Tumor cell and immune cell profiles in primary human glioblastoma: Impact on patient outcome.

The distribution and role of tumor-infiltrating leucocytes in glioblastoma (GBM) remain largely unknown. Here, we investigated the cellular composition of 55 primary (adult) GBM samples by flow cytometry and correlated the tumor immune profile with patient features at diagnosis and outcome. GBM single-cell suspensions were stained at diagnosis (n = 44) and recurrence following radiotherapy and chemotherapy (n = 11) with a panel of 8-color monoclonal antibody combinations for the identification and enumeration of (GFAP+ CD45- ) tumor and normal astrocytic cells, infiltrating myeloid cells -i.e. microglial and blood-derived tumor-associated macrophages (TAM), M1-like, and M2-like TAM, neutrophils. and myeloid-derived suppressor cells (MDSC)- and tumor-infiltrating lymphocytes (TIL) -i.e. CD3+ T-cells and their TCD4+ , TCD8+ , TCD4- CD8- , and (CD25+ CD127lo ) regulatory (T-regs) subsets, (CD19+ CD20+ ) B-cells, and (CD16+ ) NK-cells-. Overall, GBM samples consisted of a major population (mean ± 1SD) of tumor and normal astrocytic cells (73% ± 16%) together with a significant but variable fraction of immune cells (24% ± 18%). Within myeloid cells, TAM predominated (13% ± 12%) including both microglial cells (10% ± 11%) and blood-derived macrophages (3% ± 5%), in addition to a smaller proportion of neutrophils (5% ± 9%) and MDSC (4% ± 8%). Lymphocytes were less represented and mostly included TCD4+ (0.5% ± 0.7%) and TCD8+ cells (0.6% ± 0.7%), together with lower numbers of TCD4- CD8- T-cells (0.2% ± 0.4%), T-regs (0.1% ± 0.2%), B-lymphocytes (0.1% ± 0.2%) and NK-cells (0.05% ± 0.05%). Overall, three distinct immune profiles were identified: cases with a minor fraction of leucocytes, tumors with a predominance of TAM and neutrophils, and cases with mixed infiltration by TAM, neutrophils, and T-lymphocytes. Untreated GBM patients with mixed myeloid and lymphoid immune infiltrates showed a significantly shorter patient overall survival versus the other two groups, in the absence of gains of the EGFR gene (p = 0.02). Here we show that immune cell infiltrates are systematically present in GBM, with highly variable levels and immune profiles. Patients with mixed myeloid and T-lymphoid infiltrates showed a worse outcome.

González-Tablas Pimenta M ，Otero Á ，Arandia Guzman DA ，Pascual-Argente D ，Ruíz Martín L ，Sousa-Casasnovas P ，García-Martin A ，Roa Montes de Oca JC ，Villaseñor-Ledezma J ，Torres Carretero L ，Almeida M ，Ortiz J ，Nieto A ，Orfao A ，Tabernero MD ... -  《-》

Quantitative analysis of immune cells within the tumor microenvironment of glioblastoma and their relevance for prognosis.

Glioblastoma (GBM) is a high malignant tumor with no effective treatment. To comprehensively characterize the landscape of immune cells in GBM and evaluate their correlation with prognosis, we developed a multispectral fluorescent imaging pipeline that included tumor-infiltrating lymphocytic markers (CD3, CD4, CD8, FOXP3, NKP46), immune checkpoint markers (PD-1, PD-L1), and markers to characterize myeloid cells (CD68, CD66b, CD163, HLA-DR), to spatially quantify 18 immune cell subsets in 21 GBM cases. We found that macrophages are the most abundant in GBM microenvironment, followed by T cells and neutrophils, while NK and NKT cells are the least. Previously unreported CD8+ Treg, PD-L1+ neutrophils, and high proportion of PD-1+ NK and PD-1+ T cells were also detected. Single high densities of PD-1+CD8+ T cells, neutrophils, and PD-L1-expressing CD68+ cells were associated with longer survival. Moreover, closer proximity of T cells to PD-L1+ macrophages or PD-L1+ neutrophils were associated with poor prognosis. Correlative analysis revealed circulating PMN-MDSC and e-MDSC were positively correlated with intratumoral M2 macrophages, while circulating NK cells were inversely associated with infiltrating CD4+ Treg cells in GBM patients. Our findings highlighted the potential roles of infiltrating immune cells in prognosis prediction and developing novel immunotherapeutic strategies for GBM patients.

