PD‐1‐induced T cell exhaustion is controlled by a Drp1‐dependent mechanism

Programmed cell death‐1 (PD‐1) signaling downregulates the T‐cell response, promoting an exhausted state in tumor‐infiltrating T cells, through mostly unveiled molecular mechanisms. Dynamin‐related protein‐1 (Drp1)‐dependent mitochondrial fission plays a crucial role in sustaining T‐cell motility, proliferation, survival, and glycolytic engagement. Interestingly, such processes are exactly those inhibited by PD‐1 in tumor‐infiltrating T cells. Here, we show that PD‐1^pos CD8⁺ T cells infiltrating an MC38 (murine adenocarcinoma)‐derived murine tumor mass have a downregulated Drp1 activity and more elongated mitochondria compared with PD‐1^neg counterparts. Also, PD‐1^pos lymphocytic elements infiltrating a human colon cancer rarely express active Drp1. Mechanistically, PD‐1 signaling directly prevents mitochondrial fragmentation following T‐cell stimulation by downregulating Drp1 phosphorylation on Ser616, via regulation of the ERK1/2 and mTOR pathways. In addition, downregulation of Drp1 activity in tumor‐infiltrating PD‐1^pos CD8⁺ T cells seems to be a mechanism exploited by PD‐1 signaling to reduce motility and proliferation of these cells. Overall, our data indicate that the modulation of Drp1 activity in tumor‐infiltrating T cells may become a valuable target to ameliorate the anticancer immune response in future immunotherapy approaches.     

DDR1 functions as an immune negative factor in colorectal cancer by regulating tumor‐infiltrating T cells through IL‐18

Immunotherapies represented by programmed cell death protein 1/programmed cell death ligand 1 (PD‐1/PD‐L1) immune checkpoint inhibitors have made great progress in the field of anticancer treatment, but most colorectal cancer patients do not benefit from immunotherapy. Discoidin domain receptor 1 (DDR1), a tyrosine kinase receptor, is activated by collagen binding and overexpressed in various malignancies. However, the role of DDR1 in colorectal cancer and immunoregulation remains unclear. In this study, we found DDR1 is highly expressed in colorectal cancer tissues and negatively associated with patient survival. We demonstrated that DDR1 promotes colorectal tumor growth only in vivo. Mechanistically, DDR1 is a negative immunomodulator in colorectal cancer and is involved in low infiltration of CD4⁺ and CD8⁺ T cells by inhibiting IL‐18 synthesis. We also reported that DDR1 enhances the expression of PD‐L1 through activating the c‐Jun amino terminal kinase (JNK) signaling pathway. In conclusion, our findings elucidate the immunosuppressive role of DDR1 in colorectal cancer, which may represent a novel target to enhance the efficacy of immunotherapy in colorectal cancer.     

CPI‐203 improves the efficacy of anti‐PD‐1 therapy by inhibiting the induced PD‐L1 overexpression in liver cancer

Hepatocellular carcinoma (HCC) is one of the commonest lethal malignancies worldwide, and often diagnosed at an advanced stage, without any curative therapy. Immune checkpoint blockers targeting the programmed death receptor 1 (PD‐1) have shown impressive antitumor activity in patients with advanced‐stage HCC, while the response rate is only 30%. Inducible PD‐L1 overexpression may result in a lack of response to cancer immunotherapy, which is attributed to a mechanism of adaptive immune resistance. Our study investigated that the overexpression of PD‐L1 promoted the invasion and migration of liver cancer cells in vitro, and the induced overexpression of PD‐L1 in the tumor microenvironment could weaken the effects of anti‐PD‐1 immunotherapy in a BALB/c mouse model of liver cancer. CPI‐203, a small‐molecule bromodomain‐containing protein 4 (BRD4) inhibitor, which can potently inhibit PD‐L1 expression in vitro and in vivo, combined with PD‐1 antibody improved the response to immunotherapy in a liver cancer model. Cell transfection and chromatin immunoprecipitation assay manifested that BRD4 plays a key role in PD‐L1 expression; CPI‐203 can inhibit PD‐L1 expression by inhibiting the BRD4 occupation of the PD‐L1 promoter region. This study indicates a potential clinical immunotherapy method to reduce the incidence of clinical resistance to immunotherapy in patients with HCC.     

