Discovery of an immunosuppressive functional metabolite from the insect-derived endophytic Aspergillus taichungensis SMU01.

Three p-terphenyl metabolites (1-3), three indole-diterpenoids (4-6), an herbicide sesquiterpene (7), a flavonoid (8), and five other small molecules containing nitrogen (9-13) were isolated from the medicinal insect (Periplaneta americana)-derived endophytic Aspergillus taichungensis SMU01. Their chemical structures were elucidated on the basis of spectroscopic data and quantum chemical computational methods. Biological activity of these isolates in the differentiation of mouse CD4+ T cell subsets was evaluated. Importantly, metabolites 2 targeting JAK-STAT signaling pathway could hold potential benefits in maintaining peripheral immune homeostasis and alleviating the progression of autoimmune diseases.

circIFNGR2 regulating ankylosing spondylitis-associated inflammation through macrophage polarization.

Macrophages activation is crucial in pathogenesis of rheumatic diseases like ankylosing spondylitis (AS). Circular RNAs (circRNAs)-induced macrophage-associated inflammation participates in many autoimmune diseases but remains elusive in AS. Here, we verified increased expression of circIFNGR2 in peripheral blood mononuclear cells from patients with AS and its expression levels were correlated with the AS severity. In vitro assays revealed that circIFNGR2 enhances macrophage proliferation, and regulates M1/M2 macrophage polarization and NF-κB/Akt pathways. We identified that circIFNGR2 promoted the expression of iNOS/TNFα and M1 polarization, and restrained M2 polarization by sponging miR-939. Additionally, the RNA-binding protein, eIF4A3, was found to enhance the production of circIFNGR2. Interestingly, miR-939 attenuated joint damage in collagen-induced arthritis mice, whereas circIFNGR2 reversed this effect. Our findings highlight the pro-inflammatory roles of eIF4A3-induced circIFNGR2 in AS by modulating macrophage-associated inflammation through miR-939.

Long-term Follow-up of Detaenial Sigmoid Neobladder Reconstruction for Paediatric Patients with Bladder and Prostate Rhabdomyosarcoma: Technique and Results from a Single High-volume Centre.

BACKGROUND: Rhabdomyosarcoma (RMS) is the most common paediatric soft-tissue sarcoma. Approximately 15-20% of RMS cases arise from the bladder and prostate (B/P). The optimal treatment strategy for B/P RMS remains unclear. OBJECTIVE: To retrospectively evaluate the applicability of our procedure performed to treat paediatric patients with B/P RMS. DESIGN, SETTING, AND PARTICIPANTS: This is a retrospective analysis from a single tertiary referral hospital. From August 2003 to March 2021, 62 children pathologically diagnosed with B/P RMS underwent radical cystectomy and orthotopic detaenial sigmoid neobladder reconstruction in our centre. SURGICAL PROCEDURE: Surgical procedures included laparoscopic radical cystectomy and detaenial sigmoid neobladder reconstruction, which is demonstrated in the accompanying video. MEASUREMENTS: Demographic, clinical, and follow-up data were collected. Perioperative and long-term oncological and functional outcomes were reported. A logistic regression analysis was also performed. RESULTS AND LIMITATIONS: All surgeries, including three intracorporeal laparoscopic surgeries, were completed successfully. Of the 62 patients, 54 were alive without evidence of disease recurrence or metastasis at the last follow-up. Five of the 14 >12-yr-old boys reported that they experienced erections. Two female patients >12 yr old reported that they menstruated. However, this was a retrospective study conducted at a single centre with limited surgeon experience. CONCLUSIONS: Our results confirmed the safety and feasibility of primary orthotopic sigmoid neobladder reconstruction after radical cystectomy for paediatric patients with B/P RMS. Good outcomes in terms of oncological control and functional recovery were achieved. The high histocompatibility and tissue adaptability of children are inspiring. PATIENT SUMMARY: We describe our stepwise technique of radical cystectomy and detaenial sigmoid neobladder reconstruction for paediatric patients with bladder and prostate rhabdomyosarcoma. With this technique, we were able to achieve good functional recovery without compromising cancer control and significantly increasing complications.

Aspertaichunol A, an Immunomodulatory Polyketide with an Uncommon Scaffold from the Insect-Derived Endophytic Aspergillus taichungensis SMU01.

