PD-1胞外段cDNA在真核细胞的表达与其功能鉴定

PD-L/PD-1是参与肿瘤免疫逃避的一条抑制性信号途径。为了用可溶性的PD 1受体阻断PD-L/PD-1的相互作用 ,将小鼠PD-1胞外段 (aa1-aa167)作为独立可溶性分子进行了真核表达 ,并对其功能进行鉴定。构建了编码小鼠PD-1胞外段cDNA(sPD 1)的真核质粒表达载体pPD-1A和编码sPD-1-GFP重组蛋白的真核质粒表达载体pPD-1B ;细胞转染实验表明其表达产物主要是分泌到细胞外的可溶性产物 (sPD-1) ,流式细胞仪检测表明sPD-1可有效结合PD-1配体 ;肿瘤细胞杀伤实验表明 ,sPD-1作用于肿瘤细胞或在脾淋巴细胞激活过程中作用于淋巴细胞 ,均可增强Hsp70-H22抗原肽复合物激活的脾淋巴细胞杀伤肿瘤细胞的作用。sPD-1真核表达载体的构建为在肿瘤局部表达抑制性共刺激分子的可溶性受体 ,拮抗肿瘤微环境中负调节因素对T细胞的抑制作用 ,增强机体的抗肿瘤能力 ,提供了一种新的肿瘤基因治疗手段     

New insights into the important roles of tumor cell-intrinsic PD-1

PD-1 (Programmed cell death protein-1) is mainly expressed in various immune cells, while its ligands PD-L1/PD-L2 (Programmed death ligand-1/Programmed death ligand-2) are mostly expressed in tumor cells. Generally, the binding of PD-L1/PD-L2 and PD-1 could lead to the tumor immune evasion. However, some recent studies showed that PD-1 could also be expressed in tumor cells and could activate mTOR (Mammalian Target of Rapamycin) or Hippo signaling pathway, therefore facilitating tumor proliferation independent of the immune system. While there was evidence that tumor cell-intrinsic PD-1 inhibited the activation of AKT and ERK1/2 pathways, thereby inhibiting tumor cell growth. Based on TCGA and CCLE database, we found that PD-1 was expressed in a variety of tumors and was associated with patient''s prognosis. Besides, we found that PD-1 may be involved in many carcinogenic signaling pathway on the basis of PD-1 gene enrichment analysis of cancer tissues and cancer cells. Our understanding of the tumor cell-intrinsic PD-1 function is still limited. This review is aimed at elaborating the potential effects of tumor cell-intrinsic PD-1 on carcinogenesis, providing a novel insight into the effects of anti-PD-1/PD-L1 immunotherapy, and helping to open a major epoch of combination therapy.     

Secretion of human soluble programmed cell death protein 1 by chimeric antigen receptor-modified T cells enhances anti-tumor efficacy

Background aimsChimeric antigen receptor (CAR) T cells have achieved favorable responses in patients with hematologic malignancies, but the outcome has been far from satisfactory in the treatment of tumors with high expression of immunosuppressive molecules. To overcome this limitation, we modified CAR T cells to secrete types of human soluble programmed cell death protein 1 (PD-1) called sPD-1 CAR T cells.MethodsTo compare the effector function between second (conventional second-generation CAR targeting CD19) and sPD-1 CAR T cells, we measured cytotoxicity, cytokine secretion and activation markers incubated with or without tumor cells expressing CD19 and/or programmed cell death ligand 1 (PD-L1). Furthermore, the anti-tumor efficacy of second and sPD-1 CAR T cells was determined using an NSG mouse model bearing NALM-6-PD-L1. Finally, the underlying mechanism was investigated by metabolic parameters and RNA sequencing analysis of different CAR T cells.ResultsCompared with second CAR T cells, sPD-1 CAR T cells enhanced killing efficiency toward CD19⁺PD-L1⁺ tumor cells in vitro. Furthermore, sPD-1 CAR T cells reduced the tumor burden and prolonged overall survival of the NSG (NOD-SCID-IL2rg) mice bearing NALM-6-PD-L1. To explore the effect of soluble PD-1 on CAR T cells, we found that sPD-1 CAR T cells exhibited higher levels of activation and ameliorative profiles of differentiation, exhaustion, glycolysis and apoptosis.ConclusionsWith constitutive soluble PD-1 secretion, sPD-1 CAR T cells have tended to eradicate tumors with a high expression of PD-L1 more effectively than second CAR T cells. This may be due to soluble PD-1 enhancing apoptosis resistance, aerobic metabolism and a more “stem” differentiation of CAR T cells. Overall, our study presents a feasible strategy to increase the efficacy of CAR T cells.     

