Inhibition of glioma growth by a GOLPH3 siRNA-loaded cationic liposomes

Glioblastoma (GBM) generates a varied immune response and understanding the immune microenvironment may lead to novel immunotherapy treatments modalities. The goal of this study was to evaluate the expression of immunologic markers of potential clinical significance in primary versus recurrent GBM and assess the relationship between these markers and molecular characteristics of GBM. Human GBM samples were evaluated and analyzed with immunohistochemistry for multiple immunobiologic markers (CD3, CD8, FoxP3, CD68, CD163, PD1, PDL1, CTLA4, CD70). Immunoreactivity was analyzed using Aperio software. Degree of strong positive immunoreactivity within the tumor was compared to patient and tumor characteristics including age, gender, MGMT promoter methylation status, and ATRX, p53, and IDH1 mutation status. Additionally, the TCGA database was used to perform similar analysis of these factors in GBM using RNA-seq by expectation–maximization. Using odds ratios, IDH1 mutated GBM had statistically significant decreased expression of CD163 and CD70 and a trend for decreased PD1, CTLA4, and Foxp3. ATRX-mutated GBMs exhibited statistically significant increased CD3 immunoreactivity, while those with p53 mutations were found to have significantly increased CTLA4 immunoreactivity. The odds of having strong CD8 and CD68 reactivity was significantly less in MGMT methylated tumors. No significant difference was identified in any immune marker between the primary and recurrent GBM, nor was a significant change in immunoreactivity identified among age intervals. TCGA analysis corroborated findings related to the differential immune profile of IDH1 mutant, p53 mutant, and MGMT unmethylated tumors. Immunobiologic markers have greater association with the molecular characteristics of the tumor than with primary/recurrent status or age.     

CD276 suppresses CAR-T cell function by promoting tumor cell glycolysis in esophageal squamous cell carcinoma

BackgroundAs an immune checkpoint that suppresses antitumor immunity, CD276 is a potential therapeutic target for cancer immunotherapy. However, the role of CD276 in esophageal squamous cell carcinoma (ESCC) has not been thoroughly examined. A greater understanding of the regulatory mechanism of CD276 may improve the clinical response and efficacy of cancer immunotherapy.MethodsThe expression of CD276 was measured by qRT-PCR, IHC and flow cytometry analysis. T cell infiltration in ESCC was measured by qRT-PCR and immunofluorescence analysis. The regulation function of CD276 in glucose metabolism was examined by metabolism assays, western blotting and small molecule inhibitors. Transfection was used for gene editing. The oncogenic function of CD276 was examined in vivo by CAR-T cell therapy model.ResultsBased on our findings, CD276 regulated the expression of the PKM2 gene in ESCC. Overexpression of CD276 induced the phosphorylation of PKM2 by the STAT3 signalling pathway to promote glucose metabolism in tumors. The accumulation of lactic acid in the tumor microenvironment has been reported to regulate the immune cells, particularly CD8+ T cells. We further analyzed the effect of CD276 on the function of T cells. Chimeric antigen receptor T cells (CAR-T) targeting human epidermal growth factor receptor 2 (HER2) were used as effector cells to detect the effect of CD276 on immunotherapy. The therapeutic effects of CAR-T cells were markedly limited by CD276 overexpression.ConclusionsOur results are the first to show that tumor-derived CD276 supports disease progression. Overexpression of CD276 promoted glucose metabolism in tumor and inhibited the function of CD8+ T cells. Therefore, strategies targeting CD276 might improve the response to cancer immunotherapy of ESCC patients.     

