). Strategies targeting TAMs to enable tumor cell killing through cellular phagocytosis have emerged as promising cancer immunotherapy. Although several phagocytosis checkpoints have been identified, the desired efficacy has not yet been achieved by blocking such checkpoints in preclinical models or clinical trials. Here, we showed that late-stage non-Hodgkin lymphoma (NHL) was resistant to therapy targeting phagocytosis checkpoint CD47 due to the compromised capacity of TAMs to phagocytose lymphoma cells. Via a high-throughput screening of the US Food and Drug Administration-approved anticancer small molecule compounds, we identified paclitaxel as a potentiator that promoted the clearance of lymphoma by directly evoking phagocytic capability of macrophages, independently of paclitaxel's chemotherapeutic cytotoxicity toward NHL cells. A combination with paclitaxel dramatically enhanced the anticancer efficacy of CD47-targeted therapy toward late-stage NHL. Analysis of TME by single-cell RNA sequencing identified paclitaxel-induced TAM populations with an upregulation of genes for tyrosine kinase signaling. The activation of Src family tyrosine kinases signaling in macrophages by paclitaxel promoted phagocytosis against NHL cells. In addition, we identified a role of paclitaxel in modifying the TME by preventing the accumulation of a TAM subpopulation that was only present in late-stage lymphoma resistant to CD47-targeted therapy. Our findings identify a novel and effective strategy for NHL treatment by remodeling TME to enable the tumoricidal roles of TAMs. Furthermore, we characterize TAM subgroups that determine the efficiency of lymphoma phagocytosis in the TME and can be potential therapeutic targets to unleash the antitumor activities of macrophages.

The GABA receptor GABRR1 is expressed on and functional in hematopoietic stem cells and megakaryocyte progenitors.

GABRR1 is a rho subunit receptor of GABA, the major inhibitory neurotransmitter in the mammalian brain. While most investigations of its function focused on the nervous system, its regulatory role in hematopoiesis has not been reported. In this study, we found GABRR1 is mainly expressed on subsets of human and mouse hematopoietic stem cells (HSCs) and megakaryocyte progenitors (MkPs). GABRR1-negative (GR-) HSCs led to higher donor-derived hematopoietic chimerism than GABRR1-positive (GR+) HSCs. GR+ but not GR- HSCs and MkPs respond to GABA in patch clamp studies. Inhibition of GABRR1 via genetic knockout or antagonists inhibited MkP differentiation and reduced platelet numbers in blood. Overexpression of GABRR1 or treatment with agonists significantly promoted MkP generation and megakaryocyte colonies. Thus, this study identifies a link between the neural and hematopoietic systems and opens up the possibility of manipulating GABA signaling for platelet-required clinical applications.

Screening for genes that regulate the differentiation of human megakaryocytic lineage cells.

Different combinations of transcription factors (TFs) function at each stage of hematopoiesis, leading to distinct expression patterns of lineage-specific genes. The identification of such regulators and their functions in hematopoiesis remain largely unresolved. In this study, we utilized screening approaches to study the transcriptional regulators of megakaryocyte progenitor (MkP) generation, a key step before platelet production. Promising candidate genes were generated from a microarray platform gene expression commons and individually manipulated in human hematopoietic stem and progenitor cells (HSPCs). Deletion of some of the candidate genes (the hit genes) by CRISPR/Cas9 led to decreased MkP generation during HSPC differentiation, while more MkPs were produced when some hit genes were overexpressed in HSPCs. We then demonstrated that overexpression of these genes can increase the frequency of mature megakaryocytic colonies by functional colony forming unit-megakaryocyte (CFU-Mk) assay and the release of platelets after in vitro maturation. Finally, we showed that the histone deacetylase inhibitors could also increase MkP differentiation, possibly by regulating some of the newly identified TFs. Therefore, identification of such regulators will advance the understanding of basic mechanisms of HSPC differentiation and conceivably enable the generation and maturation of megakaryocytes and platelets in vitro.