Aspertaichunol A (1), a polyketide with a novel scaffold, was isolated from the medicinal insect (Periplaneta americana)-derived endophytic Aspergillus taichungensis SMU01. Its structure was assigned on the basis of spectroscopic data and quantum chemical computational methods. Notably, 1 possesses an uncommon tricyclo[6.2.0.02,6]decane motif and an unusual bridgehead double bond (anti-Bredt system). A plausible biosynthetic pathway, involving a key intramolecular [2+2] cycloaddition and a reductive cyclization, is postulated. Finally, the immunomodulatory activity of 1 at 20 nM was evaluated by promoting Th9 cell differentiation from naive CD4+CD62L+ T cells.

IL-9/STAT3/fatty acid oxidation-mediated lipid peroxidation contributes to Tc9 cell longevity and enhanced antitumor activity.

CD8+ T cell longevity regulated by metabolic activity plays important roles in cancer immunotherapy. Although in vitro-polarized, transferred IL-9-secreting CD8+ Tc9 (cytotoxic T lymphocyte subset 9) cells exert greater persistence and antitumor efficacy than Tc1 cells, the underlying mechanism remains unclear. Here, we show that tumor-infiltrating Tc9 cells display significantly lower lipid peroxidation than Tc1 cells in several mouse models, which is strongly correlated with their persistence. Using RNA-sequence and functional validation, we found that Tc9 cells exhibited unique lipid metabolic programs. Tc9 cell-derived IL-9 activated STAT3, upregulated fatty acid oxidation and mitochondrial activity, and rendered Tc9 cells with reduced lipid peroxidation and resistance to tumor- or ROS-induced ferroptosis in the tumor microenvironment. IL-9 signaling deficiency, inhibiting STAT3, or fatty acid oxidation increased lipid peroxidation and ferroptosis of Tc9 cells, resulting in impaired longevity and antitumor ability. Similarly, human Tc9 cells also exhibited lower lipid peroxidation than Tc1 cells and tumor-infiltrating CD8+ T cells expressed lower IL9 and higher lipid peroxidation- and ferroptosis-related genes than circulating CD8+ T cells in patients with melanoma. This study indicates that lipid peroxidation regulates Tc9 cell longevity and antitumor effects via the IL-9/STAT3/fatty acid oxidation pathway and regulating T cell lipid peroxidation can be used to enhance T cell-based immunotherapy in human cancer.

RARγ activation sensitizes human myeloma cells to carfilzomib treatment through the OAS-RNase L innate immune pathway.

Proteasome inhibitors (PIs) such as bortezomib (Btz) and carfilzomib (Cfz) are highly efficacious for patients with multiple myeloma (MM). However, relapses are frequent, and acquired resistance to PI treatment emerges in most patients. Here, we performed a high-throughput screen of 1855 Food and Drug Administration (FDA)-approved drugs and identified all-trans retinoic acid (ATRA), which alone has no antimyeloma effect, as a potent drug that enhanced MM sensitivity to Cfz-induced cytotoxicity and resensitized Cfz-resistant MM cells to Cfz in vitro. ATRA activated retinoic acid receptor (RAR)γ and interferon-ß response pathway, leading to upregulated expression of IRF1. IRF1 in turn initiated the transcription of OAS1, which synthesized 2-5A upon binding to double-stranded RNA (dsRNA) induced by Cfz and resulted in cellular RNA degradation by RNase L and cell death. Similar to ATRA, BMS961, a selective RARγ agonist, could also (re)sensitize MM cells to Cfz in vitro, and both ATRA and BMS961 significantly enhanced the therapeutic effects of Cfz in established MM in vivo. In support of these findings, analyses of large datasets of patients' gene profiling showed a strong and positive correlation between RARγ and OAS1 expression and patient's response to PI treatment. Thus, this study highlights the potential for RARγ agonists to sensitize and overcome MM resistance to Cfz treatment in patients.

CD36-mediated ferroptosis dampens intratumoral CD8+ T cell effector function and impairs their antitumor ability.

Understanding the mechanisms underlying how T cells become dysfunctional in a tumor microenvironment (TME) will greatly benefit cancer immunotherapy. We found that increased CD36 expression in tumor-infiltrating CD8+ T cells, which was induced by TME cholesterol, was associated with tumor progression and poor survival in human and murine cancers. Genetic ablation of Cd36 in effector CD8+ T cells exhibited increased cytotoxic cytokine production and enhanced tumor eradication. CD36 mediated uptake of fatty acids by tumor-infiltrating CD8+ T cells in TME, induced lipid peroxidation and ferroptosis, and led to reduced cytotoxic cytokine production and impaired antitumor ability. Blocking CD36 or inhibiting ferroptosis in CD8+ T cells effectively restored their antitumor activity and, more importantly, possessed greater antitumor efficacy in combination with anti-PD-1 antibodies. This study reveals a new mechanism of CD36 regulating the function of CD8+ effector T cells and therapeutic potential of targeting CD36 or inhibiting ferroptosis to restore T cell function.