Interaction of PD-L1 on tumor cells with PD-1 on tumor-specific T cells as a mechanism of immune evasion: implications for tumor immunotherapy

Programmed death receptor ligand 1 (PD-L1, also called B7-H1) is a recently described B7 family member. In contrast to B7-1 and B7-2, PD-L1 does not interact with either CD28 or CTLA-4. To date, one specific receptor has been identified that can be ligated by PD-L1. This receptor, programmed death receptor 1 (PD-1), has been shown to negatively regulate T-cell receptor (TCR) signaling. Upon ligating its receptor, PD-L1 has been reported to decrease TCR-mediated proliferation and cytokine production. PD-1 gene–deficient mice developed autoimmune diseases, which early led to the hypothesis of PD-L1 regulating peripheral tolerance. In contrast to normal tissues, which show minimal surface expression of PD-L1 protein, PD-L1 expression was found to be abundant on many murine and human cancers and could be further up-regulated upon IFN- stimulation. Thus, PD-L1 might play an important role in tumor immune evasion. This review discusses the currently available data concerning negative T-cell regulation via PD-1, the blockade of PD-L1/PD-1 interactions, and the implications for adoptive T-cell therapies.     

Programmed death-1 targeting can promote allograft survival

The recently identified CD28 homolog and costimulatory molecule programmed death-1 (PD-1) and its ligands, PD-L1 and PD-L2, which are homologs of B7, constitute an inhibitory regulatory pathway of potential therapeutic use in immune-mediated diseases. We examined the expression and functions of PD-1 and its ligands in experimental cardiac allograft rejection. In initial studies, we found that most normal tissues and cardiac isografts had minimal expression of PD-1, PD-L1, or PD-L2, but intragraft induction of all three molecules occurred during development of cardiac allograft rejection. Intragraft expression of all three genes was maintained despite therapy with cyclosporin A or rapamycin, but was prevented in the early posttransplant period by costimulation blockade using CD154 or anti-inducible costimulator mAb. We prepared PD-L1.Ig and PD-L2.Ig fusion proteins and showed that each bound to activated PD-1(+) T cells and inhibited T cell functions in vitro, thereby allowing us to test the effects of PD-1 targeting on allograft survival in vivo. Neither agent alone modulated allograft rejection in wild-type recipients. However, use of PD-L1.Ig administration in CD28(-/-) recipients, or in conjunction with immunosuppression in fully MHC-disparate combinations, markedly prolonged cardiac allograft survival, in some cases causing permanent engraftment, and was accompanied by reduced intragraft expression of IFN-gamma and IFN-gamma-induced chemokines. PD-L1.Ig use also prevented development of transplant arteriosclerosis post-CD154 mAb therapy. These data show that when combined with limited immunosuppression, or in the context of submaximal TCR or costimulatory signals, targeting of PD-1 can block allograft rejection and modulate T and B cell-dependent pathologic immune responses in vivo.     