Targeting CD47 as a cancer therapeutic strategy: the cutaneous T-cell lymphoma experience.

PURPOSE OF REVIEW: To describe the relevance of CD47 in the tumor microenvironment and summarize data on anti-CD47 therapies, including its role in cutaneous T-cell lymphoma (CTCL). RECENT FINDINGS: CD47 is expressed on all normal cells and targets SIRPα on the surface of myeloid cells. However, CD47 is found to be overexpressed on cancer cells. CD47-SIRPα interaction inhibits macrophage phagocytosis, allowing cancer cells to escape immune surveillance. Current focus in immunotherapy has been targeted toward inhibiting CD47-SIRPα interaction via anti-CD47 antibodies. This activates innate immunity, promoting cancer cell destruction by macrophages. It also activates adaptive immunity resulting in antigen-presentation, mostly by dendritic cells, leading to antitumor cytotoxic reactions. Current CD47 antagonists undergoing clinical trials include Hu5F9 (an anti-CD47 antibody that directly inhibits the CD47-SIRPα interaction) and TTI-621, (a fusion protein composed of CD47 binding domain of human SIRPα and linked to the Fc region of IgG1). These agents have continued to show strong efficacy against solid and hematological tumors. SUMMARY: In the CTCL tumor microenvironment, increased immune checkpoint inhibition expression via CD47 bound to SIRPα correlates with a more advanced disease state. Continued success in treating these patients requires further studies on CD47 antagonists, specifically when combined with other antibodies.

Evaluation of Novel Anticaries Adhesive in a Secondary Caries Animal Model.

We investigated the anticaries properties of an adhesive containing dimethylaminododecyl methacrylate (DMADDM) in vivo via a secondary caries animal model. Cavities were prepared in the maxillary first molars of Wistar rats. DMADDM-containing adhesives were applied on one side and commercial adhesives on the opposite side as a control. After a 3-week feeding period to induce secondary caries, the molars were harvested for the evaluation of the secondary caries. Lesion depth (LD) and mineral loss (ML) were measured via a micro-CT method, and a modified Keyes scoring method yielded scores for the caries lesions. Statistical analysis was divided into 2 parts: a correlation analysis between 2 evaluations with one-way ANOVA and a least-significant differences (LSD) test, and an evaluation of anticaries adhesives with a paired samples t test. The results showed that: (1) secondary caries was successfully produced in rats; (2) there was a correlation between the modified Keyes scoring method and micro-CT in the evaluation of the secondary caries; (3) the adhesive containing DMADDM significantly reduced both LD and ML (according to micro-CT), and also lowered the scores (based on the modified Keyes scoring method). This suggests that the novel DMADDM adhesive could perform an anticaries function in vivo via the secondary caries animal model which was also developed and testified in research. Secondary caries is one of the major reasons leading to the failure of caries restoration treatment. As a solution, anticaries adhesives perform well in biofilm inhibition in vitro. However, the lack of secondary caries animal models limits the evaluation of anticaries adhesives in vivo.

Macrophages eat cancer cells using their own calreticulin as a guide: roles of TLR and Btk.

Macrophage-mediated programmed cell removal (PrCR) is an important mechanism of eliminating diseased and damaged cells before programmed cell death. The induction of PrCR by eat-me signals on tumor cells is countered by don't-eat-me signals such as CD47, which binds macrophage signal-regulatory protein α to inhibit phagocytosis. Blockade of CD47 on tumor cells leads to phagocytosis by macrophages. Here we demonstrate that the activation of Toll-like receptor (TLR) signaling pathways in macrophages synergizes with blocking CD47 on tumor cells to enhance PrCR. Bruton's tyrosine kinase (Btk) mediates TLR signaling in macrophages. Calreticulin, previously shown to be an eat-me signal on cancer cells, is activated in macrophages for secretion and cell-surface exposure by TLR and Btk to target cancer cells for phagocytosis, even if the cancer cells themselves do not express calreticulin.

