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⁺CD127^lo) 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.

中文翻译：

---

原发性人胶质母细胞瘤中的肿瘤细胞和免疫细胞特征：对患者预后的影响

肿瘤浸润白细胞在胶质母细胞瘤（GBM）中的分布和作用仍然很大程度上未知。在这里，我们通过流式细胞术研究了 55 个原发性（成人）GBM 样本的细胞组成，并将肿瘤免疫特征与诊断和结果时的患者特征相关联。GBM 单细胞悬浮液在诊断时 (n = 44) 以及放疗和化疗后复发 (n = 11) 时使用一组 8 色单克隆抗体组合进行染色，用于识别和计数 (GFAP + ^CD45 − ⁾肿瘤和正常肿瘤星形胶质细胞、浸润性骨髓细胞，即小胶质细胞和血液来源的肿瘤相关巨噬细胞 (TAM)、M1 样和 M2 样 TAM、中性粒细胞。和骨髓源性抑制细胞 (MDSC) - 和肿瘤浸润淋巴细胞 (TIL) - 即 CD3 ⁺ T 细胞及其 TCD4 ⁺、 TCD8 ⁺、 TCD4 ^- CD8 ^-和 (CD25 ⁺ CD127 ^lo ) 调节性 (T-regs) ) 亚群、(CD19 ⁺ CD20 ⁺ ) B 细胞和 (CD16 ⁺ ) NK 细胞—。总体而言，GBM 样本由肿瘤和正常星形胶质细胞 (73% ± 16%) 的主要群体（平均值 ± 1SD）以及显着但可变的免疫细胞 (24% ± 18%) 组成。在骨髓细胞中，TAM 占主导地位 (13% ± 12%)，包括小胶质细胞 (10% ± 11%) 和血源性巨噬细胞 (3% ± 5%)，此外还有较小比例的中性粒细胞 (5% ± 9%)。 %) 和 MDSC (4% ± 8%)。淋巴细胞较少，主要包括 TCD4 ⁺ (0.5% ± 0.7%) 和 TCD8 ⁺细胞 (0.6% ± 0.7%)，以及较少数量的 TCD4 ^– CD8 ^– T 细胞 (0.2% ± 0.4%)、T- reg (0.1% ± 0.2%)、B 淋巴细胞 (0.1% ± 0.2%) 和 NK 细胞 (0.05% ± 0.05%)。总体而言，确定了三种不同的免疫特征：具有少量白细胞的病例，以 TAM 和中性粒细胞为主的肿瘤，以及 TAM、中性粒细胞和 T 淋巴细胞混合浸润的病例。在没有EGFR基因增益的情况下，未经治疗的具有混合骨髓和淋巴免疫浸润的 GBM 患者与其他两组相比，患者总生存期显着缩短( p  = 0.02)。在这里，我们表明免疫细胞浸润系统地存在于 GBM 中，其水平和免疫特征存在很大差异。混合性骨髓和 T 淋巴浸润的患者表现出更差的结果。