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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.

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. Antigen-specific IL9-secreting CD4 Th9 and CD8 Tc9 cells have been previously characterized for their anti-tumour properties. Here, the authors show that ex vivo polarized Th9/Tc9 human CAR-T cells display increased anti-tumor activity in pre-clinical haematological and solid cancer models compared to conventional IL-2 activated CAR-T cells.

Multiple myeloma (MM) is a hematologic malignancy characterized by uncontrolled proliferation of plasma cells in the bone marrow. MM patients with aggressive progression have poor survival, emphasizing the urgent need for identifying new therapeutic targets. Here, we show that the leukocyte immunoglobulin-like receptor B1 (LILRB1), a transmembrane receptor conducting negative immune response, is a top-ranked gene associated with poor prognosis in MM patients. LILRB1 deficiency inhibits MM progression in vivo by enhancing the ferroptosis of MM cells. Mechanistic studies reveal that LILRB1 forms a complex with the low-density lipoprotein receptor (LDLR) and LDLR adapter protein 1 (LDLRAP1) to facilitate LDL/cholesterol uptake. Loss of LILRB1 impairs cholesterol uptake but activates the de novo cholesterol synthesis pathway to maintain cellular cholesterol homeostasis, leading to the decrease of anti-ferroptotic metabolite squalene. Our study uncovers the function of LILRB1 in regulating cholesterol metabolism and protecting MM cells from ferroptosis, implicating LILRB1 as a promising therapeutic target for MM patients. Patients with multiple myeloma need new therapeutic targets. The authors identify the transmembrane receptor LILRB1 as a potential target as it protects myeloma cells from ferroptosis by facilitating cholesterol uptake and maintaining cholesterol homeostasis and squalene levels.

Because cytokine-priming signals direct CD8 ⁺ T cells to acquire unique profiles that affect their ability to mediate specific immune responses, here we generated IL-9–skewed CD8 ⁺ T (Tc9) cells by priming with Th9-polarized condition. Compared with type-I CD8 ⁺ cytotoxic T (Tc1) cells, Tc9 secreted different cytokines and were less cytolytic in vitro but surprisingly elicited greater antitumor responses against advanced tumors in OT-I/B16-OVA and Pmel-1/B16 melanoma models. After adoptive transfer, Tc9 cells persisted longer and differentiated into IFN-γ– and granzyme-B (GrzB)–producing cytolytic Tc1-like effector cells. Phenotypic analysis revealed that adoptively transferred Tc9 cells secreted IL-2 and were KLRG-1 ˡᵒʷ and IL-7Rα ʰⁱᵍʰ, suggesting that they acquired a signature of “younger” phenotype or became long-term lived cells with capacity of self-renewal. Our results also revealed that Tc9-mediated therapeutic effect critically depended on IL-9 production in vivo. These findings have clinical implications for the improvement of CD8 ⁺ T-cell-based adoptive immunotherapy of cancers.

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.