Synergistic effect of honokiol and 5-fluorouracil on apoptosis of oral squamous cell carcinoma cells.

BACKGROUND: 5-Fluorouracil (5-FU) is an essential chemotherapeutic agent for oral squamous cell carcinoma (OSCC). However, toxic side effects have limited its role in OSCC therapy. The aim of this study was to explore whether combination therapy with 5-FU and honokiol (HNK), a small natural organic molecule shown to induce apoptosis in OSCC cells, could enhance the anticancer activity of 5-FU without notably increasing its toxicity. METHODS: 5-FU and/or HNK were used to treat OSCC cells both in vitro and in vivo. The therapeutic effect and underlying mechanisms were evaluated by cell viability assay, flow cytometry, OSCC xenograft mouse model, and Western blot. Tumor tissue apoptosis was detected by terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay. Toxicity was assessed following hematoxylin and eosin staining. RESULTS: Exposure to HNK + 5-FU produced a synergistic cytotoxic effect on OSCC cells. Both HNK and 5-FU could induce apoptosis through the mitochondria-mediated intrinsic pathway, and their specific signaling pathways were different. In the mouse OSCC xenograft model, treatment with 5-FU + HNK substantively retarded tumor growth, as compared to treatment with either drug individually. TUNEL analysis further confirmed that the superior in vivo antitumor efficacy of 5-FU + HNK was associated with enhanced stimulation of cell apoptosis. Notably, HNK did not increase the toxicity of 5-FU. CONCLUSION: These findings suggest that HNK and 5-FU exert a synergistic therapeutic effect on OSCC by inducing apoptosis. HNK might thus enhance the clinical therapeutic efficacy of 5-FU without increasing its toxicity.

Anti-Bacteria and Microecosystem-Regulating Effects of Dental Implant Coated with Dimethylaminododecyl Methacrylate.

The effects of dimethylaminododecyl methacrylate (DMADDM) modified titanium implants on bacterial activity and microbial ecosystem of saliva-derived biofilm were investigated for the first time. Titanium discs were coated with DMADDM solutions at mass fractions of 0 mg/mL (control), 1, 5 and 10 mg/mL, respectively. Biomass accumulation and metabolic activity of biofilms were tested using crystal violet assay and MTT (3-(4,5-Dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. 16S rRNA gene sequencing was performed to measure the microbial community. Live/dead staining and scanning electron microscopy (SEM) were used to value the structure of biofilm. The results showed that the higher mass fraction of DMADDM the coating solution had, the significantly lower the values of metabolic activity and accumulated biofilms got, as well as fewer live cells and less extracellular matrix. Moreover, 5 mg/mL of DMADDM was the most effective concentration, as well as 10 mg/mL. In microecosystem-regulation, the DMADDM modified titanium implant decreased the relative abundance of Neisseria and Actinomyces and increased the relative abundance of Lactobacillus, a probiotic for peri-implant diseases. In conclusion, via inhibiting growth and regulating microecosystem of biofilm, this novel titanium implant coating with DMADDM was promising in preventing peri-implant disease in an 'ecological manner'.

Effect of Antimicrobial Denture Base Resin on Multi-Species Biofilm Formation.

