Targeted deletion of CD244 on monocytes promotes differentiation into anti-tumorigenic macrophages and potentiates PD-L1 blockade in melanoma.

BACKGROUND: In the myeloid compartment of the tumor microenvironment, CD244 signaling has been implicated in immunosuppressive phenotype of monocytes. However, the precise molecular mechanism and contribution of CD244 to tumor immunity in monocytes/macrophages remains elusive due to the co-existing lymphoid cells expressing CD244. METHODS: To directly assess the role of CD244 in tumor-associated macrophages, monocyte-lineage-specific CD244-deficient mice were generated using cre-lox recombination and challenged with B16F10 melanoma. The phenotype and function of tumor-infiltrating macrophages along with antigen-specific CD8 T cells were analyzed by flow cytometry and single cell RNA sequencing data analysis, and the molecular mechanism underlying anti-tumorigenic macrophage differentiation, antigen presentation, phagocytosis was investigated ex vivo. Finally, the clinical feasibility of CD244-negative monocytes as a therapeutic modality in melanoma was confirmed by adoptive transfer experiments. RESULTS: CD244fl/flLysMcre mice demonstrated a significant reduction in tumor volume (61% relative to that of the CD244fl/fl control group) 14 days after tumor implantation. Within tumor mass, CD244fl/flLysMcre mice also showed higher percentages of Ly6Clow macrophages, along with elevated gp100+IFN-γ+ CD8 T cells. Flow cytometry and RNA sequencing data demonstrated that ER stress resulted in increased CD244 expression on monocytes. This, in turn, impeded the generation of anti-tumorigenic Ly6Clow macrophages, phagocytosis and MHC-I antigen presentation by suppressing autophagy pathways. Combining anti-PD-L1 antibody with CD244-/- bone marrow-derived macrophages markedly improved tumor rejection compared to the anti-PD-L1 antibody alone or in combination with wild-type macrophages. Consistent with the murine data, transcriptome analysis of human melanoma tissue single-cell RNA-sequencing dataset revealed close association between CD244 and the inhibition of macrophage maturation and function. Furthermore, the presence of CD244-negative monocytes/macrophages significantly increased patient survival in primary and metastatic tumors. CONCLUSION: Our study highlights the novel role of CD244 on monocytes/macrophages in restraining anti-tumorigenic macrophage generation and tumor antigen-specific T cell response in melanoma. Importantly, our findings suggest that CD244-deficient macrophages could potentially be used as a therapeutic agent in combination with immune checkpoint inhibitors. Furthermore, CD244 expression in monocyte-lineage cells serve as a prognostic marker in cancer patients.

Protein tyrosine phosphatases in lymphocyte activation and autoimmunity.

Lymphocyte activation must be tightly regulated to ensure sufficient immunity to pathogens and prevent autoimmunity. Protein tyrosine phosphatases (PTPs) serve critical roles in this regulation by controlling the functions of key receptors and intracellular signaling molecules in lymphocytes. In some cases, PTPs inhibit lymphocyte activation, whereas in others they promote it. Here we discuss recent progress in elucidating the roles and mechanisms of action of PTPs in lymphocyte activation. We also review the accumulating evidence that genetic alterations in PTPs are involved in human autoimmunity.

SLAMF7 is critical for phagocytosis of haematopoietic tumour cells via Mac-1 integrin.