Identification of an immunogenic DKK1 long peptide for immunotherapy of human multiple myeloma.

Dickkopf-1 (DKK1), broadly expressed by tumor cells from human multiple myeloma (MM) and other cancers but absent from most normal tissues, may be an ideal target for immunotherapy. Our previous studies have shown that DKK1 (peptide)-specific cytotoxic T lymphocytes can effectively lyse primary MM cells in vitro. To develop DKK1-based vaccines that can be easily and inexpensively made and used by all patients, we identified a DKK1 long peptide (LP), DKK13-76-LP, that contains 74 amino acids and epitopes that can potentially bind to all major MHC class I and II molecules. Using HLA-A*0201- and HLA-DR*4-transgenic mouse models, we found that DKK1-specific CD4+ and CD8+ T-cell responses, detected by DKK1 short peptide (P20 and P66v)-HLA-A*0201 tetramer staining and cytotoxic assay for CD8+ T cells or by carboxyfluorescein diacetate succinimidyl ester (CSFE) dilution and IFN-g secretion for CD4+ T cells, respectively, can be induced in vivo by immunizing mice with the DKK13-76-LP. In addition, DKK13-76-LP also induced anti-DKK1 humoral immunity in the transgenic mice and the DKK1 antibodies were functional. Finally, DKK13-76-LP stimulated human blood T cells ex vivo to generate DKK1-specific CD4+ and CD8+ T-cell responses from 8 out of 10 MM patients with different MHC backgrounds. The generated DKK1-specific CD8+ cells efficiently lysed autologous MM cells from these patients. Thus, these results confirm the immunogenicity of the DKK13-76-LP in eliciting DKK1-specific CD4+ and CD8+ T-cell responses in vitro and in vivo, and suggest that the DKK13-76-LP can be used for immunotherapy of MM and other cancers.

Hsa_circ_0000652 Aggravates Inflammation by Activation of Macrophages and Enhancement of OX40/OX40L Interaction in Ankylosing Spondylitis.

Circular RNAs (circRNAs) have emerged as important roles in various inflammatory processes of rheumatic diseases. However, their expression profiles and influences in the pathogenesis of ankylosing spondylitis (AS) remain unclear. In this study, we revealed the differential expression profiles of circRNAs in peripheral blood mononuclear cells (PBMCs) in AS by circRNA sequencing. We screened the differentially expressed circRNAs in AS and verified that hsa_circ_0000652 was upregulated and had potential to be a biomarker of progression. Functionally, hsa_circ_0000652 promoted proliferation and cytokine production in macrophages and inhibited apoptosis. Through dual-luciferase assays and RNA pull-down assays, we demonstrated that hsa_circ_0000652 acted as a competing endogenous RNA (ceRNA) by binding with hsa-miR-1179 and regulated OX40L, which is characterized as a co-stimulatory molecule and found to be upregulated in AS patients. As a result, hsa_circ_0000652 aggravated the inflammation in the coculture system containing CD4+ T cells and macrophages via OX40/OX40L interaction. Our findings suggest that hsa_circ_0000652 was upregulated in AS patients and may serve as a pro-inflammatory factor in macrophages and a positive regulator of OX40/OX40L by sponging hsa-miR-1179.

Enhanced CAR-T activity against established tumors by polarizing human T cells to secrete interleukin-9.

CAR-T cell therapy is effective for hematologic malignancies. However, considerable numbers of patients relapse after the treatment, partially due to poor expansion and limited persistence of CAR-T cells in vivo. Here, we demonstrate that human CAR-T cells polarized and expanded under a Th9-culture condition (T9 CAR-T) have an enhanced antitumor activity against established tumors. Compared to IL2-polarized (T1) cells, T9 CAR-T cells secrete IL9 but little IFN-γ, express central memory phenotype and lower levels of exhaustion markers, and display robust proliferative capacity. Consequently, T9 CAR-T cells mediate a greater antitumor activity than T1 CAR-T cells against established hematologic and solid tumors in vivo. After transfer, T9 CAR-T cells migrate effectively to tumors, differentiate to IFN-γ and granzyme-B secreting effector memory T cells but remain as long-lived and hyperproliferative T cells. Our findings are important for the improvement of CAR-T cell-based immunotherapy for human cancers.