Our aims of the research were to study the antimicrobial effect of dimethylaminododecyl methacrylate (DMADDM) modified denture base resin on multi-species biofilms and the biocompatibility of this modified dental material. Candida albicans (C. albicans), Streptococcus mutans (S. mutans), Streptococcus sanguinis (S. sanguinis), as well as Actinomyces naeslundii (A. naeslundii) were used for biofilm formation on denture base resin. Colony forming unit (CFU) counts, microbial viability staining, and 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) array were used to evaluate the antimicrobial effect of DMADDM. C. albicans staining and Real-time PCR were used to analyze the morphology and expression of virulence genes of C. albicans in biofilm. Lactate dehydrogenase (LDH) array and Real-time PCR were conducted to examine the results after biofilm co-cultured with epithelial cell. Hematoxylin and eosin (HE) staining followed by histological evaluation were used to study the biocompatibility of this modified material. We found that DMADDM containing groups reduced both biomass and metabolic activity of the biofilm significantly. DMADDM can also inhibit the virulence of C. albicans by means of inhibiting the hyphal development and downregulation of two virulence related genes. DMADDM significantly reduced the cell damage caused by multi-species biofilm according to the LDH activity and reduced the expression of IL-18 gene of the cells simultaneously. The in vivo histological evaluation proved that the addition of DMADDM less than 6.6% in denture material did not increase the inflammatory response (p > 0.05). Therefore, we proposed that the novel denture base resin containing DMADDM may be considered as a new promising therapeutic system against problems caused by microbes on denture base such as denture stomatitis.

Function and mechanism of bispecific antibodies targeting SARS-CoV-2.

As the dynamic evolution of SARS-CoV-2 led to reduced efficacy in monoclonal neutralizing antibodies and emergence of immune escape, the role of bispecific antibodies becomes crucial in bolstering antiviral activity and suppressing immune evasion. This review extensively assesses a spectrum of representative bispecific antibodies targeting SARS-CoV-2, delving into their characteristics, design formats, mechanisms of action, and associated advantages and limitations. The analysis encompasses factors influencing the selection of parental antibodies and strategies for incorporating added benefits in bispecific antibody design. Furthermore, how different classes of parental antibodies contribute to augmenting the broad-spectrum neutralization capability within bispecific antibodies is discussed. In summary, this review presents analyses and discussions aimed at offering valuable insights for shaping future strategies in bispecific antibody design to effectively confront the challenges posed by SARS-CoV-2 and propel advancements in antiviral therapeutic development.

Human anti-PSCA CAR macrophages possess potent antitumor activity against pancreatic cancer.

Due to the limitations of autologous chimeric antigen receptor (CAR)-T cells, alternative sources of cellular immunotherapy, including CAR macrophages, are emerging for solid tumors. Human induced pluripotent stem cells (iPSCs) offer an unlimited source for immune cell generation. Here, we develop human iPSC-derived CAR macrophages targeting prostate stem cell antigen (PSCA) (CAR-iMacs), which express membrane-bound interleukin (IL)-15 and truncated epidermal growth factor receptor (EGFR) for immune cell activation and a suicide switch, respectively. These allogeneic CAR-iMacs exhibit strong antitumor activity against human pancreatic solid tumors in vitro and in vivo, leading to reduced tumor burden and improved survival in a pancreatic cancer mouse model. CAR-iMacs appear safe and do not exhibit signs of cytokine release syndrome or other in vivo toxicities. We optimized the cryopreservation of CAR-iMac progenitors that remain functional upon thawing, providing an off-the-shelf, allogeneic cell product that can be developed into CAR-iMacs. Overall, our preclinical data strongly support the potential clinical translation of this human iPSC-derived platform for solid tumors, including pancreatic cancer.

Enhanced treatment of breast cancer brain metastases with oncolytic virus expressing anti-CD47 antibody and temozolomide.

Limited therapeutic options are available for patients with breast cancer brain metastases (BCBM), and thus there is an urgent need for novel treatment approaches. We previously engineered an effective oncolytic herpes simplex virus 1 (oHSV) expressing a full-length anti-CD47 monoclonal antibody (mAb) with a human IgG1 scaffold (OV-αCD47-G1) that was used to treat both ovarian cancer and glioblastoma. Here, we demonstrate that the combination of OV-αCD47-G1 and temozolomide (TMZ) improve outcomes in preclinical models of BCBM. The combination of TMZ with OV-αCD47-G1 synergistically increased macrophage phagocytosis against breast tumor cells and led to greater activation of NK cell cytotoxicity. In addition, the combination of OV-αCD47-G1 with TMZ significantly prolonged the survival of tumor-bearing mice when compared with TMZ or OV-αCD47-G1 alone. Combination treatment with the mouse counterpart of OV-αCD47-G1, termed OV-A4-IgG2b, also enhanced mouse macrophage phagocytosis, NK cell cytotoxicity, and survival in an immunocompetent model of mice bearing BCBM compared with TMZ or OV-A4-IgG2b alone. Collectively, these results suggest that OV-αCD47-G1 combined with TMZ should be explored in patients with BCBM.