Local immune checkpoint blockade therapy by an adenovirus encoding a novel PD-L1 inhibitory peptide inhibits the growth of colon carcinoma in immunocompetent mice

A novel peptide that interferes with the PD-1/PD-L1 immune checkpoint pathway, termed PD-L1 inhibitory peptide 3 (PD-L1ip3), was computationally designed, experimentally validated for its specific binding to PD-L1, and evaluated for its antitumor effects in cell culture and in a mouse colon carcinoma syngeneic murine model. In several cell culture studies, direct treatment with PD-L1ip3, but not a similar peptide with a scrambled sequence, substantially increased death of CT26 colon carcinoma cells when co-cultured with murine CD8⁺ T cells primed by CT26 cell antigens. In a syngeneic mouse tumor model, the growth of CT26 tumor cells transduced with the PD-L1ip3 gene by an adenovirus vector was significantly slower than that of un-transduced CT26 cells in immunocompetent mice. This tumor growth attenuation was further enhanced by the coadministration of the peptide form of PD-L1ip3 (10 mg/kg/day). The current study suggests that this peptide can stimulate host antitumor immunity via blockade of the PD-1/PD-L1 pathway, thereby increasing CD8⁺ T cell-induced death of colon carcinoma cells. The tumor site-specific inhibition of PD-L1 by an adenovirus carrying the PD-L1ip3 gene, together with direct peptide treatment, may be used as a local immune checkpoint blockade therapy to inhibit colon carcinoma growth.     

Differential role of programmed death-ligand 1 [corrected] and programmed death-ligand 2 [corrected] in regulating the susceptibility and chronic progression of experimental autoimmune encephalomyelitis

Programmed death-1 (PD-1) is a negative costimulatory molecule, and blocking the interaction of PD-1 with its ligands, PD-L1 (B7-H1) and PD-L2 (B7-DC), enhances autoimmune disease in several animal models. We have studied the role of PD-1 ligands in disease susceptibility and chronic progression in experimental autoimmune encephalomyelitis (EAE). In BALB/c mice immunized with myelin oligodendrocyte glycoprotein (MOG) peptide 35-55, PD-L1 but not PD-L2 blockade significantly increased EAE incidence. In B10.S mice immunized with myelin proteolipid protein (PLP) peptide 139-151, both PD-L1 and PD-L2 blockade markedly enhanced EAE severity. In prediabetic NOD mice immunized with PLP48-70, PD-L2 blockade worsened EAE but did not induce diabetes, whereas PD-L1 blockade precipitated diabetes but did not worsen EAE, suggesting different regulatory roles of these two ligands in EAE and diabetes. B6 mice immunized with MOG35-55 developed chronic persistent EAE, and PD-L2 blockade in the chronic phase exacerbated EAE, whereas PD-L1 blockade did not. In contrast, SJL/J mice immunized with PLP139-151 developed chronic relapsing-remitting EAE, and only PD-L1 blockade during remission precipitated EAE relapse. The strain-specific effects of PD-1 ligand blockade did not correlate with the expression of PD-L1 and PD-L2 on dendritic cells and macrophages in lymphoid tissue, or on inflammatory cells in the CNS. However, EAE enhancement is correlated with less prominent Th2 cytokine induction after specific PD-1 ligand blockade. In conclusion, PD-L1 and PD-L2 differentially regulate the susceptibility and chronic progression of EAE in a strain-specific manner.     

Contribution of the PD-L1/PD-1 pathway to T-cell exhaustion: an update on implications for chronic infections and tumor evasion

Recent clinical data support ideas of Programmed death receptor-ligand 1 (PD-L1; also called B7-H1, CD274) playing an important role in immune evasion of tumor cells. Expression of PD-L1 on tumors strongly correlates with the survival of cancer patients. PD-L1 on tumors interacts with the co-inhibitory molecule Programmed death receptor-1 (PD-1, CD279) on T cells mediating decreased TCR-mediated proliferation and cytokine production. In animal tumor models, blockade of PD-L1/PD-1 interactions resulted in an improved tumor control. In addition, exhausted T cells during chronic viral infections could be revived by PD-L1 blockade. Thus, targeting PD-L1/PD-1 interactions might improve the efficacy of adoptive cell therapies (ACT) of chronic infections as well as cancers. Obstacles for a general blockade of PD-L1 might be its role in mediating peripheral tolerance. This review discusses the currently available data concerning the role of PD-L1 in tumor immune evasion and envisions possibilities for implementation into ACT for cancer patients. This article is a symposium paper from the conference “Cancer Immunotherapy 2006 Meets Strategies for Immune Therapy,” held in Mainz, Germany, on 4–5 May 2006.     