Cancer cells elude anti-tumour immunity through multiple mechanisms, including upregulated expression of ligands for inhibitory immune checkpoint receptors. Phagocytosis by macrophages plays a critical role in cancer control. Therapeutic blockade of signal regulatory protein (SIRP)-α, an inhibitory receptor on macrophages, or of its ligand CD47 expressed on tumour cells, improves tumour cell elimination in vitro and in vivo, suggesting that blockade of the SIRPα-CD47 checkpoint could be useful in treating human cancer. However, the pro-phagocytic receptor(s) responsible for tumour cell phagocytosis is(are) largely unknown. Here we find that macrophages are much more efficient at phagocytosis of haematopoietic tumour cells, compared with non-haematopoietic tumour cells, in response to SIRPα-CD47 blockade. Using a mouse lacking the signalling lymphocytic activation molecule (SLAM) family of homotypic haematopoietic cell-specific receptors, we determined that phagocytosis of haematopoietic tumour cells during SIRPα-CD47 blockade was strictly dependent on SLAM family receptors in vitro and in vivo. In both mouse and human cells, this function required a single SLAM family member, SLAMF7 (also known as CRACC, CS1, CD319), expressed on macrophages and tumour cell targets. In contrast to most SLAM receptor functions, SLAMF7-mediated phagocytosis was independent of signalling lymphocyte activation molecule-associated protein (SAP) adaptors. Instead, it depended on the ability of SLAMF7 to interact with integrin Mac-1 (refs 18, 19, 20) and utilize signals involving immunoreceptor tyrosine-based activation motifs. These findings elucidate the mechanism by which macrophages engulf and destroy haematopoietic tumour cells. They also reveal a novel SAP adaptor-independent function for a SLAM receptor. Lastly, they suggest that patients with tumours expressing SLAMF7 are more likely to respond to SIRPα-CD47 blockade therapy.

The phosphatase PTP-PEST promotes secondary T cell responses by dephosphorylating the protein tyrosine kinase Pyk2.

PTP-PEST (encoded by Ptpn12) is an intracellular protein tyrosine phosphatase belonging to the same family as LYP. LYP inhibits secondary T cell responses by suppressing Src family protein tyrosine kinases and is implicated in human autoimmunity. To determine the function of PTP-PEST in T cells, we generated mice with a conditionally deleted allele of Ptpn12. By removing PTP-PEST in T cells, we determined that PTP-PEST was not necessary for T cell development or primary responses. However, PTP-PEST was required for secondary T cell responses, anergy prevention, and autoimmunity induction. PTP-PEST specifically regulated the phosphorylation of Pyk2, a substrate of the Src family kinase Fyn. It also promoted the formation of T cell homoaggregates, which are known to enhance T cell activation. Thus, PTP-PEST controls Pyk2 activity and is a positive regulator of secondary T cell activation. These data illustrate the critical role of protein tyrosine phosphatases in T cell regulation.

Important roles of protein tyrosine phosphatase PTPN12 in tumor progression.

Protein tyrosine phosphatases (PTPs), which are ubiquitously expressed in hematopoietic and non-hematopoietic cells, are critical for regulating cell proliferation as well as differentiation in the physiology of multicellular organisms. PTPs regulate the intracellular signaling mechanism of immune cells via dephosphorylation of multiple targets and are associated with the onset of various autoimmune diseases through genomic alterations. PTPs also affect disease through their role in innate and/or acquired immunity. By modulating multiple substrates, PTPN12, a member of the proline-, glutamic acid-, serine- and threonine-rich (PEST) family of PTPs, is an important regulator of cell migration and adhesion. According to its newly identified roles and functions, PTPN12 is considered a promising therapeutic target against critical diseases, including cancer, diabetes, metabolic disease and autoimmune diseases. In this review, we provide an overview of PTPs and discuss the critical roles of PTPN12/PTP-PEST in tumor progression.

Synthesis of Either C2- or C4'-Alkylated Derivatives of Honokiol and Their Biological Evaluation for Anti-inflammatory Activity.

Honokiol, a biphenolic neolignan isolated from Magnolia officinalis, was reported to have a promising anti-inflammatory activity for the treatment of various diseases. There are many efforts on the synthesis and structure-activity relationship of honokiol derivatives. However, regioselective O-alkylation of honokiol remains a challenge and serves as a tool to provide not only some derivatives but also chemical probes for target identification and mode of action. In this study, we examined the reaction condition for regioselective O-alkylation, in which C2 and C4'-alkylated analogs of honokiol were synthesized and evaluated for inhibitory activity on nitric oxide production and cyclooxygenase-2 expression. Furthermore, we successfully synthesized a potential photoaffinity probe consisting of biotin and benzophenone based on a C4'-alkylated derivative.

Synthesis of Natural Homoisoflavonoids Having Either 5,7-Dihydroxy-6-methoxy or 7-Hydroxy-5,6-dimethoxy Groups.

Naturally occurring homoisoflavonoids containing either 5,7-dihydroxy-6-methoxy or 7-hydroxy-5,6-dimethoxy groups such as the antiangiogenic homoisoflavanone, cremastranone, were synthesized via three or four linear steps from the known 4-chromenone. This facile synthesis includes chemoselective 1,4-reduction of 4-chromenone and selective deprotection of 3-benzylidene-4-chromanone a containing C7-benzyloxy group.

