Immune cell constitution in the tumor microenvironment predicts the outcome in diffuse large B-cell lymphoma.
肿瘤微环境中的免疫细胞构成可预测弥漫性大 B 细胞淋巴瘤的结局。
- 作者列表："Autio M","Leivonen SK","Brück O","Mustjoki S","Jørgensen JM","Karjalainen-Lindsberg ML","Beiske K","Holte H","Pellinen T","Leppä S
:Tumor microenvironment (TME) and limited immune surveillance play important roles in lymphoma pathogenesis. Here, we aimed to characterize immunological profiles of diffuse large B-cell lymphoma (DLBCL), and predict the outcome in response to immunochemotherapy. We profiled the expression of 730 immune-related genes in tumor tissues of 81 patients with DLBCL utilizing the Nanostring platform, and used multiplex immunohistochemistry to characterize T-cell phenotypes, including cytotoxic T-cells (CD8, Granzyme B, OX40, Ki67), T-cell immune checkpoint (CD3, CD4, CD8, PD1, TIM3, LAG3), as well as regulatory T-cells and Th1 effector cells (CD3, CD4, FOXP3, TBET) in 188 patients. We observed a high degree of heterogeneity at the transcriptome level. Correlation matrix analysis identified gene expression signatures with highly correlating genes - the main cluster containing genes for cytolytic factors, immune checkpoint molecules, T-cells and macrophages, together entitled as a TME immune cell signature. Immunophenotyping of the distinct cell subsets revealed that a high proportion of immune checkpoint positive T-cells translated to unfavorable survival. Together, our results demonstrate that the immunological profile of DLBCL TME is heterogeneous and clinically meaningful. This highlights the potential impact of T-cell immune checkpoint in regulating survival and resistance to immunochemotherapy.
: 肿瘤微环境 (TME) 和有限免疫监视在淋巴瘤发病机制中起重要作用。在此，我们旨在描述弥漫性大 B 细胞淋巴瘤 (DLBCL) 的免疫学特征，并预测免疫化疗的结局。我们利用 Nanostring 平台分析了 81 例 DLBCL 患者肿瘤组织中 730 个免疫相关基因的表达，并使用多重免疫组化来表征 T 细胞表型, 包括细胞毒性 T 细胞 (CD8 、颗粒酶 B 、 OX40 、 Ki67) 、 T 细胞免疫检查点 (CD3 、 CD4 、 CD8 、 PD1 、 TIM3 、 LAG3) 、以及 188 例患者的调节性 T 细胞和 Th1 效应细胞 (CD3 、 CD4 、 FOXP3 、 TBET)。我们在转录组水平观察到高度异质性。相关矩阵分析确定了具有高度相关基因的基因表达标签 -- 主要包含细胞溶解因子、免疫检查点分子、 T 细胞和巨噬细胞的基因簇，共同命名为 TME 免疫细胞标签。不同细胞亚群的免疫表型发现，高比例的免疫检查点阳性 T 细胞转化为不利的存活。总之，我们的结果证明 DLBCL TME 的免疫学特征是异质性的，具有临床意义。这突出了 T 细胞免疫检查点在调节生存和免疫化疗抵抗中的潜在影响。
METHODS::Macrophages (MΦ) play a critical role in tumor growth, immunosuppression and inhibition of adaptive immune responses in cancer. Hence, targeting signaling pathways in MΦs that promote tumor immunosuppression will provide therapeutic benefit. PI3Kγ has been recently established by our group and others as a novel immuno-oncology target. Herein, we report that a macrophage Syk-PI3K axis drives polarization of immunosuppressive MΦs which establish an immunosuppressive tumor microenvironment in in vivo syngeneic tumor models. Genetic or pharmacological inhibition of Syk and/or PI3Kγ in MΦs promotes a pro-inflammatory MΦphenotype, restores CD8+ T cell activity, destabilizes HIF under hypoxia, and stimulates an antitumor immune response. Assay for Transposase-accessible Chromatin using Sequencing (ATAC-seq) analyses on the bone marrow derived macrophages (BMDMs) show that inhibition of Syk kinase promotes activation and binding of NF-κB motif in SykMC-KO BMDMs, thus stimulating immunostimulatory transcriptional programming in MΦs to suppress tumor growth. Finally, we have developed in silico the "first in class" dual Syk/PI3K inhibitor, SRX3207, for the combinatorial inhibition of Syk and PI3K in one small molecule. This chemotype demonstrates efficacy in multiple tumor models and represents a novel combinatorial approach to activate antitumor immunity.
METHODS::Dysfunction of invariant natural killer T (iNKT) cells in tumor microenvironment hinders their anti-tumor efficacy, and the underlying mechanisms remain unclear. Here we report that iNKT cells increase lipid biosynthesis after activation, and that is promoted by PPARγ and PLZF synergically through enhancing transcription of Srebf1. Among those lipids, cholesterol is required for the optimal IFN-γ production from iNKT cells. Lactic acid in tumor microenvironment reduces expression of PPARγ in intratumoral iNKT cells and consequently diminishes their cholesterol synthesis and IFN-γ production. Importantly, PPARγ agonist pioglitazone, a thiazolidinedione drug for type 2 diabetes, successfully restores IFN-γ production in tumor-infiltrating iNKT cells from both human patients and mouse models. Combination of pioglitazone and alpha-galactosylceramide treatments significantly enhances iNKT cell-mediated anti-tumor immune responses and prolongs survival of tumor-bearing mice. Our studies provide a strategy to augment the anti-tumor efficacy of iNKT cell-based immunotherapies via promoting their lipid biosynthesis.
METHODS::Near-infrared photoimmunotherapy (NIR-PIT) is a newly developed and selective cancer treatment that induces necrotic and immunogenic cell death and utilizes a monoclonal antibody conjugated to a photo-absorber dye, IR700DX, activated by NIR light. Although CD44 is surface cancer marker associated with drug resistance, anti-CD44-IR700 NIR-PIT results in inhibited cell growth and prolonged survival in multiple tumor types. Meanwhile, anti-CD25-IR700-targeted NIR-PIT has been reported to achieve selective and local depletion of FOXP3+CD25+CD4+ regulatory T cells (Tregs), which are primary immunosuppressive cells in the tumor microenvironment (TME), resulting in activation of local antitumor immunity. Combined NIR-PIT with CD44- and CD25-targeted agents has the potential to directly eliminate tumor cells and also amplify the immune response by removing FOXP3+CD25+CD4+ Tregs from the TME. We investigated the difference in therapeutic effects of CD44-targeted NIR-PIT alone, CD25-targeted NIR-PIT alone, and the combination of CD44- and CD25-targeted NIR-PIT in several syngeneic tumor models, including MC38-luc, LL/2, and MOC1. The combined NIR-PIT showed significant tumor growth inhibition and prolonged survival compared with CD44-targeted NIR-PIT alone in all tumor models and showed prolonged survival compared with CD25-targeted NIR-PIT alone in MC38-luc and LL/2 tumors. Combined CD44/CD25 NIR-PIT also resulted in some complete remissions, whereas this was not achieved with either type of NIR-PIT alone. Therefore, combined NIR-PIT simultaneously targeting cancer antigens and immunosuppressive cells in the TME may be more effective than either type of NIR-PIT alone and may have potential to induce prolonged immune responses in treated tumors.