MK‐6, a novel not‐α IL‐2, elicits a potent antitumor activity by improving the effector to regulatory T cell balance

IL‐2 is a pleiotropic cytokine that regulates immune cell homeostasis. Its immunomodulatory function has been used clinically as an active immunotherapy agent for metastatic cancers. However, severe adverse effects, including the vascular leak syndrome and the preferential stimulation of anti‐immunogenic Treg rather than effector T cells, have been obstacles. We newly designed a mutein IL‐2, Mutakine‐6 (MK‐6), with reduced IL‐2Rα–binding capability. MK‐6 induced comparable cell growth potential toward IL‐2Rβγ–positive T cells but was far less efficient in in vitro Treg proliferation and STAT5 activation. Unlike IL‐2, in vivo administration of MK‐6 produced minimal adverse effects. Using CT26 and B16F10‐syngeneic tumor models, we found MK‐6 was highly efficacious on tumor regression. Serum albumin conjugation to MK‐6 prolonged in vivo half‐life and accumulated in CT26 tumors, showing enhanced antitumor effect. Tumor‐infiltrating leukocytes analysis revealed that albumin‐fused MK‐6 increased the ratio of effector CD8⁺ T cells to CD4⁺ Treg cells. These results demonstrated that MK‐6 is an efficient immunomodulator potentially used for improved immunotherapy with decreased adverse effects and attenuated Treg stimulation.     

Melatonin inhibits EMT and PD‐L1 expression through the ERK1/2/FOSL1 pathway and regulates anti‐tumor immunity in HNSCC

Melatonin is an endogenous hormone with various biological functions and possesses anti‐tumor properties in multiple malignancies. Immune evasion is one of the most important hallmarks of head and neck squamous cell carcinoma (HNSCC) and is closely related to tumor progression. However, as an immune modulator under physiological conditions, the roles of melatonin in tumor immunity in HNSCC remains unclear. In this study, we found that the endogenous melatonin levels in patients with HNSCC were lower than those in patients with benign tumors in head and neck. Importantly, lower melatonin levels were related to lymph node metastasis among patients with HNSCC. Moreover, melatonin significantly suppressed programmed death‐ligand 1 (PD‐L1) expression and inhibited epithelial–mesenchymal transition (EMT) of HNSCC through the ERK1/2/FOSL1 pathway in vitro and in vivo. In SCC7/C3H syngeneic mouse models, anti‐programmed death‐1 (PD‐1) antibody combined with melatonin significantly inhibited tumor growth and modulated anti‐tumor immunity by increasing CD8⁺ T cell infiltration and decreasing the regulatory T cell (Treg) proportion in the tumor microenvironment. Taken together, melatonin inhibited EMT and downregulated PD‐L1 expression in HNSCC through the ERK1/2/FOSL1 pathway and exerted synergistic effects with anti‐PD‐1 antibody in vivo, which could provide promising strategies for HNSCC treatment.     

Nasopharyngeal cancer cell‐derived exosomal PD‐L1 inhibits CD8+ T‐cell activity and promotes immune escape