Upregulation of circulating PD-L1/PD-1 is associated with poor post-cryoablation prognosis in patients with HBV-related hepatocellular carcinoma

Background

The programmed cell death-1 receptor/programmed cell death-1 ligand (PD-1/PD-L1) pathway plays a crucial role in tumor evasion from host immunity. This study was designed to evaluate the association between circulating PD-L1/PD-1 and prognosis after cryoablation in patients with HBV-related hepatocellular carcinoma (HCC).

Methodology/Principal Findings

In the present study, 141 HBV-related HCC patients were enrolled and of those 109 patients received cryoablation. Circulating PD-L1/PD-1 expression was tested by flow cytometry, and 23 patients were simultaneously evaluated for intratumoral PD-L1 expression by immunohistochemical staining. Circulating PD-1/PD-L1 expression was associated with severity of diseases in patients with HCC, and the circulating PD-L1 expression was closely correlated with intratumoral PD-L1 expression. Of the clinical parameters, PD-1/PD-L1 expression was associated with tumor size, blood vessel invasion and BCLC staging. Moreover, PD-1/PD-L1 expression dropped after cryoablation while being elevated at the time of tumor recurrence. Patients with higher expression of circulating PD-L1, as well as circulating PD-1, had a significantly shorter overall survival and tumor-free survival than those with lower expression. Multivariate analysis confirmed that circulating PD-L1 could serve as an independent predictor of overall survival and tumor-recurrence survival in HCC patients after cryoablation.

Conclusions/Significance

Upregulation of circulating PD-L1/PD-1 is associated with poor post-cryoablation prognosis in patients with HBV-related hepatocellular carcinoma.     

Blockade of B7-H1 suppresses the development of chronic intestinal inflammation

A newly identified costimulatory molecule, programmed death-1 (PD-1), provides a negative signal that is essential for immune homeostasis. However, it has been suggested that its ligands, B7-H1 (PD-L1) and B7-dendritic cells (B7-DC; PD-L2), could also costimulate T cell proliferation and cytokine secretion. Here we demonstrate the involvement of PD-1/B7-H1 and B7-DC interaction in the development of colitis. We first examined the expression profiles of PD-1 and its ligands in both human inflammatory bowel disease and a murine chronic colitis model induced by adoptive transfer of CD4(+)CD45RB(high) T cells to SCID mice. Second, we assessed the therapeutic potential of neutralizing anti-B7-H1 and/or B7-DC mAbs using this colitis model. We found significantly increased expression of PD-1 on T cells and of B7-H1 on T, B, and macrophage/DCs in inflamed colon from both inflammatory bowel disease patients and colitic mice. Unexpectedly, the administration of anti-B7-H1, but not anti-B7-DC, mAb after transfer of CD4(+)CD45RB(high) T cells suppressed wasting disease with colitis, abrogated leukocyte infiltration, and reduced the production of IFN-gamma, IL-2, and TNF-alpha, but not IL-4 or IL-10, by lamina propria CD4(+) T cells. These data suggest that the interaction of PD-1/B7-H1, but not PD-1/B7-DC, might be involved in intestinal mucosal inflammation and also show a possible role of interaction between B7-H1 and an as yet unidentified receptor for B7-H1 in inducing T cell activation.     

Refolding and characterization of recombinant human GST-PD-1 fusion protein expressed in Escherichia coli

Programmed death-1 (PD-1) is a costimulatory molecule of CD28 family expressed onactivated T, B and myeloid cells. The engagement of PD-1 with its two ligands, PD-L1 and PD-L2, inhibitsproliferation of T cell and production of a series of its cytokines. The blockade of PD-1 pathway is involvedin antiviral and antitumoral immunity. In this study, human PD-1 cDNA encoding extracellular domain wasamplified and cloned into expression plasmid pGEX-Sx-3. The fusion protein GST-PD-1 was effectivelyexpressed in E. coli BL21 (DE3) as inclusion bodies and a denaturation and refolding procedure was per-formed to obtain bioactive soluble GST-PD-I. Fusion protein of above 95% purity was acquired by a conve-nient two-step purification using GST affinity and size exclusion columns. Furthermore, a PD-L1-dependentin vitro bioassay method was set up to characterize GST-PD-1 bioactivity. The results suggested that GST-PD-1 could competently block the interaction between PD-Ll and PD-l and increase the production of IL-2 and IFN-γ of phytohemagglutinin-activated T cells.     