BackgroundCancer immunotherapy using immune checkpoint blockade (ICB) has revolutionized cancer treatment. However, patients with multiple myeloma (MM) rarely respond to ICB. Accumulating evidence indicates that the complicated tumor microenvironment (TME) significantly impacts the efficacy of ICB therapy. Therefore, investigating how TME components in MM influence ICB treatment is urgent.MethodsWe employed two well-established murine myeloma models, 5TGM1 and Vk*MYC, by intravenously injecting 5TGM1 or Vk*MYC cells into mice, respectively, to determine ICB therapeutic efficacy in MM. Total mouse IgG or Ig2b ELISA or QuickGel split beta SPE kits and in vivo bioluminescent imaging were used to monitor MM tumor burden. Cytometry by time of flight (CyTOF) was used to quantify MM TME components. T cell proliferation and function were detected using flow cytometry. Peptide-Fc fusion proteins were used to deplete myeloid-derived suppressor cells (MDSCs). MMDTR, Foxp3DTR, CD4 KO and CD8 KO mice were used to elucidate the underlying mechanisms. Gene expression levels in human MM were analyzed using Gene Expression Omnibus public datasets.ResultsWe found that programmed cell death protein 1 (PD-1) antibody treatment had a therapeutic effect in 5TGM1 mice; it was ineffective in Vk*MYC mice. CyTOF indicated that the bone marrow (BM) of both models was inflamed, suggesting that immune suppressive cells might be inhibiting the reactivation of T cells in the BM. We observed higher numbers of MDSCs, regulatory T (Treg) cells, and tumor-associated macrophage (TAMs) in myeloma BM compared with that of tumor-free mice. Specifically, depleting MDSCs, but not Treg cells or TAMs, sensitized Vk*MYC mice and enhanced the response of 5TGM1 mice to PD-1 ICB, which was dependent on CD8+ but not CD4+ T cells. MDSCs, especially M-MDSCs and CD84+ MDSCs, significantly inhibited the activation and cytotoxic cytokine production of CD8+ T cells in vitro. Moreover, database profiling of patient BM revealed a negative correlation between MDSCs signature genes and cytotoxic CD8+ T cell signature genes, with post-maintenance patients with myeloma displaying a higher ratio of cytotoxic CD8+ T cell to MDSCs signature genes compared with pretreated patients.ConclusionOur study highlights the potential of MDSCs depletion in enhancing the sensitivity of patients with myeloma to PD-1 ICB therapy.

Lysophosphatidic acid (LPA) is a naturally occurring phospholipid that regulates cell proliferation, survival, and migration. However, its role on human multiple myeloma (MM) cells is largely unknown. In this study, we show that LPA, which is highly elevated in MM patients, plays an important role in protecting human MM cells against proteasome inhibitor (PI)-induced apoptosis. LPA bound to its receptor LPAR2 activated its downstream MEK1/2-ERK1/2 signaling pathway and enhanced oxidative phosphorylation (OXPHOS) in mitochondria in MM cells. Increased OXPHOS activity produced more NAD + and ATP, reduced proteasome activity, and enhanced protein folding and refolding in endoplasmic reticulum (ER), leading to induction of MM resistance to PIs. Importantly, inhibiting LPAR2 activity or knocking out LPAR2 in MM cells significantly enhanced MM sensitivity to PI-induced apoptosis in vitro and in vivo. Interestingly, primary MM cells from LPA-high patients were more resistant to PI-induced apoptosis than MM cells from LPA-low patients. Thus, our study indicates that LPA-LPAR2-mediated signaling pathways play an important role in MM sensitivity to PIs and targeting LPA or LPAR2 may potentially be used to (re)sensitize patients to PI-based therapy.

Background Cholesterol metabolism reprogramming is a hallmark of cancer cells that exhibit cholesterol addiction by absorbing low-density lipoprotein (LDL) to generate cholesterol for growth. Yet the underlying mechanisms remain unclear. Methods We began by identifying Niemann-Pick C1 (NPC1) as a key cholesterol uptake gene linked to cancer progression through clinical data analysis. Using three tumor models, we showed that NPC1 promotes tumor growth by suppressing pyroptosis. Finally, we demonstrated that the NPC1 inhibitor U18666A effectively inhibits tumor growth, supporting its therapeutic potential. Results Here we report that NPC1, a key player in cholesterol transport, protects cancer cells from pyroptosis across multiple cancer types. NPC1 expression was highly elevated in human cancers and negatively correlated with patient survival. NPC1 deficiency led to reduced cancer growth and enhanced sensitivity to pyroptosis under pyroptotic stress. NPC1 protects cancer cells from pyroptosis by maintaining cholesterol homeostasis and facilitating LDL-mediated cholesterol uptake, leading to enhanced geranylgeranyl pyrophosphate synthesis for cancer cell survival. Moreover, NPC1 inhibitor U18666A induced cancer cell pyroptosis and was highly therapeutic, either alone or combined with chemotherapeutics, against human hematologic and solid cancers in xenograft mouse models. Conclusion This study reveals that NPC1 may be a potential therapeutic target for the treatment of human cancers.

