TIM-3/Galectin-9 interaction and glutamine metabolism in AML cell lines, HL-60 and THP-1.

BACKGROUND: T cell immunoglobulin and mucin-domain containing-3 (TIM-3) is a cell surface molecule that was first discovered on T cells. However, recent studies revealed that it is also highly expressed in acute myeloid leukemia (AML) cells and it is related to AML progression. As, Glutamine appears to play a prominent role in malignant tumor progression, especially in their myeloid group, therefore, in this study we aimed to evaluate the relation between TIM-3/Galectin-9 axis and glutamine metabolism in two types of AML cell lines, HL-60 and THP-1. METHODS: Cell lines were cultured in RPMI 1640 which supplemented with 10% FBS and 1% antibiotics. 24, 48, and 72 h after addition of recombinant Galectin-9 (Gal-9), RT-qPCR analysis, RP-HPLC and gas chromatography techniques were performed to evaluate the expression of glutaminase (GLS), glutamate dehydrogenase (GDH) enzymes, concentration of metabolites; Glutamate (Glu) and alpha-ketoglutarate (α-KG) in glutaminolysis pathway, respectively. Western blotting and MTT assay were used to detect expression of mammalian target of rapamycin complex (mTORC) as signaling factor, GLS protein and cell proliferation rate, respectively. RESULTS: The most mRNA expression of GLS and GDH in HL-60 cells was seen at 72 h after Gal-9 treatment (p = 0.001, p = 0.0001) and in THP-1 cell line was observed at 24 h after Gal-9 addition (p = 0.001, p = 0.0001). The most mTORC and GLS protein expression in HL-60 and THP-1 cells was observed at 72 and 24 h after Gal-9 treatment (p = 0.0001), respectively. MTT assay revealed that Gal-9 could promote cell proliferation rate in both cell lines (p = 0.001). Glu concentration in HL-60 and α-KG concentration in both HL-60 (p = 0.03) and THP-1 (p = 0.0001) cell lines had a decreasing trend. But, Glu concentration had an increasing trend in THP-1 cell line (p = 0.0001). CONCLUSION: Taken together, this study suggests TIM-3/Gal-9 interaction could promote glutamine metabolism in HL-60 and THP-1 cells and resulting in AML development.

Caveolin-mediated cytosolic delivery of spike nanoparticle enhances antitumor immunity of neoantigen vaccine for hepatocellular carcinoma.

Rationale: Although neoantigen-based cancer vaccines have shown promise in various solid tumors, limited immune responses and clinical outcomes have been reported in patients with advanced disease. Cytosolic transport of neoantigen and adjuvant is required for the activation of intracellular Toll-like receptors (TLRs) and cross-presentation to prime neoantigen-specific CD8+T cells but remains a significant challenge. Methods: In this study, we aimed to develop a virus-like silicon vaccine (V-scVLPs) with a unique spike topological structure, capable of efficiently co-delivering a hepatocellular carcinoma (HCC)-specific neoantigen and a TLR9 agonist to dendritic cells (DCs) to induce a robust CD8+T cell response to prevent orthotopic tumor growth. We evaluated the antitumor efficacy of V-scVLPs by examining tumor growth and survival time in animal models, as well as analyzing tumor-infiltrating CD8+T cells and cytokine responses in the tumor microenvironment (TME). To evaluate the synergistic efficacy of V-scVLPs in combination with α-TIM-3 in HCC, we used an orthotopic HCC mouse model, a lung metastasis model, and a tumor rechallenge model after hepatectomy. Results: We found that V-scVLPs can efficiently co-deliver the hepatocellular carcinoma (HCC)-specific neoantigen and the TLR9 agonist to DCs via caveolin-mediated endocytosis. This advanced delivery strategy results in efficient lymph node draining of V-scVLPs to activate lymphoid DC maturation for promoting robust CD8+T cells and central memory T cells responses, which effectively prevents orthotopic HCC tumor growth. However, in the established orthotopic liver tumor models, the inhibitory receptor of TIM-3 was significantly upregulated in tumor-infiltrating CD8+T cells after immunization with V-scVLPs. Blocking the TIM-3 signaling further restored the antitumor activity of V-scVLPs-induced CD8+T cells, reduced the proportion of regulatory T cells, and increased the levels of cytokines to alter the tumor microenvironment to efficiently suppress established orthotopic HCC tumor growth, and inhibit lung metastasis as well as recurrence after hepatectomy. Conclusion: Overall, the developed novel spike nanoparticles with efficient neoantigen and adjuvant intracellular delivery capability holds great promise for future clinical translation to improve HCC immunotherapy.