外周血PD-1及相关免疫指标在鼻咽癌患者放化疗过程中的变化及其意义

目的：检测鼻咽癌患者接受放化疗过程中T细胞表面PD-1表达、T细胞亚群等免疫指标的水平,探讨不同治疗时间段内患者的免疫功能。方法：收集2015年4月至2015年9月在福建省肿瘤医院经病理确诊的30例鼻咽癌患者的血液标本,通过流式细胞术动态监测患者在不同治疗时段（治疗前、新辅助化疗后、放疗结束）的免疫指标水平,包括T细胞上PD-1及T细胞亚群、NK细胞、B细胞等。结果：与治疗前相比,新辅助化疗后外周血CD3+细胞、CD4+T细胞、CD8+CD28+T细胞及CD4+/CD8+比值显著升高,而CD19（+ 即B细胞）及CD3-CD16+CD56（+ 即NK细胞）细胞水平显著下降（均P<0.05）。放化疗结束后,外周血CD4+细胞、CD8+CD28+T细胞、CD4+/CD8+比值下调至较治疗前更低的水平（均P<0.05）,而NK细胞的比例则表现为上调（P<0.05）。与治疗前相比,放疗后T细胞PD-1的表达水平显著升高（P<0.05）。结论：鼻咽癌患者外周血T细胞亚群比例在新辅助化疗后上调,而放疗结束时明显下降,提示放疗结束时患者的免疫功能受到显著抑制;T细胞PD-1的表达水平在放疗结束时显著上调,意味着放化疗结束时可能正是使用PD-1抑制剂的良机,抗PD-1维持治疗有望产生更高效持久的抗肿瘤效应。     

Upregulation of HLA-II related to LAG-3+CD4+ T cell infiltration is associated with patient outcome in human glioblastoma

Glioblastoma (GBM) is the most common malignant diffuse glioma of the brain. Although immunotherapy with immune checkpoint inhibitors (ICIs), such as programmed cell death protein (PD)-1/PD ligand-1 inhibitors, has revolutionized the treatment of several cancers, the clinical benefit in GBM patients has been limited. Lymphocyte-activation gene 3 (LAG-3) binding to human leukocyte antigen-II (HLA-II) plays an essential role in triggering CD4⁺ T cell exhaustion and could interfere with the efficiency of anti-PD-1 treatment; however, the value of LAG-3–HLA-II interactions in ICI immunotherapy for GBM patients has not yet been analyzed. Therefore, we aimed to investigate the expression and regulation of HLA-II in human GBM samples and the correlation with LAG-3⁺CD4⁺ T cell infiltration. Human leukocyte antigen-II was highly expressed in GBM and correlated with increased LAG-3⁺CD4⁺ T cell infiltration in the stroma. Additionally, HLA-II^HighLAG-3^High was associated with worse patient survival. Increased interleukin-10 (IL-10) expression was observed in GBM, which was correlated with high levels of HLA-II and LAG-3⁺ T cell infiltration in stroma. HLA-II^HighIL-10^High GBM associated with LAG-3⁺ T cells infiltration synergistically showed shorter overall survival in patients. Combined anti-LAG-3 and anti-IL-10 treatment inhibited tumor growth in a mouse brain GL261 tumor model. In vitro, CD68⁺ macrophages upregulated HLA-II expression in GBM cells through tumor necrosis factor-α (TNF-α). Blocking TNF-α-dependent inflammation inhibited tumor growth in a mouse GBM model. In summary, T cell–tumor cell interactions, such as LAG-3–HLA-II, could confer an immunosuppressive environment in human GBM, leading to poor prognosis in patients. Therefore, targeting the LAG-3–HLA-II interaction could be beneficial in ICI immunotherapy to improve the clinical outcome of GBM patients.     

Prognostic value of programmed death-1, programmed death-ligand 1, programmed death-ligand 2 expression,and CD8(+) T cell density in primary tumors and metastatic lymph nodes from patients with stage T1-4N+M0 gastric adenocarcinoma