MIF as a biomarker and therapeutic target for overcoming resistance to proteasome inhibitors in human myeloma.

Multiple myeloma (MM) remains largely incurable despite significant advances in biotherapy and chemotherapy. The development of drug resistance is a major problem in MM management. Macrophage migration inhibitory factor (MIF) expression was significantly higher in purified MM cells from relapsed patients than those with sustained response, and MM patients with high MIF had significantly shorter progression-free survival (PFS) and overall survival (OS). MM cell lines also express high levels of MIF, and knocking out MIF made them more sensitive to proteasome inhibitor (PI)-induced apoptosis not observed with other chemotherapy drugs. Mechanistic studies showed that MIF protects MM cells from PI-induced apoptosis by maintaining mitochondrial function via suppression of superoxide production in response to PIs. Specifically, MIF, in the form of a homotrimer, acts as a chaperone for superoxide dismutase 1 (SOD1) to suppress PI-induced SOD1 misfolding and to maintain SOD1 activity. MIF inhibitor 4-iodo-6-phenylpyrimidine and homotrimer disrupter ebselen, which do not kill MM cells, enhanced PI-induced SOD1 misfolding and loss of function, resulting in significantly more cell death in both cell lines and primary MM cells. More importantly, inhibiting MIF activity in vivo displayed synergistic antitumor activity with PIs and resensitized PI-resistant MM cells to treatment. In support of these findings, gene-profiling data showed a significantly negative correlation between MIF and SOD1 expression and response to PI treatment in patients with MM. This study shows that MIF plays a crucial role in MM sensitivity to PIs and suggests that targeting MIF may be a promising strategy to (re)sensitize MM to the treatment.

Enhanced Lipid Accumulation and Metabolism Are Required for the Differentiation and Activation of Tumor-Associated Macrophages.

Tumor-associated macrophages (TAM) are important tumor-promoting cells. However, the mechanisms underlying how the tumor and its microenvironment reprogram these cells remain elusive. Here we report that lipids play a crucial role in generating TAMs in the tumor microenvironment (TME). Macrophages from both human and murine tumor tissues were enriched with lipids due to increased lipid uptake by macrophages. TAMs expressed elevated levels of the scavenger receptor CD36, accumulated lipids, and used fatty acid oxidation (FAO) instead of glycolysis for energy. High levels of FAO promoted mitochondrial oxidative phosphorylation, production of reactive oxygen species, phosphorylation of JAK1, and dephosphorylation of SHP1, leading to STAT6 activation and transcription of genes that regulate TAM generation and function. These processes were critical for TAM polarization and activity, both in vitro and in vivo. In summary, we highlight the importance of lipid metabolism in the differentiation and function of protumor TAMs in the TME. SIGNIFICANCE: This study highlights the role of lipid metabolism in the differentiation and function of TAMs and suggests targeting TAM fatty acid oxidation as a potential therapeutic modality for human cancers.

Cholesterol Induces CD8+ T Cell Exhaustion in the Tumor Microenvironment.

Tumor-infiltrating T cells often lose their effector function; however, the mechanisms are incompletely understood. We report that cholesterol in the tumor microenvironment induces CD8+ T cell expression of immune checkpoints and exhaustion. Tumor tissues enriched with cholesterol and cholesterol content in tumor-infiltrating CD8+ T cells were positively and progressively associated with upregulated T cell expression of PD-1, 2B4, TIM-3, and LAG-3. Adoptively transferred CD8+ T cells acquired cholesterol, expressed high levels of immune checkpoints, and became exhausted upon entering a tumor. Tumor culture supernatant or cholesterol induced immune checkpoint expression by increasing endoplasmic reticulum (ER) stress in CD8+ T cells. Consequently, the ER stress sensor XBP1 was activated and regulated PD-1 and 2B4 transcription. Inhibiting XBP1 or reducing cholesterol in CD8+ T cells effectively restored antitumor activity. This study reveals a mechanism underlying T cell exhaustion and suggests a new strategy for restoring T cell function by reducing cholesterol to enhance T cell-based immunotherapy.

TNF-α enhances Th9 cell differentiation and antitumor immunity via TNFR2-dependent pathways.