Depletion and Reversal of Hepatocellular Carcinoma Inducing CTL through ER Stress-Dependent PERK-CHOP Signaling Pathway.

Aims: In this report, it was investigated that hepatoma cells can cause downregulation of cytotoxic T lymphocyte (CTL) function and tea polyphenols (TPs) can reverse downregulation of CTL function. Methods: The expression of GRP78, PD-1, and TIM-3 was detected by western blotting in CTLL-2 cocultured with Hepa1-6 cells. Moreover, perforin (PRF1) and granzyme B (GzmB) protein levels and ER morphology were examined by ELISA and TEM, respectively. After 4-phenylbutyric acid (4-PBA) or tunicamycin (TM) treatment, programmed cell death protein 1 (PD-1), and mucin domain 3 (TIM-3), PRF1, and GzmB were measured by western blotting and ELISA. After sh-CHOP or GSK2656157 (PERK inhibitor) stimulation, the activation of the PERK-CHOP pathway was detected in CTLL-2 cells. Finally, changes in PD-1, TIM-3, PRF1, and GzmB levels were detected to verify the reversal of CTL depletion by TP. Results: The expression of GRP78, PD-1, and TIM-3 clearly increased, and swelling was observed for the endoplasmic reticulum (ER) in CTLL-2 cells cocultured with hepatoma cells. Concurrently, the levels of PRF1 and GzmB decreased. CTLL-2 depletion was induced after stimulation with TM and differed from 4-PBA stimulation. Treatment with sh-CHOP or GSK2656157 caused a decrease in PD-1 and TIM-3 expression, whereas the expression of PRF1 and GzmB clearly increased. After adding TP, the function of CTLs increased markedly. Conclusion: Hepatoma cells induced the depletion of CTLs through the ER stress PERK-CHOP pathway, and TP reversed this depletion by downregulating ER stress.

Intratumoral PD-1+CD8+ T cells associate poor clinical outcomes and adjuvant chemotherapeutic benefit in gastric cancer.

BACKGROUND: Although PD-1 has been reported to be a marker of T-cell exhaustion in several malignancies, the biological role of PD-1+CD8+ T cells in gastric cancer (GC) remains unclear. Herein, we aimed to investigate the role of PD-1+CD8+ T cells in the tumour microenvironment and its clinical significance in GC. DESIGNS: This study included 441 tumour microarray specimens and 60 Flow cytometry specimens of GC patients from Zhongshan Hospital, and 250 GC patients from the Asian Cancer Research Group. RESULTS: Here, we demonstrated that PD-1+CD8+ T cells functioned as an independent adverse prognosticator in GC. In addition, an abundance of intratumoral PD-1+CD8+ T cells indicated worse chemotherapeutic responsiveness to fluorouracil in Stage III GC patients. Mechanistically, PD-1+CD8+ T cell high infiltration indicated an exhausted phenotype of global CD8+ T cells in GC tissues, which was characterised by elevated immune checkpoint expression including CTLA-4 and TIM-3, whereas decreased expression of perforin. Furthermore, PD-1+CD8+ T cell high-infiltration patients with Stage III GC held elevated activity of several therapeutic signal pathways. CONCLUSIONS: Our study highlighted that PD-1+CD8+ T cell abundance predicts inferior prognosis in GC, and may serve as a novel predictive biomarker to guide therapeutic option.

Dahuang Fuzi Baijiang decoction restricts progenitor to terminally exhausted T cell differentiation in colorectal cancer.