Bi-functional CpG-STAT3 decoy oligonucleotide triggers multilineage differentiation of acute myeloid leukemia in mice.

Acute myeloid leukemia (AML) cells resist differentiation stimuli despite high expression of innate immune receptors, such as Toll-like receptor 9 (TLR9). We previously demonstrated that targeting Signal Transducer and Activator of Transcription 3 (STAT3) using TLR9-targeted decoy oligodeoxynucleotide (CpG-STAT3d) increases immunogenicity of human and mouse AML cells. Here, we elucidated molecular mechanisms of inv(16) AML reprogramming driven by STAT3-inhibition/TLR9-activation in vivo. At the transcriptional levels, AML cells isolated from mice after intravenous administration of CpG-STAT3d or leukemia-targeted Stat3 silencing and TLR9 co-stimulation, displayed similar upregulation of myeloid cell differentiation (Irf8, Cebpa, Itgam) and antigen-presentation (Ciita, Il12a, B2m)-related genes with concomitant reduction of leukemia-promoting Runx1. Single-cell transcriptomics revealed that CpG-STAT3d induced multilineage differentiation of AML cells into monocytes/macrophages, erythroblastic and B cell subsets. As shown by an inducible Irf8 silencing in vivo, IRF8 upregulation was critical for monocyte-macrophage differentiation of leukemic cells. TLR9-driven AML cell reprogramming was likely enabled by downregulation of STAT3-controlled methylation regulators, such as DNMT1 and DNMT3. In fact, the combination of DNA methyl transferase (DNMT) inhibition using azacitidine with CpG oligonucleotides alone mimicked CpG-STAT3d effects, resulting in AML cell differentiation, T cell activation, and systemic leukemia regression. These findings highlight immunotherapeutic potential of bi-functional oligonucleotides to unleash TLR9-driven differentiation of leukemic cells by concurrent STAT3 and/or DNMT inhibition.

A novel class of inhibitors that disrupts the stability of integrin heterodimers identified by CRISPR-tiling-instructed genetic screens.

The plasma membrane is enriched for receptors and signaling proteins that are accessible from the extracellular space for pharmacological intervention. Here we conducted a series of CRISPR screens using human cell surface proteome and integrin family libraries in multiple cancer models. Our results identified ITGAV (integrin αV) and its heterodimer partner ITGB5 (integrin ß5) as the essential integrin α/ß pair for cancer cell expansion. High-density CRISPR gene tiling further pinpointed the integral pocket within the ß-propeller domain of ITGAV for integrin αVß5 dimerization. Combined with in silico compound docking, we developed a CRISPR-Tiling-Instructed Computer-Aided (CRISPR-TICA) pipeline for drug discovery and identified Cpd_AV2 as a lead inhibitor targeting the ß-propeller central pocket of ITGAV. Cpd_AV2 treatment led to rapid uncoupling of integrin αVß5 and cellular apoptosis, providing a unique class of therapeutic action that eliminates the integrin signaling via heterodimer dissociation. We also foresee the CRISPR-TICA approach to be an accessible method for future drug discovery studies.

Blockade of the Immune Checkpoint CD47 by TTI-621 Potentiates the Response to Anti-PD-L1 in Cutaneous T-Cell Lymphoma.