Programmed cell death ligand 1 (PD‐L1) is an immune surface protein that binds to programmed cell death 1 (PD‐1) and allows tumors to evade T‐cell immunity. This study aims to define the role of PD‐L1 shuttled by tumor cell‐derived exosomes in the immune escape of nasopharyngeal carcinoma (NPC). PD‐L1 expression was determined in the exosomes isolated from the plasma of NPC patients or from NPC cells. It was found that PD‐L1 was highly expressed in the exosomes from the plasma of NPC patients and also in the exosomes from NPC cells. PD‐L1/PD‐1 binding was identified in the presence or absence of interferon‐gamma (IFN‐γ) or anti‐PD‐L1 antibody. PD‐L1 expression was elevated following IFN‐γ treatment. Binding of PD‐L1 to PD‐1 was augmented by IFN‐γ and blocked by anti‐PD‐L1 antibody. Following this, CD8+ T cells were sorted out from peripheral blood samples to assess the binding between exosomal PD‐L1 and PD‐1 on the CD8+ T‐cell surface, and to measure the percentage of Ki‐67‐positive T cells. The results indicated that exosomal PD‐L1 bound to the PD‐1 on CD8+ T‐cell surface, leading to a reduced percentage of Ki‐67‐positive CD8+ T cells and downregulated production of cytokines. In vivo data confirmed that exosomal PD‐L1 promoted NPC tumor growth in mice by suppressing CD8+ T‐cell activity. In conclusion, NPC cell‐derived exosomes deliver PD‐L1 to bind to PD‐1 on the CD8+ T‐cell surface, through which cytotoxic CD8+ T‐cell function was attenuated and the immune escape was thus promoted in NPC.     

Immune microenvironment in patients with mismatch‐repair‐proficient oligometastatic colorectal cancer exposed to chemotherapy: the randomized MIROX GERCOR cohort study

In the era of immune checkpoint inhibitors, understanding the metastatic microenvironment of proficient mismatch repair/microsatellite stable (pMMR/MSS) colorectal cancer (CRC) is of paramount importance to both prognostication and the development of more effective novel therapies. In this study, primary and paired metastasis tissue samples were collected from patients with resectable metastatic CRC treated with adjuvant FOLFOX or peri‐operative chemotherapy in the MIROX phase III prospective study. In total, 74 cancer tissues were stained for CD3, CD8, Forkhead box protein 3 (FOXP3), programmed cell death protein‐1 (PD‐1, invasive front, stromal, intra‐epithelial compartments), and programmed death‐ligand 1 (PD‐L1, tumor, immune cells). The immune profiling of primary CRC had a limited value to predict the immune context of paired metastases for all markers but CD3+. The expression of CD8 and PD‐L1 was higher in metastases after neoadjuvant FOLFOX. In metastases, both CD3 T cells at the invasive front and PD‐L1 expressions on immune cells were predictive of better disease‐free survival. These results show that the effect of FOLFOX on modifying the immune microenvironment in resected CRC metastases and measurement of PD‐L1 expression and tumor‐infiltrating CD8 T cells in pMMR/MSS metastatic tissue samples could improve treatment strategies of metastatic CRC patients.     

CircPCBP2 promotes the stemness and chemoresistance of DLBCL via targeting miR‐33a/b to disinhibit PD‐L1

Diffuse large B‐cell lymphoma (DLBCL) is the most common lymphoid malignancy with a high relapse rate of up to 40%. The prognosis of the disease needs improvement and requires a understanding of its molecular mechanism. We investigated the mechanisms of DLBCL development and its sensitivity to chemotherapy by focusing on circPCBP2/miR‐33a/b/PD‐L1 axis. Human DLBCL specimens and cultured cancer cell lines were used. Features of circPCBP2 were systematically characterized through Sanger sequencing, Actinomycin D, RNase R treatment, and FISH. The expression levels of circPCBP2, miR‐33a/b, PD‐L1, stemness‐related markers, ERK/AKT and JAK2/STAT3 signaling were measured using qRT‐PCR, western blotting, and immunohistochemistry. Stemness of DLBCL cells was assessed through spheroid formation assay and flow cytometry. Cell viability and apoptosis upon cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP) treatment were determined using MTT assay and flow cytometry, respectively. Interactions of circPCBP2‐miR‐33a/b and miR‐33a/b‐PD‐L1 were validated using dual luciferase activity assay and RNA‐RIP. Nude mouse xenograft model was used to assess the function of circPCBP2 in DLBCL growth in vivo. circPCBP2 was upregulated in human DLBCL specimens and cultured DLBCL cells while miR‐33a/b was reduced. Knockdown of circPCBP2 or miR‐33a/b overexpression inhibited the stemness of DLBCL cells and promoted cancer cell apoptosis upon CHOP treatment. circPCBP2 directly bound with miR‐33a/b while miR‐33a/b targeted PD‐L1 3’‐UTR. circPCBP2 disinhibited PD‐L1 signaling via sponging miR‐33a/b. miR‐33a/b inhibitor and activating PD‐L1 reversed the effects of circPCBP2 knockdown and miR‐33a/b mimics, respectively. circPBCP2 knockdown restrained DLBCL growth in vivo and potentiated the anti‐tumor effects of CHOP. In conclusion, circPCBP2 enhances DLBCL cell stemness but suppresses its sensitivity to CHOP via sponging miR‐33a/b to disinhibit PD‐L1 expression. circPCBP2/miR‐33a/b/PD‐L1 axis could serve as a diagnosis marker or therapeutic target for DLBCL.     