Obesity increases the risk of colorectal cancer (CRC) by 30%. The obese tumor microenvironment compromises antitumor immunity by eliciting exhausted T cells (Tex). Hypothesizing that Dahuang Fuzi Baijiang decoction (DFB) is a combined classical prescription from the "Synopsis of Prescriptions of the Golden Chamber". We first determined that DFB regresses tumor growth in high-fat diet-induced obese mice by expanding the TIM3- subset with intermediate expression of programmed cell death-1 (PD-1int TIM3- ) and restricting the PD-1hi TIM3+ subset. Transcription factor 1 (TCF1) is highly expressed in the PD-1int TIM3- subset but is absent in PD-1hi TIM3+ cells. We next confirmed that progenitor PD-1int TCF+ cells robustly produce tumor necrosis factor-α (TNFα) and interferon-γ, whereas terminally differentiated PD-1int TCF+ cells have defects in generating TNFα. With transgenic ob/ob mice, we found that DFB produces cooperative efficacy with anti-PD-1 (αPD-1) by limiting the PD-1hi Tim3+ subset and amplifying the PD-1int TCF+ population. Finally, we defined the recombinant chemokine C-C-motif receptor 2 (CCR2)+ CD8+ subset as terminal Tex and identified that the differentiation from progenitor to terminal Tex is driven, at least in part, by the chemokine (C-C motif) ligand 2 (CCL2)/CCR2 axis. The CCR2 inhibitor enhances the response to αPD-1 by promoting the counts of progenitor Tex. Altogether, DFB dampens CCL2 and preserves progenitor Tex in the obese microenvironment to restrain CRC progression. These findings provide unambiguous evidence that the traditional Chinese formula DFB can prevent tumor progression by modulating adaptive immunity and establish a strong rationale for further clinical verification.

Phytochemicals in Cancer Immune Checkpoint Inhibitor Therapy.

The interaction of immune checkpoint molecules in the tumor microenvironment reduces the anti-tumor immune response by suppressing the recognition of T cells to tumor cells. Immune checkpoint inhibitor (ICI) therapy is emerging as a promising therapeutic option for cancer treatment. However, modulating the immune system with ICIs still faces obstacles with severe immunogenic side effects and a lack of response against many cancer types. Plant-derived natural compounds offer regulation on various signaling cascades and have been applied for the treatment of multiple diseases, including cancer. Accumulated evidence provides the possibility of efficacy of phytochemicals in combinational with other therapeutic agents of ICIs, effectively modulating immune checkpoint-related signaling molecules. Recently, several phytochemicals have been reported to show the modulatory effects of immune checkpoints in various cancers in in vivo or in vitro models. This review summarizes druggable immune checkpoints and their regulatory factors. In addition, phytochemicals that are capable of suppressing PD-1/PD-L1 binding, the best-studied target of ICI therapy, were comprehensively summarized and classified according to chemical structure subgroups. It may help extend further research on phytochemicals as candidates of combinational adjuvants. Future clinical trials may validate the synergetic effects of preclinically investigated phytochemicals with ICI therapy.

Regulation of Th1 T Cell Differentiation by Iron via Upregulation of T Cell Immunoglobulin and Mucin Containing Protein-3 (TIM-3).