Metabolic circuits in neural stem cells.

Metabolic activity indicative of cellular demand is emerging as a key player in cell fate decision. Numerous studies have demonstrated that diverse metabolic pathways have a critical role in the control of the proliferation, differentiation and quiescence of stem cells. The identification of neural stem/progenitor cells (NSPCs) and the characterization of their development and fate decision process have provided insight into the regenerative potential of the adult brain. As a result, the potential of NSPCs in cell replacement therapies for neurological diseases is rapidly growing. The aim of this review is to discuss the recent findings on the crosstalk among key regulators of NSPC development and the metabolic regulation crucial for the function and cell fate decisions of NSPCs. Fundamental understanding of the metabolic circuits in NSPCs may help to provide novel approaches for reactivating neurogenesis to treat degenerative brain conditions and cognitive decline.

The phosphatase PTP-PEST/PTPN12 regulates endothelial cell migration and adhesion, but not permeability, and controls vascular development and embryonic viability.

Protein-tyrosine phosphatase (PTP)-PEST (PTPN12) is ubiquitously expressed. It is essential for normal embryonic development and embryonic viability in mice. Herein we addressed the involvement of PTP-PEST in endothelial cell functions using a combination of genetic and biochemical approaches. By generating primary endothelial cells from an inducible PTP-PEST-deficient mouse, we found that PTP-PEST is not needed for endothelial cell differentiation and proliferation or for the control of endothelial cell permeability. Nevertheless, it is required for integrin-mediated adhesion and migration of endothelial cells. PTP-PEST-deficient endothelial cells displayed increased tyrosine phosphorylation of Cas, paxillin, and Pyk2, which were previously also implicated in integrin functions. By eliminating PTP-PEST in endothelial cells in vivo, we obtained evidence that expression of PTP-PEST in endothelial cells is required for normal vascular development and embryonic viability. Therefore, PTP-PEST is a key regulator of integrin-mediated functions in endothelial cells seemingly through its capacity to control Cas, paxillin, and Pyk2. This function explains at least in part the essential role of PTP-PEST in embryonic development and viability.

PEST family phosphatases in immunity, autoimmunity, and autoinflammatory disorders.

The proline-, glutamic acid-, serine- and threonine-rich (PEST) family of protein tyrosine phosphatases (PTPs) includes proline-enriched phosphatase (PEP)/lymphoid tyrosine phosphatase (LYP), PTP-PEST, and PTP-hematopoietic stem cell fraction (HSCF). PEP/LYP is a potent inhibitor of T-cell activation, principally by suppressing the activity of Src family protein tyrosine kinases (PTKs). This function seems to be dependent, at least in part, on the ability of PEP to bind C-terminal Src kinase (Csk), a PTK also involved in inactivating Src kinases. Interestingly, a polymorphism of LYP in humans (R620W) is a significant risk factor for autoimmune diseases including type 1 diabetes, rheumatoid arthritis, and lupus. The R620W mutation may be a 'gain-of-function' mutation. In non-hematopoietic cells, PTP-PEST is a critical regulator of adhesion and migration. This effect correlates with the aptitude of PTP-PEST to dephosphorylate cytoskeletal proteins such as Cas, focal adhesion associated-kinase (FAK), Pyk2, and PSTPIP. While not established, a similar function may also exist in immune cells. Additionally, overexpression studies provided an indication that PTP-PEST may be a negative regulator of lymphocyte activation. Interestingly, mutations in a PTP-PEST- and PTP-HSCF-interacting protein, PSTPIP1, were identified in humans with pyogenic sterile arthritis, pyoderma gangrenosum, and acne (PAPA) syndrome and familial recurrent arthritis, two autoinflammatory diseases. These mutations abrogate the ability of PSTPIP1 to bind PTP-PEST and PTP-HSCF, suggesting that these two PTPs may be negative regulators of inflammation.

Inflammasome as a promising therapeutic target for cancer.