BackgroundTargeted immunotherapy with monoclonal antibodies (mAbs) is an effective and safe method for the treatment of malignancies. Development of mAbs with improved cytotoxicity, targeting new and known tumor-associated antigens, therefore continues to be an active research area. We reported that Dickkopf-1 (DKK1) is a good target for immunotherapy of human cancers based on its wide expression in different cancers but not in normal tissues. As DKK1 is a secreted protein, mAbs binding directly to DKK1 have limited effects on cancer cells in vivo.MethodsThe specificity and antibody-binding capacity of DKK1-A2 mAbs were determined using indirect ELISA, confocal imaging, QIFIKIT antibody-binding capacity and cell surface binding assays. The affinity of mAbs was determined using a surface plasmon resonance biosensor. A flow cytometry-based cell death was performed to detect tumor cell apoptosis. Antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) assays were used to evaluate the ability of DKK1-A2 mAbs to mediate ADCC and CDC activities against tumor cells in vitro. Flow cytometry data were collected with an FACSymphony A3 cell analyzer and analyzed with FlowJo V.10.1 software. Human cancer xenograft mouse models were used to determine the in vivo therapeutic efficacy and the potential safety and toxicity of DKK1-A2 mAbs. In situ TUNEL assay was performed to detect apoptosis in tumors and mouse organs.ResultsWe generated novel DKK1-A2 mAbs that recognize the DKK1 P20 peptide presented by human HLA-A*0201 (HLA-A2) molecules (DKK1-A2 complexes) that are naturally expressed by HLA-A2+DKK1+ cancer cells. These mAbs directly induced apoptosis in HLA-A2+DKK1+ hematologic and solid cancer cells by activating the caspase-9 cascade, effectively lysed the cancer cells in vitro by mediating CDC and ADCC and were therapeutic against established cancers in their xenograft mouse models. As DKK1 is not detected in most human tissues, DKK1-A2 mAbs neither bound to or killed HLA-A2+ blood cells in vitro nor caused tissue damage in tumor-free or tumor-bearing HLA-A2-transgenic mice.ConclusionOur study suggests that DKK1-A2 mAbs may be a promising therapeutic agent to treat human cancers.

NVP-BKM120 is a novel phosphatidylinositol 3-kinase (PI3K) inhibitor and is currently being investigated in phase I clinical trials in solid tumors. This study aimed to evaluate the therapeutic efficacy of BKM120 in multiple myeloma (MM). BKM120 induces cell growth inhibition and apoptosis in both MM cell lines and freshly isolated primary MM cells. However, BKM120 only shows limited cytotoxicity toward normal lymphocytes. The presence of MM bone marrow stromal cells, insulin-like growth factor, or interleukin-6 does not affect BKM120-induced tumor cell apoptosis. More importantly, BKM120 treatment significantly inhibits tumor growth in vivo and prolongs the survival of myeloma-bearing mice. In addition, BKM120 shows synergistic cytotoxicity with dexamethasone in dexamethasone-sensitive MM cells. Low doses of BKM120 and dexamethasone, each of which alone has limited cytotoxicity, induce significant cell apoptosis in MM.1S and ARP-1. Mechanistic study shows that BKM120 exposure causes cell cycle arrest by upregulating p27 (Kip1) and downregulating cyclin D1 and induces caspase-dependent apoptosis by downregulating antiapoptotic XIAP and upregulating expression of cytotoxic small isoform of Bim, BimS. In summary, our findings demonstrate the in vitro and in vivo anti-MM activity of BKM120 and suggest that BKM120 alone or together with other MM chemotherapeutics, particularly dexamethasone, may be a promising treatment for MM.