Iron plays an important role in host-pathogen interactions, in being an essential element for both pathogen and host metabolism, but also by impacting immune cell differentiation and anti-microbial effector pathways. Iron has been implicated to affect the differentiation of T lymphocytes during inflammation, however, so far the underlying mechanism remained elusive. In order to study the role of iron in T cell differentiation we here investigated how dietary iron supplementation affects T cell function and outcome in a model of chronic infection with the intracellular bacterium Salmonella enterica serovar typhimurium (S. Typhimurium). Iron loading prior to infection fostered bacterial burden and, unexpectedly, reduced differentiation of CD4+ T helper cells type 1 (Th1) and expression of interferon-gamma (IFNγ), a key cytokine to control infections with intracellular pathogens. This effect could be traced back to iron-mediated induction of the negative immune checkpoint regulator T cell immunoglobulin and mucin domain-containing protein 3 (TIM-3), expressed on the surface of this T cell subset. In vitro experiments demonstrated that iron supplementation specifically upregulated mRNA and protein expression of TIM-3 in naïve Th cells in a dose-depdendent manner and hindered priming of those T cells towards Th1 differentiation. Importantly, administration of TIM-3 blocking antibodies to iron-loaded mice infected with S. Typhimurium virtually restored Th1 cell differentiation and significantly improved bacterial control. Our data uncover a novel mechanism by which iron modulates CD4+ cell differentiation and functionality and hence impacts infection control with intracellular pathogens. Specifically, iron inhibits the differentiation of naive CD4+ T cells to protective IFNγ producing Th1 lymphocytes via stimulation of TIM-3 expression. Finally, TIM-3 may serve as a novel drug target for the treatment of chronic infections with intracellular pathogens, specifically in iron loading diseases.

Differential expression of Tim-3, PD-1, and CCR5 on peripheral T and B lymphocytes in hepatitis C virus-related hepatocellular carcinoma and their impact on treatment outcomes.

BACKGROUND AND OBJECTIVE: Activation of the immune checkpoints and expression of chemokines and chemokine receptors have been reported to promote HCC progression. This study aimed to assess the differential expression of Tim-3, PD-1, and CCR5 on peripheral blood lymphocytes from patients with HCV-related HCC and correlate their expression with the treatment outcomes. PATIENTS AND METHODS: The study incorporated 40 patients with chronic HCV-related HCC and 40 healthy controls. Patients were radiologically assessed for hepatic focal lesions and portal vein thrombosis. Response to HCC treatment and overall survival (OS) outcomes were determined. The expression of Tim-3, PD-1, and CCR5 among CD19+, CD4+, and CD8+ lymphocytes was assessed by flow cytometry. RESULTS: Higher frequencies of CD4+ and CD8+ cells expressing each of Tim-3 and PD-1 and PD-1+CD19+ cells were observed in the HCV-related HCC patients in comparison with controls. The highest expression of Tim-3 and PD-1 was by the CD8+ cells. Strong relations were detected among PD-1+CD19+, PD-1+CD4+ and PD-1+CD8+ cells. Elevated levels of PD-1+ lymphocytes were significantly associated with poor treatment response and shorter OS. CONCLUSION: Modulation of the expression of immune checkpoints as Tim-3 and PD-1, and of CCR5 on T cells is somehow related to HCC. CD8+ T cells expressing PD-1 were the most relevant to HCC prognosis (OS and treatment response) and could represent a promising target for immune therapy against HCC. Future studies need to focus on exploring PD-1+ B cells and Tim-3+CD4+ cells, which seem to play a significant role in the pathogenesis of HCC.

Immunologic Features of Patients With Advanced Hepatocellular Carcinoma Before and During Sorafenib or Anti-programmed Death-1/Programmed Death-L1 Treatment.

INTRODUCTION: Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-related death worldwide. Today, a promising treatment strategy is focused on the enhancement of antitumor immune responses by immune checkpoint modification. However, as only 20% of patients with HCC are responders, identification of predictive factors is urgently required. Therefore, for the first time, the features of the intrahepatic and circulating immune system in patients with advanced-stage HCC, before and during the treatment, were analyzed. METHODS: We collected fresh HCC biopsies, along with adjacent tumor-free liver tissues and peripheral blood samples, from 21 patients with advanced HCC. Furthermore, we performed an extensive immunomonitoring of patients with HCC treated with sorafenib or programmed death (PD)-1/PD-L1 pathway blockade using multiparametric flow cytometry. RESULTS: We observed that regardless of the treatment, low baseline intratumoral CD4/CD8 T-cell ratio was associated with better overall survival (P = 0.0002). The baseline frequency of intratumoral PD-1 CD8 T cells was significantly lower in patients responding to sorafenib treatment than in the nonresponders (P = 0.0117), and the frequency of circulating PD-1 T cells increased with tumor progression (P = 0.0329). By contrast, responders to PD-1/PD-L1 pathway blockade showed a trend of high baseline frequency of intratumoral PD-1 CD8 T cells. Moreover, we observed a trend of LAG3 and TIM3 upregulation on circulating T cells in nonresponding patients to PD-1/PD-L1 pathway blockade. DISCUSSION: Immunosuppressive state, characterized by an enhanced intratumoral CD4/CD8 T-cell ratio, was associated with poor prognosis. Additionally, our results suggest that the frequency of intratumoral PD-1 CD8 T cells may serve as a biomarker to identify which individuals will benefit from which treatment and support the use of combination strategies.