Programmed death-1 (PD-1):PD-ligand 1 interactions inhibit TCR-mediated positive selection of thymocytes

Positive selection during thymocyte development is driven by the affinity and avidity of the TCR for MHC-peptide complexes expressed in the thymus. In this study, we show that programmed death-1 (PD-1), a member of the B7/CD28 family of costimulatory receptors, inhibits TCR-mediated positive selection through PD-1 ligand 1 (PD-L1):PD-1 interactions. Transgenic mice that constitutively overexpress PD-1 on CD4+CD8+ thymocytes display defects in positive selection in vivo. Using an in vitro model system, we find that PD-1 is up-regulated following TCR engagement on CD4+CD8+ murine thymocytes. Coligation of TCR and PD-1 on CD4+CD8+ thymocytes with a novel PD-1 agonistic mAb inhibits the activation of ERK and up-regulation of bcl-2, both of which are downstream mediators essential for positive selection. Inhibitory signals through PD-1 can overcome the ability of positive costimulators, such as CD2 and CD28, to facilitate positive selection. Finally, defects in positive selection that result from PD-1 overexpression in thymocytes resolve upon elimination of PD-L1, but not PD-1 ligand 2, expression. PD-L1-deficient mice have increased numbers of CD4+CD8+ and CD4+ thymocytes, indicating that PD-L1 is involved in normal thymic selection. These data demonstrate that PD-1:PD-L1 interactions are critical to positive selection and play a role in shaping the T cell repertoire.     

Dendritic cell programming by cytomegalovirus stunts naive T cell responses via the PD-L1/PD-1 pathway

Early during infection, CMV targets dendritic cells (DC) and alters their functions. Herein we show that CMV-infected DC maintain the ability to present both virus-derived and exogenous Ags, but that they actively induce tolerance or anergy in Ag-specific T cells. CMV accomplishes this by selectively maintaining high-level expression of the negative costimulatory molecule programmed death ligand-1 (PD-L1), while commensurately down-regulating positive costimulatory molecules and MHC on the DC surface. Consequently, CD4 and CD8 T cells activated by these infected DC have a stunted phenotype, characterized by poor proliferation, effector function. and recall responses. Blocking PD-L1, but not PD-L2, during direct priming of naive T cells by infected DC significantly restores Ag-specific T cell functions. Using systems where direct and cross-priming of T cells can be distinguished revealed that PD-L1/PD-1 signaling contributes only when naive T cells are primed directly by infected DC, and not upon cross-presentation of viral Ags by uninfected DC. These data suggest that murine CMV programs infected DC during acute infection to inhibit early host adaptive antiviral responses by tipping the balance between negative and positive cosignals.     

RNA interference targeting programmed death receptor-1 improves immune functions of tumor-specific T cells

Adoptive cell transfer (ACT), either using rapidly expanded tumor infiltrating lymphocytes or T-cell receptor transduced peripheral blood lymphocytes, can be considered one of the most promising approaches in cancer immunotherapy. ACT results in the repopulation of the host with high frequencies of tumor-specific T cells; however, optimal function of these cells within the tumor micro-environment is required to reach long-term tumor clearance. We and others have shown that ongoing anti-tumor immune responses can be impaired by the expression of ligands, such as PD-L1 (B7-H1) on tumor cells. Such inhibitory molecules can affect T cells at the effector phase via their receptor PD-1. PD-L1/PD-1 interaction has indeed been shown crucial in inducing T-cell anergy and maintaining peripheral tolerance. In order to maximize anti-tumor responses, antibodies that target the PD-1/PD-L1 axis are currently in phase I/II trials. Alternatively, a more refined approach could be the selective targeting of PD-1 in tumor-specific T cells to obtain long-term resistance against PD-1-mediated inhibition. We addressed whether this goal could be achieved by means of retroviral siRNA delivery. Effective siRNA sequences resulting in the reduction of surface PD-1 expression led to improved murine as well as human T-cell immune functions in response to PD-L1 expressing melanoma cells. These data suggest that blockade of PD-1-mediated T-cell inhibition through siRNA forms a promising approach to achieve long-lasting enhancement of tumor-specific T-cell function in adoptive T-cell therapy protocols.     