Tumor specific Th9 cells are potential effector cells for adoptive therapy of human cancers. TNF family members OX40L, TL1A and GITRL have been shown to promote the induction of Th9 cells and antitumor immunity. However, the role of TNF-α, the prototype of the TNF superfamily cytokines, in Th9 cell differentiation and their antitumor efficacy is not defined. Here, we showed that TNF-α potently promoted naïve CD4+ T cells to differentiate into Th9 cells in vitro. Furthermore, the addition of TNF-α during Th9 cell differentiation increased T cell survival and proliferation. More importantly, the adoptive transfer of TNF-α-treated Th9 cells induced more potent antitumor effects than regular Th9 cells in mouse tumor model. TNF-α signals via two cell surface receptors, TNFR1 and TNFR2. Mechanistic studies revealed that TNF-α drove Th9 cell differentiation through TNFR2 but not TNFR1. In addition, under Th9 polarizing condition, TNF-α activated STAT5 and NF-κB pathways in T cells in a TNFR2-dependent manner. Inhibition of STAT5 and NF-κB pathways by their specific inhibitors impaired TNF-α-induced Th9 cell differentiation. Our results identified TNF-α as a new powerful inducer of Th9 cells and clarified the molecular mechanisms underlying TNF-α-induced Th9 cell differentiation.

E-cadherin expression on multiple myeloma cells activates tumor-promoting properties in plasmacytoid DCs.

Plasmacytoid dendritic cells (pDCs) play a key role in antiviral responses by producing type-1 IFNs. However, recent studies showed that pDCs induce immune suppression and promote tumor growth in human ovarian cancer and myeloma. The molecular mechanisms underlying pDC acquisition of these properties are unknown. Here we show that human pDCs activated by CpG inhibited growth and induced apoptosis in myeloma cells via secreted IFN-α, but direct contact with myeloma cells converted pDCs into tumor-promoting cells by suppressing pDC IFN-α production. E-cadherin, expressed on both myeloma cells and pDCs, mediated these effects via a homophilic interaction - activation of E-cadherin signaling upregulated and activated TNFAIP3 to interact with TLR9, resulting in TLR9 ubiquitination and degradation, and inhibition of IFN-α production in pDCs. These findings were supported by an in vivo study in which pDC depletion induced tumor regression and better survival in the Vk*MYC myeloma mouse model. Furthermore, IFNAR1 expression level positively correlated to overall survival of patients with multiple myeloma (MM), and the IFN-α level in patient bone marrow was significantly lower than that in marrow of healthy individuals. This study reveals a novel mechanism underlying how MM tumors educate pDCs in their microenvironment and provides new targets for improving the treatment of MM.

Th9 Cells Represent a Unique Subset of CD4+ T Cells Endowed with the Ability to Eradicate Advanced Tumors.

The antitumor effector T helper 1 (Th1) and Th17 cells represent two T cell paradigms: short-lived cytolytic Th1 cells and "stem cell-like" memory Th17 cells. We report that Th9 cells represent a third paradigm-they are less-exhausted, fully cytolytic, and hyperproliferative. Only tumor-specific Th9 cells completely eradicated advanced tumors, maintained a mature effector cell signature with cytolytic activity as strong as Th1 cells, and persisted as long as Th17 cells in vivo. Th9 cells displayed a unique Pu.1-Traf6-NF-κB activation-driven hyperproliferative feature, suggesting a persistence mechanism rather than an antiapoptotic strategy. Th9 antitumor efficacy depended on interleukin-9 and upregulated expression of Eomes and Traf6. Thus, tumor-specific Th9 cells are a more effective CD4+ T cell subset for adoptive cancer therapy.

Cholesterol negatively regulates IL-9-producing CD8+ T cell differentiation and antitumor activity.

CD8+ T cells can be polarized into IL-9-secreting (Tc9) cells. We previously showed that adoptive therapy using tumor-specific Tc9 cells generated stronger antitumor responses in mouse melanoma than classical Tc1 cells. To understand why Tc9 cells exert stronger antitumor responses, we used gene profiling to compare Tc9 and Tc1 cells. Tc9 cells expressed different levels of cholesterol synthesis and efflux genes and possessed significantly lower cholesterol content than Tc1 cells. Unique to Tc9, but not other CD8+ or CD4+ T cell subsets, manipulating cholesterol content in polarizing Tc9 cells significantly affected IL-9 expression and Tc9 differentiation and antitumor response in vivo. Mechanistic studies showed that IL-9 was indispensable for Tc9 cell persistence and antitumor effects, and cholesterol or its derivatives inhibited IL-9 expression by activating liver X receptors (LXRs), leading to LXR Sumoylation and reduced p65 binding to Il9 promoter. Our study identifies cholesterol as a critical regulator of Tc9 cell differentiation and function.