Advantages of Molecular Weight Identification during Native MS Screening.

Native mass spectrometry detection of ligand-protein complexes allowed rapid detection of natural product binders of apo and calcium-bound S100A4 (a member of the metal binding protein S100 family), T cell/transmembrane, immunoglobulin (Ig), and mucin protein 3, and T cell immunoreceptor with Ig and ITIM (immunoreceptor tyrosine-based inhibitory motif) domains precursor protein from extracts and fractions. Based on molecular weight common hits were detected binding to all four proteins. Seven common hits were identified as apigenin 6-C-ß-D-glucoside 8-C-α-L-arabinoside, sweroside, 4',5-dihydroxy-7-methoxyflavanone-6-C-rutinoside, loganin acid, 6-C-glucosylnaringenin, biochanin A 7-O-rutinoside and quercetin 3-O-rutinoside. Mass guided isolation and NMR identification of hits confirmed the mass accuracy of the ligand in the ligand-protein MS complexes. Thus, molecular weight ID from ligand-protein complexes by electrospray ionization Fourier transform mass spectrometry allowed rapid dereplication. Native mass spectrometry using electrospray ionization Fourier transform mass spectrometry is a tool for dereplication and metabolomics analysis.

Epigallocatechin-3-gallate Prevents Triptolide-Induced Hepatic Injury by Restoring the Th17/Treg Balance in Mice.

Drug-induced liver injury (DILI) is the most common cause of acute liver failure. Disruption of the Th17/Treg balance can lead to hepatic inflammation, which causes the main symptoms of DILI. Here we investigate the protective mechanisms of (-)-Epigallocatechin-3-gallate (EGCG) on triptolide (TP)-induced DILI that shows the Th17/Treg imbalance. Pretreatment with EGCG (5[Formula: see text]mg/kg) for 10 days before TP (0.5[Formula: see text]mg/kg) administration in mice significantly reduced the increased alanine aminotransferase (ALT) level ([Formula: see text]) induced by TP treatment. The hepatic histology analysis further proved that EGCG protected mice from TP-induced liver injury. The imbalance of Th17/Treg was induced by TP treatment, as shown by the upregulation of TLR4 and downregulation of Tim3 expression. EGCG pretreatment can maintain the expression of TLR4 and Tim3 at normal levels to restore the Th17/Treg imbalance. In addition, EGCG can block the TP-induced expression of the downstream targets of TLR4, including MyD88, NF[Formula: see text]B, and retinoid related orphan receptor (ROR-[Formula: see text]t), while EGCG can restore the TP inhibition of forkhead/winged-helix family transcriptional repressor p3 (FoxP3) that is the downstream target of Tim3. Consequently, EGCG pretreatment can effectively inhibit the Th17-related pro-inflammatory cytokine (e.g. IL-17 and IL-6) upregulation induced by TP treatment. However, TP inhibition of Treg-related anti-inflammatory cytokine IL-10 production was restored by EGCG pretreatment. Taken together, these results suggest that EGCG possesses significant protective properties against TP-induced hepatic inflammatory injury, and that these properties are carried out via the restoration of the Th17/Treg imbalance by the inhibition of the TLR4 signaling pathway and the enhanced activation of the Tim3 signaling pathway.

Adaptive resistance to therapeutic PD-1 blockade is associated with upregulation of alternative immune checkpoints.