PD-1/PD-L1 immune checkpoint: Potential target for cancer therapy

Recent studies show that cancer cells are sometimes able to evade the host immunity in the tumor microenvironment. Cancer cells can express high levels of immune inhibitory signaling proteins. One of the most critical checkpoint pathways in this system is a tumor-induced immune suppression (immune checkpoint) mediated by the programmed cell death protein 1 (PD-1) and its ligand, programmed death ligand 1 (PD-L1). PD-1 is highly expressed by activated T cells, B cells, dendritic cells, and natural killer cells, whereas PD-L1 is expressed on several types of tumor cells. Many studies have shown that blocking the interaction between PD-1 and PD-L1 enhances the T-cell response and mediates antitumor activity. In this review, we highlight a brief overview of the molecular and biochemical events that are regulated by the PD-1 and PD-L1 interaction in various cancers.     

Deubiquitinating enzyme OTUB1 promotes cancer cell immunosuppression via preventing ER-associated degradation of immune checkpoint protein PD-L1

Upregulation of programmed death ligand 1 (PD-L1) helps tumor cells escape from immune surveillance, and therapeutic antibodies targeting PD-1/PD-L1 have shown better patient outcomes only in several types of malignancies. Recent studies suggest that the clinical efficacy of anti-PD-1/PD-L1 treatments is associated with PD-L1 levels; however, the underlying mechanism of high PD-L1 protein levels in cancers is not well defined. Here, we report that the deubiquitinase OTUB1 positively regulates PD-L1 stability and mediates cancer immune responses through the PD-1/PD-L1 axis. Mechanistically, we demonstrate that OTUB1 interacts with and removes K48-linked ubiquitin chains from the PD-L1 intracellular domain in a manner dependent on its deubiquitinase activity to hinder the degradation of PD-L1 through the ERAD pathway. Functionally, depletion of OTUB1 markedly decreases PD-L1 abundance, reduces PD-1 protein binding to the tumor cell surface, and causes increased tumor cell sensitivity to human peripheral blood mononuclear cells (PBMCs)-mediated cytotoxicity. Meanwhile, OTUB1 ablation-induced PD-L1 destabilization facilitates more CD8⁺ T cells infiltration and increases the level of IFN-γ in serum to enhance antitumor immunity in mice, and the tumor growth suppression by OTUB1 silencing could be reversed by PD-L1 overexpression. Furthermore, we observe a significant correlation between PD-L1 abundance and OTUB1 expression in human breast carcinoma. Our study reveals OTUB1 as a deubiquitinating enzyme that influences cancer immunosuppression via regulation of PD-L1 stability and may be a potential therapeutic target for cancer immunotherapy.Subject terms: Proteins, Immune evasion     