Distinct roles of programmed death ligand 1 alternative splicing isoforms in colorectal cancer

Although anti–programmed death‐1 (PD‐1)/programmed death ligand 1 (PD‐L1) immunotherapy has achieved great success in some cancers, most colorectal cancer (CRC) patients remain unresponsive. Therefore, further clarification of the underlying mechanisms is needed to improve the therapy. In this study, we explored the distinct functions of different PD‐L1 alternative splicing isoforms in CRC. We investigated the biological functions in PD‐L1 knocked down/knockout cells, which were verified through overexpression of PD‐L1 isoforms a, b, and c. The roles of PD‐L1 isoforms in immune surveillance resistance was also analyzed. Meanwhile, we performed RNA‐seq to screen the downstream molecules regulated by PD‐L1 isoforms. Finally, we detected PD‐L1 and PD‐L1 isoforms levels in a cohort of serum samples, two cohorts of CRC tissue samples, and analyzed the correlation of PD‐L1 isoforms with PD‐1 blockade therapy response in two clinical CRC cases. The results indicated that PD‐L1 knockout inhibited proliferation, migration, and invasion, and isoform b exerted a more significant inhibitory effect on T cells than the other two isoforms. Moreover, isoform c could promote CRC progression through regulating epithelial‐mesenchymal transition. Clinical data showed that CRC patients with positive PD‐L1 expression were associated with poorer overall survival. High serum PD‐L1 level was associated with poor prognosis. The level of isoform b or c was negatively associated with prognosis, and a higher level of isoform b was associated with a good response to anti–PD‐1 therapy. In conclusion, isoform b should be considered as a biomarker for clinical responsiveness to anti–PD‐1/PD‐L1 immunotherapy; isoform c had a prometastatic role and is a new potential target for CRC therapy.     

IL‐27 improves adoptive CD8+ T cells’ antitumor activity via enhancing cell survival and memory T cell differentiation

IL‐27 is an anti‐inflammatory cytokine that triggers enhanced antitumor immunity, particularly cytotoxic T lymphocyte responses. In the present study, we sought to develop IL‐27 into a therapeutic adjutant for adoptive T cell therapy using our well‐established models. We have found that IL‐27 directly improved the survival status and cytotoxicity of adoptive OT‐1 CD8⁺ T cells in vitro and in vivo. Meanwhile, IL‐27 treatment programs memory T cell differentiation in CD8⁺ T cells, characterized by upregulation of genes associated with T cell memory differentiation (T‐bet, Eomes, Blimp1, and Ly6C). Additionally, we engineered the adoptive OT‐1 CD8⁺ T cells to deliver IL‐27. In mice, the established tumors treated with OT‐1 CD8⁺ T‐IL‐27 were completely rejected, which demonstrated that IL‐27 delivered via tumor antigen–specific T cells enhances adoptive T cells’ cancer immunity. To our knowledge, this is the first application of CD8⁺ T cells as a vehicle to deliver IL‐27 to treat tumors. Thus, this study demonstrates IL‐27 is a feasible approach for enhancing CD8⁺ T cells’ antitumor immunity and can be used as a therapeutic adjutant for T cell adoptive transfer to treat cancer.     