Background: Anti-programmed death-1/programmed death-ligand 1 (PD-1/PD-L1) immunotherapy has been proved to be effective on gastric cancer in ongoing clinical trials. However, the value of PD-L1 in predicting responses of patients with gastric cancer to anti-PD-1/PD-L1 immunotherapy is controversial. Some studies suggested that intra- and inter-tumoral heterogeneity of PD-L1 expression might explain the controversy. This study aimed to analyze the expression of PD-L1, PD-L2, and PD-1 as well as CD8(+) T-cell density in primary tumors and lymph nodes from patients with stage T1-4N+M0 gastric adenocarcinoma to explore the heterogeneity of PD-1 signaling pathway molecules. Methods: In primary tumors and metastatic as well as non-metastatic lymph nodes from patients with stage T1-4N+M0 gastric adenocarcinoma, we detected PD-L1 and PD-L2 expression with immunohistochemistry. CD8(+) T-cell density in primary tumors and PD-1 expression on CD8(+) T cells were detected with immunofluorescence. Uni-variate analysis was used to determine the prognostic values of them. Cox proportional hazard regression model was used to identify independent risk factors that affect patients' overall survival and disease-free survival. Results: Among 119 eligible patients who had undergone surgical resection, the positive rate of PD-L1 was higher in metastatic lymph nodes than in primary tumors (45.4％ vs. 38.7％,P= 0.005); the positive rate of PD-1 on CD8(+) T cells was significantly higher in primary tumors and metastatic lymph nodes than in tumor-free lymph nodes (both P < 0.001). The intensity of PD-1 expression on CD8(+) T cells in primary tumors and in metastatic lymph nodes were stronger than that in tumor-free lymph nodes from the same patient. Beside, the positive rate of PD-L2 did not show any differences between primary tumors and metastatic lymph nodes. In multivariate analysis, PD-L1 expression, PD-L2 expression, a low density of CD8(+) T cells in primary tumors, and PD-1 expression on CD8(+) T cells in primary tumors were associated with poor prognosis.Conclusion: The expression of PD-L1 is heterogeneous in primary tumors and in metastatic lymph nodes from patients with stage T1-4N+M0 gastric adenocarcinoma, which might explain the inconsistent results in assessing the prognostic value of PD-L1 expression in previous studies.     

ILC2s在肿瘤免疫调控中的研究进展

Ⅱ型固有淋巴细胞(Type 2 innate lymphoid cells, ILC2s)是一类独特的免疫效应细胞,在抗感染、调控炎症和维持免疫稳态中起着重要作用。近期研究表明ILC2s也参与调节肿瘤免疫,在肿瘤微环境中发现了ILC2s,但是目前ILC2s在肿瘤免疫反应和免疫治疗中的作用尚未阐明。有研究表明白细胞介素33(IL-33)可以激活肿瘤微环境中的ILC2s, ILC2s表达抑制性检查点受体PD-1,PD-1阻断剂直接作用于ILC2s,减轻ILC2s细胞固有的PD-1抑制,最终增加了肿瘤组织CD8⁺细胞毒性T淋巴细胞(Cytotoxic T lymphocyte, CTL)浸润,增强了肿瘤免疫反应,活化的肿瘤ILC2s可以作为抗PD-1免疫疗法的靶标。本文就目前ILC2s参与调控肿瘤免疫可能的机制,探讨新的肿瘤免疫检查点抑制剂靶点进行综述。     

Integration of autologous dendritic cell-based immunotherapy in the primary treatment for patients with newly diagnosed glioblastoma multiforme: a pilot study

Despite resection, radiochemotherapy, and maintenance temozolomide chemotherapy (TMZm), the prognosis of patients with glioblastoma multiforme (GBM) remains poor. We integrated immunotherapy in the primary standard treatment for eight pilot adult patients (median age 50 years) with GBM, to assess clinical and immunological feasibility and toxicity in preparation of a phase I/II protocol HGG-2006. After maximum, safe resection, leukapheresis was performed before radiochemotherapy, and four weekly vaccinations with autologous GBM lysate-loaded monocyte-derived dendritic cells were given after radiochemotherapy. Boost vaccines with lysates were given during TMZm. During the course of vaccination, immunophenotyping showed a relative increase in CD8+CD25+ cells in six of the seven patients, complying with the prerequisites for implementation of immunotherapy in addition to postoperative radiochemotherapy. In five patients, a more than twofold increase in tumor antigen-reacting IFN-γ-producing T cells on Elispot was seen at the fourth vaccination compared with before vaccination. In three of these five patients this more than twofold increase persisted after three cycles of TMZm. Quality of life during vaccination remained excellent. Progression-free survival at six months was 75%. Median overall survival for all patients was 24 months (range: 13–44 months). The only serious adverse event was an ischemic stroke eight months postoperatively. We conclude that tumor vaccination, fully integrated within the standard primary postoperative treatment for patients with newly diagnosed GBM, is feasible and well tolerated. The survival data were used to power a currently running phase I/II trial.     