Inflammasomes are the major mechanistic complexes that include members of the NOD-like receptor (NLRs) or AIM2-like receptors (ALRs) families, which are affiliated with the innate immune system. Once NLRs or ALRs are activated by pathogen-associated molecular patterns (PAMPs) or damage-associated molecular patterns (DAMPs), the caspase-1 or -11 is activated by binding with NLRs or ALRs via its own unique cytosolic domains. As a result, caspase-1 or -11 enhances the production of IL-1ß and IL-18, which results in inflammation via the recruitment of immune cells, such as macrophages, and the promotion of programmed cell death mechanisms such as pyroptosis. In addition, the consistent cascades of inflammasomes would precede both minor and severe autoimmune diseases and cancers. The clinical relevance of inflammasomes in multiple forms of cancer highlights their therapeutic promise as molecular targets. To closely analyze the physiological roles of inflammasomes in cancers, here, we describe the fundamental knowledge regarding the current issues of inflammasomes in relevant cancers, and discuss possible therapeutic values in targeting these inflammasomes for the prevention and treatment of cancer.

Krill Oil-Incorporated Liposomes As An Effective Nanovehicle To Ameliorate The Inflammatory Responses Of DSS-Induced Colitis.

BACKGROUND: Phosphatidylcholine (PC) and Omega-3 fatty acid (Omega-3) are promising therapeutic molecules for treating inflammatory bowel disease (IBD). PURPOSE: Based on the IBD therapeutic potential of nanoparticles, we herein sought to develop Omega-3-incorporated PC nanoparticles (liposomes) as an orally administrable vehicle for treating IBD. METHODS: Liposomes prepared with or without Omega-3 incorporation were compared in terms of colloidal stability and anitiinflammatory effects. RESULTS: The incorporation of free Omega-3 (alpha-linolenic acid, eicosapentaenoic acid or docosahexaenoic acid) into liposomes induced time-dependent membrane fusion, resulting in particle size increase from nm to µm during storage. In contrast, krill oil incorporation into liposomes (KO liposomes) did not induce the fusion and the particle size maintained <250 nm during storage. KO liposomes also maintained colloidal stability in simulated gastrointestinal conditions and exhibited a high capacity to entrap the IBD drug, budesonide (BDS). KO liposomes greatly suppressed the lipopolysaccharide-induced production of pro-inflammatory cytokines in cultured macrophages and completely restored inflammation-impaired membrane barrier function in an intestinal barrier model. In mice subjected to dextran sulfate sodium-induced colitis, oral administration of BDS-entrapped KO liposomes suppressed tumor necrosis factor-α production (by 84.1%), interleukin-6 production (by 35.3%), and the systemic level of endotoxin (by 96.8%), and slightly reduced the macroscopic signs of the disease. CONCLUSION: Taken together, KO liposomes may have great potential as a nanovehicle for oral delivery of IBD drugs.

Distinct features of dendritic cell-based immunotherapy as cancer vaccines.

Dendritic cells (DCs) are the most professional antigen presenting cells that play important roles in connection between innate and adaptive immune responses. Numerous studies revealed that the functions of DCs are related with the capture and processing of antigen as well as the migration to lymphoid tissues for the presenting antigens to T cells. These unique features of DCs allow them to be considered as therapeutic vaccines that can induce immune responses and anti-tumor activity. Here, we discuss and understand the immunological basis of DCs and presume the possibilities of DC-based vaccines for the promising cancer therapy.

Potentiation of Th1-Type Immune Responses to Mycobacterium tuberculosis Antigens in Mice by Cationic Liposomes Combined with De-O-Acylated Lipooligosaccharide.

Tuberculosis (TB) is an infectious disease caused by Mycobacterium tuberculosis. Bacillus Calmette-Guérin (BCG) vaccine is the only TB vaccine currently available, but it is not sufficiently effective in preventing active pulmonary TB or adult infection. With the purpose of developing an improved vaccine against TB that can overcome the limitations of the current BCG vaccine, we investigated whether adjuvant formulations containing de-O-acylated lipooligosaccharide (dLOS) are capable of enhancing the immunogenicity and protective efficacy of TB subunit vaccine. The results revealed that dLOS/dimethyl dioctadecyl ammonium bromide (DDA) adjuvant formulation significantly increased both humoral and Th1-type cellular responses to TB subunit vaccine that are composed of three antigens, Ag85A, ESAT-6, and HspX. The adjuvanted TB vaccine also effectively induced Th1-type response in a BCG-primed mouse model, suggesting a potential as a booster vaccine. Finally, dLOS/DDA-adjuvanted TB vaccine showed protective efficacy against M. tuberculosis infection in vitro and in vivo. These data indicate that dLOS/DDA adjuvant enhances the Th1-type immunity and protective efficacy of TB subunit vaccine suggesting that it would be a promising adjuvant candidate for development of a booster vaccine.