Multiplex immune protein profiling of fine‐needle aspirates from patients with non‐small‐cell lung cancer reveals signatures associated with PD‐L1 expression and tumor stage

Biomarker signatures identified through minimally invasive procedures already at diagnosis of non‐small‐cell lung cancer (NSCLC) could help to guide treatment with immune checkpoint inhibitors (ICI). Here, we performed multiplex profiling of immune‐related proteins in fine‐needle aspirate (FNA) samples of thoracic lesions from patients with NSCLC to assess PD‐L1 expression and identify related protein signatures. Transthoracic FNA samples from 14 patients were subjected to multiplex antibody‐based profiling by proximity extension assay (PEA). PEA profiling employed protein panels relevant to immune and tumor signaling and was followed by Qlucore^® Omics Explorer analysis. All lesions analyzed were NSCLC adenocarcinomas, and PEA profiles could be used to monitor 163 proteins in all but one sample. Multiple key immune signaling components (including CD73, granzyme A, and chemokines CCL3 and CCL23) were identified and expression of several of these proteins (e.g., CCL3 and CCL23) correlated to PD‐L1 expression. We also found EphA2, a marker previously linked to inferior NSCLC prognosis, to correlate to PD‐L1 expression. Our identified protein signatures related to stage included, among others, CXCL10 and IL12RB1. We conclude that transthoracic FNA allows for extensive immune and tumor protein profiling with assessment of putative biomarkers of important for ICI treatment selection in NSCLC.     

Concomitant overexpression of mir‐182‐5p and mir‐182‐3p raises the possibility of IL‐17–producing Treg formation in breast cancer by targeting CD3d,ITK, FOXO1, and NFATs: A meta‐analysis and experimental study

T cells are polarized toward regulatory T cells (Tregs) in tumor microenvironment by the shuttling of microRNAs that target T cell–activating signaling pathways. We evaluated the expression of the miR‐182 cluster (miR‐96, 182, and 183) in peripheral blood mononuclear cells (PBMCs) of patients with breast cancer (BC), and T cell polarization by the expression of FOXO1, NFATs, ITK, TCR/CD3 complex, and IL‐2/IL‐2RA. Twenty‐six microRNAs overexpressed in tumor tissues and sera of these patients were extracted by a meta‐analysis. Then, the expression of the miR‐182 cluster was investigated in PBMCs and sera of these patients and correlated with their targets in PBMCs. Finally, miR‐182 was cloned into Jurkat cells to evaluate its effects on T cell polarization. FOXO1, CD3d, ITK, NFATc3, NFATc4, and IL‐2RA were targeted by miR‐182, due to which their expression decreased in PBMCs of patients. Although IL‐6, IL‐17, and TGF‐β increased after miR‐182 transduction, IL‐2 dramatically decreased. We revealed CD4⁺FOXP3⁺ T cell differentiation in the miR‐182–transduced group. Although miR‐182 has inhibitory effects on T cells by the inhibition of FOXO1, TCR/CD3 complex, NFATs, and IL‐2/IL‐2RA signaling pathways, it increases FOXP3, TGF‐β, and IL‐17 expression to possibly drive T cell deviation toward the transitional state of IL‐17–producing Tregs and Treg formation in the end.     

Dual inhibition of TGF‐β and PD‐L1: a novel approach to cancer treatment

Transforming growth factor‐β (TGF‐β) and programmed death ligand 1 (PD‐L1) initiate signaling pathways with complementary, nonredundant immunosuppressive functions in the tumor microenvironment (TME). In the TME, dysregulated TGF‐β signaling suppresses antitumor immunity and promotes cancer fibrosis, epithelial‐to‐mesenchymal transition, and angiogenesis. Meanwhile, PD‐L1 expression inactivates cytotoxic T cells and restricts immunosurveillance in the TME. Anti‐PD‐L1 therapies have been approved for the treatment of various cancers, but TGF‐β signaling in the TME is associated with resistance to these therapies. In this review, we discuss the importance of the TGF‐β and PD‐L1 pathways in cancer, as well as clinical strategies using combination therapies that block these pathways separately or approaches with dual‐targeting agents (bispecific and bifunctional immunotherapies) that may block them simultaneously. Currently, the furthest developed dual‐targeting agent is bintrafusp alfa. This drug is a first‐in‐class bifunctional fusion protein that consists of the extracellular domain of the TGF‐βRII receptor (a TGF‐β ‘trap’) fused to a human immunoglobulin G1 (IgG1) monoclonal antibody blocking PD‐L1. Given the immunosuppressive effects of the TGF‐β and PD‐L1 pathways within the TME, colocalized and simultaneous inhibition of these pathways may potentially improve clinical activity and reduce toxicity.     