Immune-mediated tumor regression induced by CpG-containing oligodeoxynucleotides.

T-cell based immunotherapy is an attractive approach for the treatment of multiple tumor types including cervical carcinoma. Immunostimulating DNA containing unmethylated cytosine-guanine (CpG) motifs have been successfully used as adjuvants to enhance immune responses to vaccines designed to trigger antitumor T-cell responses. Using a murine model of cervical carcinoma, we report here that repeated administration of synthetic oligodeoxynucleotides bearing CpG motifs (CpG-ODNs) without the need of vaccination into animals bearing large, established tumors resulted in significant antitumor effects. Both tumor regressions and extended survival resulting from CpG-ODN therapy required the participation of CD8+ T cells. On the other hand, CD4+ T cells were not only not required, but also appeared to inhibit the therapeutic effect of CpG-ODN. Tumor regression correlated with increased infiltration of CD8+ T cells into the tumors and with enhanced expression of MHC class I and II antigens by the tumor cells. Together, these results indicate that CpG therapy could be promising as a single agent for the treatment of some tumors such as cervical carcinoma.     

基于免疫抑制性受体的血液肿瘤靶向免疫治疗研究

T细胞免疫功能缺陷导致多数血液肿瘤患者不能产生有效的抗肿瘤免疫应答,从而逃避宿主免疫系统的攻击,即肿瘤细胞免疫逃逸.近年来认为肿瘤免疫抑制最为重要的机制之一是CD8+T细胞的功能耗竭,主要为免疫细胞异常表达程序性死亡分子1(PD-1)和细胞毒性T淋巴细胞相关抗原(CTLA-4)等免疫抑制所介导,而阻断此通路可以使T细胞部分或全部恢复功能.文章介绍近年来血液肿瘤中PD-1和CTLA-4及二者在介导T细胞免疫耐受中的作用及其在靶向治疗研究中的进展,为血液肿瘤的免疫靶向治疗提供新的思路.     

基于PD-1/PD-L1靶点的肿瘤免疫治疗研究进展

近年来的研究显示,肿瘤免疫治疗在恶性肿瘤的治疗上成效显著,疗效优于传统的化疗和放疗.程序性死亡受体-1(programmed death 1,PD-1)和程序性死亡受体配体-1(programmed death-ligand 1,PD-L1)这对免疫共抑制分子作为肿瘤免疫治疗的靶点备受关注,PD-L1在一些肿瘤细胞中的...     

外周T细胞淋巴瘤患者中PD-1的表达对CD45RA+、CD45RO+T细胞的影响

目的:探讨外周T细胞淋巴瘤(PTCL)患者淋巴瘤组织中PD-1的表达与外周血中初始和记忆T细胞水平的关系.方法:20例PTCL患者采用免疫组化法检测淋巴瘤组织中PD-1的表达,采用流式细胞术检测CHOP方案化疗前后外周血中CD4+CD45RA+、CD4+CD45RO+、CD8+CD45RA+和CD8+CD45RO+T细胞的比例,分析PD-1的表达与T细胞亚群的关系.结果:PD-1、PD-L1蛋白在PTCL患者中表达升高,PD-1阳性患者疗效较差.PD-1阳性患者中CD4+T细胞、CD8+T细胞明显低于PD-1阴性患者;PD-1阴性组患者CD4+CD45RO+、CD8+CD45RA+、 CD8+CD45RO+T细胞明显高于PD-1阳性患者,差异有统计学意义.结论:PTCL患者中存在PD-1/PD-L1蛋白的表达异常,PD-1阳性患者存在更明显的T细胞免疫功能缺陷.     