Comparison of the adjuvanticity of two adjuvant formulations containing de-O-acylated lipooligosaccharide on Japanese encephalitis vaccine in mice.

Adjuvants are essential vaccine components used to enhance, accelerate, and/or prolong adaptive immunity against specific vaccine antigens. In this study, we compared the adjuvanticity of two adjuvant formulations containing de-O-acylated lipooligosaccharide (dLOS), a toll-like receptor 4 agonist, on the Japanese encephalitis (JE) vaccine in mice. Mice were immunized once or twice at a two-week interval with inactivated JE vaccine in the absence or presence of adjuvant. We found that both the alum- and the liposome-based formulation induced significantly faster and higher serum IgG antibody responses as compared with the non-adjuvanted vaccine after either one or two immunizations. The antibody titers of the mouse immune sera correlated with 50% plaque reduction neutralization test (PRNT50) antibody titers. In addition, the dLOS/liposome formulation was more effective in inducing a Th1-type immune response than the dLOS/alum formulation, as suggested by a strong antigen-specific interferon (IFN)-γ response. Based on these results, we suggest that both alum- and liposome-based adjuvant formulations containing dLOS may be used for the development of JE vaccines with improved immunogenicity.

Cytokine Signaling in Tumor Progression.

Cytokines are molecules that play critical roles in the regulation of a wide range of normal functions leading to cellular proliferation, differentiation and survival, as well as in specialized cellular functions enabling host resistance to pathogens. Cytokines released in response to infection, inflammation or immunity can also inhibit cancer development and progression. The predominant intracellular signaling pathway triggered by cytokines is the JAK-signal transducer and activator of transcription (STAT) pathway. Knockout mice and clinical human studies have provided evidence that JAK-STAT proteins regulate the immune system, and maintain immune tolerance and tumor surveillance. Moreover, aberrant activation of the JAK-STAT pathways plays an undeniable pathogenic role in several types of human cancers. Thus, in combination, these observations indicate that the JAK-STAT proteins are promising targets for cancer therapy in humans. The data supporting this view are reviewed herein.

2-Hydroxyoleic acid-inserted liposomes as a multifunctional carrier of anticancer drugs.

Studies have shown that insertion of oleic acid into lipid bilayers can modulate the membrane properties of liposomes so as to improve their function as drug carriers. Considering that 2-hydroxyoleic acid (2OHOA), a potential antitumor agent currently undergoing clinical trials, is a derivative of oleic acid, we explored the possibility of developing 2OHOA-inserted liposomes as a multifunctional carrier of antitumor drugs in the present study. The insertion of 2OHOA into lipid bilayers was confirmed by surface charge determination and differential scanning calorimetry. 2OHOA insertion greatly decreased the order of dimyristoylphosphatidylcholine packing, produced a nanosized (<100 nm) dispersion, and improved the colloidal stability of liposomes during storage. Moreover, 2OHOA-inserted liposome forms exhibited greater growth inhibitory activity against cancer cells compared with free 2OHOA, and the growth-inhibitory activity of liposomal 2OHOA was selective for tumor cells. 2OHOA insertion greatly increased the liposome-incorporated concentration of hydrophobic model drugs, including mitoxantrone, paclitaxel, and all-trans retinoic acid (ATRA). The in vitro anticancer activity of ATRA-incorporated/2OHOA-inserted liposomes was significantly higher than that of ATRA-incorporated conventional liposomes. In a B16-F10 melanoma syngeneic mouse model, the tumor growth rate was significantly delayed in mice treated with ATRA-incorporated/2OHOA-inserted liposomes compared with that in the control group. Immunohistochemical analyses revealed that the enhanced antitumor activity of ATRA-incorporated/2OHOA-inserted liposomes was due, at least in part, to increased induction of apoptosis. Collectively, our findings indicate that 2OHOA-inserted liposomes exhibit multiple advantages as antitumor drug carriers, including the ability to simultaneously deliver two anticancer drugs - 2OHOA and incorporated drug - to the tumor tissue.