Wang L ，He Z ，Fan S ，Mo L ，Li Y ，Yuan X ，Xu B ，Mou Y ，Yin Y ... -  《-》

Altered Metabolism in Glioblastoma: Myeloid-Derived Suppressor Cell (MDSC) Fitness and Tumor-Infiltrating Lymphocyte (TIL) Dysfunction.

Di Ianni N ，Musio S ，Pellegatta S 《INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES》

Single-Cell Atlas Reveals Complexity of the Immunosuppressive Microenvironment of Initial and Recurrent Glioblastoma.

The Glioblastoma (GBM) immune microenvironment plays a critical role in tumor development, progression, and prognosis. A comprehensive understanding of the intricate milieu and its interactions remains unclear, and single-cell analysis is crucially needed. Leveraging mass cytometry (CyTOF), we analyzed immunocytes from 13 initial and three recurrent GBM samples and their matched peripheral blood mononuclear cells (pPBMCs). Using a panel of 30 markers, we provide a high-dimensional view of the complex GBM immune microenvironment. Hematoxylin and eosin staining and polychromatic immunofluorescence were used for verification of the key findings. In the initial and recurrent GBMs, glioma-associated microglia/macrophages (GAMs) constituted 59.05 and 27.87% of the immunocytes, respectively; programmed cell death-ligand 1 (PD-L1), T cell immunoglobulin domain and mucin domain-3 (TIM-3), lymphocyte activation gene-3 (LAG-3), interleukin-10 (IL-10) and transforming growth factor-β (TGFβ) demonstrated different expression levels in the GAMs among the patients. GAMs could be subdivided into different subgroups with different phenotypes. Both the exhausted T cell and regulatory T (Treg) cell percentages were significantly higher in tumors than in pPBMCs. The natural killer (NK) cells that infiltrated into the tumor lesions expressed higher levels of CXC chemokine receptor 3 (CXCR3), as these cells expressed lower levels of interferon-γ (IFNγ). The immune microenvironment in the initial and recurrent GBMs displayed similar suppressive changes. Our study confirmed that GAMs, as the dominant infiltrating immunocytes, present great inter- and intra-tumoral heterogeneity and that GAMs, increased exhausted T cells, infiltrating Tregs, and nonfunctional NK cells contribute to local immune suppressive characteristics. Recurrent GBMs share similar immune signatures with the initial GBMs except the proportion of GAMs decreases.

Fu W ，Wang W ，Li H ，Jiao Y ，Huo R ，Yan Z ，Wang J ，Wang S ，Wang J ，Chen D ，Cao Y ，Zhao J ... -  《Frontiers in Immunology》

Crosstalk Between Tumor-Associated Microglia/Macrophages and CD8-Positive T Cells Plays a Key Role in Glioblastoma.

Glioblastoma is considered to be the most malignant disease of the central nervous system, and it is often associated with poor survival. The immune microenvironment plays a key role in the development and treatment of glioblastoma. Among the different types of immune cells, tumor-associated microglia/macrophages (TAM/Ms) and CD8-positive (CD8+) T cells are the predominant immune cells, as well as the most active ones. Current studies have suggested that interaction between TAM/Ms and CD8+ T cells have numerous potential targets that will allow them to overcome malignancy in glioblastoma. In this review, we summarize the mechanism and function of TAM/Ms and CD8+ T cells involved in glioblastoma, as well as update on the relationship and crosstalk between these two cell types, to determine whether this association alters the immune status during glioblastoma development and affects optimal treatment. We focus on the molecular factors that are crucial to this interaction, and the role that this crosstalk plays in the biological processes underlying glioblastoma treatment, particularly with regard to immune therapy. We also discuss novel therapeutic targets that can aid in resolving reticular connections between TAM/Ms and CD8+ T cells, including depletion and reprogramming TAM/Ms and novel TAM/Ms-CD8+ T cell cofactors with potential translational usage. In addition, we highlight the challenges and discuss future perspectives of this crosstalk between TAM/Ms and CD8+ T cells.

Tu S ，Lin X ，Qiu J ，Zhou J ，Wang H ，Hu S ，Yao Y ，Wang Y ，Deng Y ，Zhou Y ，Shao A ... -  《Frontiers in Immunology》