Distribution and prognostic relevance of tumor-infiltrating lymphocytes (TILs) and PD-1/PD-L1 immune checkpoints in human brain metastases

The activation of immune cells by targeting checkpoint inhibitors showed promising results with increased patient survival in distinct primary cancers. Since only limited data exist for human brain metastases, we aimed at characterizing tumor infiltrating lymphocytes (TILs) and expression of immune checkpoints in the respective tumors.Two brain metastases cohorts, a mixed entity cohort (n = 252) and a breast carcinoma validation cohort (n = 96) were analyzed for CD3+, CD8+, FOXP3+, PD-1+ lymphocytes and PD-L1+ tumor cells by immunohistochemistry. Analyses for association with clinico-epidemiological and neuroradiological parameters such as patient survival or tumor size were performed.TILs infiltrated brain metastases in three different patterns (stromal, peritumoral, diffuse). While carcinomas often show a strong stromal infiltration, TILs in melanomas often diffusely infiltrate the tumors. Highest levels of CD3+ and CD8+ lymphocytes were seen in renal cell carcinomas (RCC) and strongest PD-1 levels on RCCs and melanomas. High amounts of TILs, high ratios of PD-1+/CD8+ cells and high levels of PD-L1 were negatively correlated with brain metastases size, indicating that in smaller brain metastases CD8+ immune response might get blocked. PD-L1 expression strongly correlated with TILs and FOXP3 expression. No significant association of patient survival with TILs was observed, while high levels of PD-L1 showed a strong trend towards better survival in melanoma brain metastases (Log-Rank p = 0.0537).In summary, melanomas and RCCs seem to be the most immunogenic entities. Differences in immunotherapeutic response between tumor entities regarding brain metastases might be attributable to this finding and need further investigation in larger patient cohorts.     

PD-1/PD-L1通路在乳腺癌治疗中的作用及意义

程序性死亡受体1（programmed death-1,PD-1,CD279）为共刺激受体CD28超家族成员,主要表达在活化T细胞、B细胞、单核细胞以及自然杀伤细胞表面。程序性死亡配体1（programmed death-ligand 1,PD-L1,CD274）为PD-1的一个重要配体,广泛表达于肿瘤细胞以及抗原呈递细胞（APC）表面。以PD-1/PD-L1为靶点的肿瘤免疫治疗为肿瘤治疗开辟了新的道路。PD-1通过与PD-L1和(或)PD-L2结合,抑制T细胞活化,诱导T细胞凋亡,在肿瘤免疫逃逸中起着重要的作用。目前PD-1/PD-L1信号通路成为免疫靶向治疗的新靶点,相关研究在非小细胞肺癌、晚期黑色素瘤等多种恶性肿瘤领域有重大进展,研究发现PD-L1 在多种肿瘤细胞包括乳腺癌中表达上调,提示 PD-1/PD-L1通路可能参与肿瘤的免疫逃逸。本文将针对PD-1/PD-L1通路在乳腺癌治疗中的作用及意义进行综述。     

鼻咽癌患者外周血共刺激分子的表达水平及其临床意义

目的:检测鼻咽癌患者外周血中T细胞表面程序性死亡分子1 (programmed death-1,PD-1)、刺激分子4-1BB的表达水平,探讨其在鼻咽癌中的临床意义.方法:选取2016年10至11月福建省肿瘤医院病理确诊的鼻咽癌患者30例作为研究对象,并选取同期本院体检中心性别、年龄相近的健康体检者30例作为对照组.通过流式细胞术检测外周血T细胞表面PD-1、4-1BB的表达情况以及T淋巴细胞亚群的比例.核磁共振扫描测量原发肿瘤体积.结果:鼻咽癌Ⅲ期患者外周血CD8+T细胞比例明显低于Ⅳ期患者[(13.1±6.2)％ vs(18.7±5.5)％,P＜0.05].与对照组相比,CD4+T细胞上PD-1阳性表达率显著上调[(8.7±6.5)％vs(3.87±3.0)％,P＜0.05].鼻咽癌患者外周血T细胞PD-1、4-1BB分子表达水平与临床分期、肿瘤负荷、性别及年龄无显著相关(P＞0.05).结论:鼻咽癌患者外周血PD-1和4-1BB两种共刺激分子在患者体内肿瘤免疫逃逸中可能存在协同效应.联合干预这两种共刺激分子的信号通路可能具有更高效持久的抗肿瘤效应,可为鼻咽癌免疫治疗提供新的思路.     