Clinical significance of BIM deletion polymorphism in chemoradiotherapy for non‐small cell lung cancer

The standard treatment for locally advanced non‐small cell lung cancer (NSCLC) is chemoradiotherapy (CRT) followed by anti‐programmed cell death‐ligand 1 (anti‐PD‐L1) treatment. BIM deletion polymorphism induces the suppression of apoptosis resulting from epidermal growth factor (EGFR)‐tyrosine kinase inhibitors in EGFR‐mutated NSCLC patients. We aimed to examine the effects of BIM polymorphism on CRT and anti‐PD‐L1/PD‐1 treatment in NSCLC patients. In this retrospective study of 1312 patients with unresectable NSCLC treated at Higashi‐Hiroshima Medical Center and Hiroshima University Hospital between April 1994 and October 2019, we enrolled those who underwent CRT or chemotherapy using carboplatin + paclitaxel or cisplatin + vinorelbine, or anti‐PD‐L1/PD‐1 treatment. Of 1312 patients, 88, 80, and 74 underwent CRT, chemotherapy, and anti‐PD‐L1/PD‐1 treatment, respectively, and 17.0%, 15.2% and 17.6% of these patients showed BIM polymorphism. Among patients receiving CRT, the progression‐free survival was significantly shorter in those with BIM deletion than in those without. In the multivariate analyses, BIM polymorphism was an independent factor of poor anti‐tumor effects. These results were not observed in the chemotherapy and anti‐PD‐L1/PD‐1 treatment groups. In in vitro experiments, BIM expression suppression using small interfering RNA in NSCLC cell lines showed a significantly suppressed anti‐tumor effect and apoptosis after irradiation but not chemotherapy. In conclusion, we showed that BIM polymorphism was a poor‐predictive factor for anti‐tumor effects in NSCLC patients who underwent CRT, specifically radiotherapy. In the implementation of CRT in patients with BIM polymorphism, we should consider subsequent treatment, keeping in mind that CRT may be insufficient.     

Suppression of high mobility group box 1 in B16F10 tumor does not inhibit the induction of neoantigen‐specific T cells

Accumulated clinical data of immune checkpoint blockades have suggested the importance of neoantigens in cancer immunity. Tumor antigens are released from dead cancer cells together with cellular components, such as damage‐associated molecular patterns (DAMPs), into the tumor microenvironment. We recently reported that high mobility group box 1 (HMGB1), a representative DAMP molecule, showed a negative impact on anti‐tumor immunity. However, a positive role of HMGB1 in the initiation of innate and subsequent adaptive immunity has also been demonstrated; thus, the effects of HMGB1 on anti‐tumor immunity have not been well understood. In this study, we identified nine immunogenic neoantigen epitopes of B16F10 murine melanoma cells and subsequently investigated the effects of suppression of HMGB1 on the induction of neoantigen‐specific immunity using HMGB1‐knockout tumors. Neoantigen‐reactive T cells were expanded in B16F10 tumor‐bearing mice, and T cell receptor repertoire analysis suggested that neoantigen‐reactive T cells were oligo‐clonally increased in B16F10 tumor bearers. An increase of neoantigen‐reactive T cells and oligoclonal expansion of the T cells were similarly detected in HMGB1‐knockout tumor‐bearing mice. The induction of neoantigen‐specific immunity under the suppression of HMGB1 in the tumor microenvironment shown in this study supports further development of combination therapy of HMGB1 suppression with neoantigen‐targeted cancer immunotherapies, including immune checkpoint blockade therapy.