Diverse macrophages polarization in tumor microenvironment.

Macrophages are traditional innate immune cells that play critical roles in the clearance of pathogens and the maintenance of tissue homeostasis. Accumulating evidence proves that macrophages affect cancer initiation and malignancy. Macrophages can be categorized into two extreme subsets, classically activated (M1) and alternatively activated (M2) macrophages based on their distinct functional abilities in response to microenvironmental stimuli. In a tumor microenvironment, tumor associated macrophages (TAMs) are considered to be of the polarized M2 phenotype that enhances tumor progression and represent a poor prognosis. Furthermore, TAMs enhance tumor angiogenesis, growth, metastasis, and immunosuppression by secreting a series of cytokines, chemokines, and proteases. The regulation of macrophage polarization is considered to be a potential future therapy for cancer management.

Control of dendritic cell migration, T cell-dependent immunity, and autoimmunity by protein tyrosine phosphatase PTPN12 expressed in dendritic cells.

Dendritic cells (DCs) capture and process antigens in peripheral tissues, migrate to lymphoid tissues, and present the antigens to T cells. PTPN12, also known as PTP-PEST, is an intracellular protein tyrosine phosphatase (PTP) involved in cell-cell and cell-substratum interactions. Herein, we examined the role of PTPN12 in DCs, using a genetically engineered mouse lacking PTPN12 in DCs. Our data indicated that PTPN12 was not necessary for DC differentiation, DC maturation, or cytokine production in response to inflammatory stimuli. However, it was needed for full induction of T cell-dependent immune responses in vivo. This function largely correlated with the need of PTPN12 for DC migration from peripheral sites to secondary lymphoid tissues. Loss of PTPN12 in DCs resulted in hyperphosphorylation of the protein tyrosine kinase Pyk2 and its substrate, the adaptor paxillin. Pharmacological inhibition of Pyk2 or downregulation of Pyk2 expression also compromised DC migration, suggesting that Pyk2 deregulation played a pivotal role in the migration defect caused by PTPN12 deficiency. Together, these findings identified PTPN12 as a key regulator in the ability of DCs to induce antigen-induced T cell responses. This is due primarily to the role of PTPN12 in DC migration from peripheral sites to secondary lymphoid organs through regulation of Pyk2.

Macrophage fusion is controlled by the cytoplasmic protein tyrosine phosphatase PTP-PEST/PTPN12.

Macrophages can undergo cell-cell fusion, leading to the formation of multinucleated giant cells and osteoclasts. This process is believed to promote the proteolytic activity of macrophages toward pathogens, foreign bodies, and extracellular matrices. Here, we examined the role of PTP-PEST (PTPN12), a cytoplasmic protein tyrosine phosphatase, in macrophage fusion. Using a macrophage-targeted PTP-PEST-deficient mouse, we determined that PTP-PEST was not needed for macrophage differentiation or cytokine production. However, it was necessary for interleukin-4-induced macrophage fusion into multinucleated giant cells in vitro. It was also needed for macrophage fusion following implantation of a foreign body in vivo. Moreover, in the RAW264.7 macrophage cell line, PTP-PEST was required for receptor activator of nuclear factor kappa-B ligand (RANKL)-triggered macrophage fusion into osteoclasts. PTP-PEST had no impact on expression of fusion mediators such as ß-integrins, E-cadherin, and CD47, which enable macrophages to become fusion competent. However, it was needed for polarization of macrophages, migration induced by the chemokine CC chemokine ligand 2 (CCL2), and integrin-induced spreading, three key events in the fusion process. PTP-PEST deficiency resulted in specific hyperphosphorylation of the protein tyrosine kinase Pyk2 and the adaptor paxillin. Moreover, a fusion defect was induced upon treatment of normal macrophages with a Pyk2 inhibitor. Together, these data argue that macrophage fusion is critically dependent on PTP-PEST. This function is seemingly due to the ability of PTP-PEST to control phosphorylation of Pyk2 and paxillin, thereby regulating cell polarization, migration, and spreading.