Despite compelling antitumour activity of antibodies targeting the programmed death 1 (PD-1): programmed death ligand 1 (PD-L1) immune checkpoint in lung cancer, resistance to these therapies has increasingly been observed. In this study, to elucidate mechanisms of adaptive resistance, we analyse the tumour immune microenvironment in the context of anti-PD-1 therapy in two fully immunocompetent mouse models of lung adenocarcinoma. In tumours progressing following response to anti-PD-1 therapy, we observe upregulation of alternative immune checkpoints, notably T-cell immunoglobulin mucin-3 (TIM-3), in PD-1 antibody bound T cells and demonstrate a survival advantage with addition of a TIM-3 blocking antibody following failure of PD-1 blockade. Two patients who developed adaptive resistance to anti-PD-1 treatment also show a similar TIM-3 upregulation in blocking antibody-bound T cells at treatment failure. These data suggest that upregulation of TIM-3 and other immune checkpoints may be targetable biomarkers associated with adaptive resistance to PD-1 blockade.

Functional restoration of exhausted CD4(+) and CD8(+) T cells in chronic viral infection by vinegar-processed flos of Daphne genkwa.

T-cell exhaustion has become an important issue in chronic infection because exhausted antigen-specific T cells show impaired abilities to eradicate persistently infected pathogens and produce effector cytokines, such as IFN-γ and TNF-α. Thus, strategies to either restore endogenous exhausted T cell responses or provide functional T cells are needed for therapeutics of chronic infection. Despite promising developments using antibodies and cell immunotherapy, there have been no reported attempts to restore exhausted T cells using treatment with materials derived from natural resources. Here, using a mouse model of chronic infection with lymphocytic choriomeningitis virus (LCMV), we found that vinegar-processed flowers (flos) of Daphne genkwa (vp-genkwa), which was composed mainly of four index components, restored exhausted CD4(+) and CD8(+) T cells significantly, as corroborated by evidence that vp-genkwa treatment enhanced functional LCMV-specific CD4(+) and CD8(+) T cells, both quantitatively and qualitatively. Furthermore, pretreatment with vp-genkwa prevented the generation of exhausted LCMV-specific CD8(+) T cells. Such restorations of exhausted LCMV-specific CD4(+) and CD8(+) T cells by vp-genkwa were closely associated with reduced viral burden in sera and tissues. More interestingly, vp-genkwa treatment induced down-regulation of negative molecules, such as PD-1 and Tim-3, in exhausted CD4(+) and CD8(+) T cells with more apparent down-regulation of Tim-3, suggesting that Tim-3 molecule may be a major target in restoring exhausted T cell responses. Collectively, these results provide valuable new insights into the use of vp-genkwa to develop a therapeutic strategy for chronic human diseases, such as hepatitis B and C virus, human immunodeficiency virus, and cancers.

Stable form of galectin-9, a Tim-3 ligand, inhibits contact hypersensitivity and psoriatic reactions: a potent therapeutic tool for Th1- and/or Th17-mediated skin inflammation.

Tim-3 is a cell surface molecule preferentially expressed in Th1 and Th17 cells. Galectin-9 is a ligand for Tim-3 and the binding of galectin-9 to Tim-3 induces apoptosis. We recently developed a stable form of galectin-9 (sGal-9) by partial deletion of the linker peptide. In this study, we characterized the therapeutic effects of sGal-9 on inflammatory reactions in contact hypersensitivity and IL-23-induced psoriatic mouse models. In contact hypersensitivity in mice, the ear swelling response was suppressed by sGal-9. In vitro treatment with sGal-9 resulted in cell apoptosis of CD4, CD8, and hepatic NK cells. sGal-9-treated mice had decreased IFN-gamma- and IL-17-producing T cells. Similarly, sGal-9 reduced epidermal thickness and dermal cellular infiltrate levels in IL-23-induced psoriasis-like skin inflammation. This was accompanied by decreased skin lesion levels of IL-17 and IL-22. sGal-9 may be a unique and useful therapeutic tool for the treatment of Th1- and/or Th17-mediated skin inflammation.