Combination Treatment of the Oral CHK1 Inhibitor,SRA737, and Low-Dose Gemcitabine Enhances the Effect of Programmed Death Ligand 1 Blockade by Modulating the Immune Microenvironment in SCLC

IntroductionDespite the enthusiasm surrounding cancer immunotherapy, most SCLC patients show very modest response to immune checkpoint inhibitor monotherapy treatment. Therefore, there is growing interest in combining immune checkpoint blockade with chemotherapy and other treatments to enhance immune checkpoint blockade efficacy. Based on favorable clinical trial results, chemotherapy and immunotherapy combinations have been recently approved by the U.S. Food and Drug Administration for frontline treatment for SCLC.Methods and ResultsHere, we show that combined treatment of SRA737, an oral CHK1 inhibitor, and anti–programmed death ligand 1 (PD-L1) leads to an antitumor response in multiple cancer models, including SCLC. We further show that combining low, non-cytotoxic doses of gemcitabine with SRA737 + anti–PD-L1/anti–PD-1 significantly increased antitumorigenic CD8+ cytotoxic T cells, dendritic cells, and M1 macrophage populations in an SCLC model. This regimen also led to a significant decrease in immunosuppressive M2 macrophage and myeloid-derived suppressor cell populations, as well as an increase in the expression of the type I interferon beta 1 gene, IFNβ, and chemokines, CCL5 and CXCL10.ConclusionsGiven that anti–PD-L1/anti–PD-1 drugs have recently been approved as monotherapy and in combination with chemotherapy for the treatment of SCLC, and that the SRA737 + low dose gemcitabine regimen is currently in clinical trials for SCLC and other malignancies, our preclinical data provide a strong rational for combining this regimen with inhibitors of the PD-L1/PD-1 pathway.     

Radiotherapy increases the permissiveness of established mammary tumors to rejection by immunomodulatory antibodies

It is becoming increasingly evident that radiotherapy may benefit from coincident or subsequent immunotherapy. In this study, we examined whether the antitumor effects of radiotherapy, in established triple-negative breast tumors could be enhanced with combinations of clinically relevant monoclonal antibodies (mAb), designed to stimulate immunity [anti-(α)-CD137, α-CD40] or relieve immunosuppression [α-programmed death (PD)-1]. While the concomitant targeting of the costimulatory molecules CD137 and CD40 enhanced the antitumor effects of radiotherapy and promoted the rejection of subcutaneous BALB/c-derived 4T1.2 tumors, this novel combination was noncurative in mice bearing established C57BL/6-derived AT-3 tumors. We identified PD-1 signaling within the AT-3 tumors as a critical limiting factor to the therapeutic efficacy of α-CD137 therapy, alone and in combination with radiotherapy. Strikingly, all mice bearing established orthotopic AT-3 mammary tumors were cured when α-CD137 and α-PD-1 mAbs were combined with single- or low-dose fractionated radiotherapy. CD8+ T cells were essential for curative responses to this combinatorial regime. Interestingly, CD137 expression on tumor-associated CD8+ T cells was largely restricted to a subset that highly expressed PD-1. These CD137+PD-1High CD8+ T cells, persisted in irradiated AT-3 tumors, expressed Tim-3, granzyme B and Ki67 and produced IFN-γ ex vivo in response to phorbol 12-myristate 13-acetate (PMA) and ionomycin stimulation. Notably, radiotherapy did not deplete, but enriched tumors of functionally active, tumor-specific effector cells. Collectively, these data show that concomitant targeting of immunostimulatory and inhibitory checkpoints with immunomodulatory mAbs can enhance the curative capacity of radiotherapy in established breast malignancy.