Cutaneous T-cell lymphoma (CTCL) is an incurable and cosmetically disfiguring disease associated with microenvironmental signals. We investigated the effects of CD47 and PD-L1 immune checkpoint blockades, as a strategy for targeting both innate and adaptive immunity. CIBERSORT analysis identified the immune-cell composition in the CTCL tumor microenvironment and the immune checkpoint expression profile for each immune-cell gene cluster from CTCL lesions. We investigated the relationship between MYC and CD47 and PD-L1 expression and found that MYC short hairpin RNA knockdown and MYC functional suppression by TTI-621 (SIRPαFc) and anti-PD-L1 (durvalumab) in CTCL cell lines reduced the expression of CD47 and PDL1 mRNA and protein as measured by qPCR and flow cytometry, respectively. In vitro, blockade of the CD47-SIRPα interaction with TTI-621 increased the phagocytic activity of macrophages against CTCL cells and enhanced CD8+ T-cell-mediated killing in a mixed leucocyte reaction. Moreover, TTI-621 synergized with anti-PD-L1 in macrophages reprogram to M1-like phenotypes and inhibited CTCL cell growth. These effects were mediated by cell death-related pathways, including apoptosis, autophagy, and necroptosis. Collectively, our findings show that CD47 and PD-L1 are critical regulators of immune surveillance in CTCL and that dual targeting of CD47 and PD-L1 will provide insight into tumor immunotherapy for CTCL.

Pro-phagocytic function and structural basis of GPR84 signaling.

GPR84 is a unique orphan G protein-coupled receptor (GPCR) that can be activated by endogenous medium-chain fatty acids (MCFAs). The signaling of GPR84 is largely pro-inflammatory, which can augment inflammatory response, and GPR84 also functions as a pro-phagocytic receptor to enhance the phagocytic activities of macrophages. In this study, we first showed that the activation of GPR84 by the synthetic agonist 6-OAU could synergize with the blockade of CD47 on cancer cells to induce phagocytosis of cancer cells by macrophages. Then, we determined a high-resolution structure of the GPR84-Gi signaling complex with 6-OAU. This structure revealed a completely occluded binding pocket for 6-OAU, the molecular basis of receptor activation involving non-conserved structural motifs of GPR84, and an unusual Gi-coupling interface. Together with computational docking and simulations studies, our structure also suggested the mechanism for the high selectivity of GPR84 for MCFAs and the potential routes of ligand binding and dissociation. Our results provide a framework for understanding GPR84 signaling and developing new drugs targeting GPR84.

Reprogramming of PD-1+ M2-like tumor-associated macrophages with anti-PD-L1 and lenalidomide in cutaneous T cell lymphoma.

Cutaneous T cell lymphoma (CTCL) is a disfiguring and incurable disease characterized by skin-homing malignant T cells surrounded by immune cells that promote CTCL growth through an immunosuppressive tumor microenvironment (TME). Preliminary data from our phase I clinical trial of anti-programmed cell death ligand 1 (anti-PD-L1) combined with lenalidomide in patients with relapsed/refractory CTCL demonstrated promising clinical efficacy. In the current study, we analyzed the CTCL TME, which revealed a predominant PD-1+ M2-like tumor-associated macrophage (TAM) subtype with upregulated NF-κB and JAK/STAT signaling pathways and an aberrant cytokine and chemokine profile. Our in vitro studies investigated the effects of anti-PD-L1 and lenalidomide on PD-1+ M2-like TAMs. The combinatorial treatment synergistically induced functional transformation of PD-1+ M2-like TAMs toward a proinflammatory M1-like phenotype that gained phagocytic activity upon NF-κB and JAK/STAT inhibition, altered their migration through chemokine receptor alterations, and stimulated effector T cell proliferation. Lenalidomide was more effective than anti-PD-L1 in downregulation of the immunosuppressive IL-10, leading to decreased expression of both PD-1 and PD-L1. Overall, PD-1+ M2-like TAMs play an immunosuppressive role in CTCL. Anti-PD-L1 combined with lenalidomide provides a therapeutic strategy to enhance antitumor immunity by targeting PD-1+ M2-like TAMs in the CTCL TME.