小鼠可溶性PD1的克隆、真核表达及生物学活性检测

旨在克隆小鼠PD1胞外区(简称mPD-1)基因,利用真核表达系统表达有活性的分泌型mPD-1蛋白,初步研究其生物学活性。克隆mPD-1基因,将其连入pcDNA3.1(+)/Fc中获得pcDNA3.1(+)-Fc/mPD-1重组表达质粒,转化至大肠杆菌DH5α,进行PCR和双酶切鉴定,并送测序。将阳性质粒转染L929细胞,利用RT-PCR和Western blotting方法鉴定mPD-1/L929稳定表达株。利用Alamar Blue法检测分泌的mPD-l蛋白对淋巴细胞增殖的影响,评价其生物学活性。结果显示,成功构建重组质粒pcDNA3.1(+)-Fc/mPD-1,转染了pcDNA3.1(+)-Fc/mPD-1的L929细胞可将mPD-l蛋白分泌至胞外。A lamar Blue检测结果显示,真核细胞分泌的mPD-1蛋白作用于混合淋巴细胞,与阴性对照相比,可明显促进淋巴细胞的增殖。本试验成功地克隆mPD-1胞外区蛋白,并在L929细胞中得到了分泌型表达。分泌的重组蛋白可有效促进淋巴细胞增殖,为进一步研究其功能和临床应用提供了条件。     

Therapy Resistance in Neoadjuvantly Treated Gastric Cancer and Cancer of the Gastroesophageal Junction is Associated with an Increased Expression of Immune Checkpoint Inhibitors—Comparison Against a Therapy Naïve Cohort

With recent studies uncovering the complex landscape of immune checkpoint regulators in gastric cancer (GC), we aimed to characterize the expression of the checkpoint proteins V-domain Ig suppressor of T-cell activation (VISTA), programmed cell death 1 ligand 1 (PD-L1), and programmed cell death protein-1 (PD-1) in a cohort of GCs following platinum-based neoadjuvant chemotherapy. A total of 141 GC samples, 93 lymph node metastases, and 15 distant metastases were assessed using immunohistochemistry. Staining results were correlated with clinicopathological patient characteristics, genetic alterations, and survival. The expression of VISTA was detected in tumor cells of 38 (30.9%) GCs and immune cells of 139 (98.6%) GCs. The expression of PD-L1 was detected in tumor cells of 27 (22.7%) GCs and immune cells of 134 (96.4%) GCs. The expression of PD-1 was only observed in lymphocyte aggregates/intratumoral lymphoid follicles of 123 (87.2%) GCs. VISTA and PD-L1 correlated in their expression and were associated with poor tumor regression. Compared with an ancient cohort of therapy naïve GCs, we observed a major increase in overall immune cell density accompanied by an over proportional increase in PD-1 and VISTA-positive immune cells. The frequency of VISTA expression in tumor cells was also found to be substantially increased. To the contrary, expression of PD-L1 was decreased in immune cells and tumor cells of neoadjuvantly treated GCs. As a result, a subset of GCs using a single (only VISTA or PD-L1) or combined (VISTA and PD-L1) immune evasion mechanisms might benefit from an anti-PD-L1/anti-VISTA–targeted therapy.     

Tumor-associated neutrophils suppress antitumor immunity of NK cells through the PD-L1/PD-1 axis

Studies have begun to emerge showing the protumor effects of tumor-associated neutrophils (TANs) in tumorigenesis, which may involve dysfunction of NK cells. However, the mechanism through which these rebellious neutrophils modulate NK cell immunity in tumor-bearing state remains unclear. In the present study, we demonstrate that neutrophils can impair the cytotoxicity and infiltration capability of NK cells, and downregulate CCR1 resulting in the weakened infiltration capability of NK cells. Moreover, neutrophils can decrease the responsiveness of NK-activating receptors, NKp46 and NKG2D. Mechanistically, enhanced PD-L1 on neutrophils and PD-1 on NK cells, and subsequent PD-L1/PD-1 interactions were the main mechanisms determining the suppression of neutrophils in NK cell immunity. G-CSF/STAT3 pathway was responsible for PD-L1 upregulation on neutrophils, while IL-18 was essential for PD-1 enhancement on NK cells. The crosstalk between neutrophils and NK cells was cell-cell interaction-dependent. These findings suggest that neutrophils can suppress the antitumor immunity of NK cells in tumor-bearing status through the PD-L1/PD-1 axis, highlighting the importance of PD-L1/PD-1 in the inhibitory effect of neutrophils on NK cells. Targeting G-CSF/STAT3 and IL-18 signaling pathway may be potential strategies to inhibit residual tumor in tumor therapy.