PO.IM01.01 · 免疫学

体内生存与流式细胞术揭示胶质母细胞瘤中双相、时间依赖性的中性粒细胞生物学特征，并经单细胞转录组学佐证

Biphasic, time-dependent neutrophil biology in glioblastoma revealed by in vivo survival and flow cytometry with single-cell transcriptomic corroboration

编号 170 展板 13 时间 4/19 02:00–05:00 区域 Section 8 主讲 Matthew Abikenari

分会场 Immune Cell Biology and Tumor-Immune Crosstalk

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作者与单位 Authors & Affiliations

Matthew Alexander Abikenari¹, John Choi¹, Justin Liu², Adam Sjoholm², George Nageeb¹, James Poe¹, Brandon Hwa-Lin Bergsneider³, Andrew Tran¹, David Bakalov⁴, Ravi Medikonda¹, Lily Kim¹, Rohit Verma³, Caren Wu¹, Kwang Bog Cho¹, Matei Banu², Michael Lim²

¹Neurosurgery, Stanford University School of Medicine, Stanford, CA,²Neurosurgery, Stanford University School of Medicine, Stanford, CA, United States, Stanford, CA,³Stanford University School of Medicine, Stanford, CA,⁴Physician Scientist Training Program, University of Utah, Salt Lake City, UT, United States, Salt Lake City, UT

摘要 Abstract

中文摘要

引言：肿瘤相关中性粒细胞（TANs）在胶质母细胞瘤（GBM）中丰度较高，但其功能具有时相依赖性。我们研究了TANs是否与外周血中性粒细胞（PBNs）存在差异转录、是否在原位获得抗原呈递功能，以及中性粒细胞清除的时机是否影响生存和肿瘤内免疫。方法：转录组学：对公开发布的近期人类GBM数据集（bulk RNA-seq和scRNA-seq；Seurat标准流程、严格质控、整合/UMAP、Wilcoxon差异表达）进行整合分析。预设模块：共刺激（CD83/CD86/CD40/ICOSLG）、MHC-II（HLA-DRB3/A、DPA1/DPB1）以及抗原加工/伴侣分子（CD74、CALR、PSME2、HLA-DMA/DMB）。体内生存：原位CT2A和GL261模型；抗Ly6G（1A8）或同型对照采用两种给药方案，早期（第−1天植入前至≥第14天）和延迟（第+8天开始）。Kaplan-Meier/log-rank；Cox模型（HR，95% CI）。离体流式（第8天）：微球标准化的光谱流式细胞术；CD45+白细胞；CD11b+髓系细胞（Ly6G+中性粒细胞；Ly6C^hi单核细胞）、表达MHC-II的F4/80+ TAMs、CD3+/CD8+ T细胞。双侧Mann-Whitney检验；必要处标注FDR。已鉴定出Ly6G表位遮蔽；清除效果通过CD11b+SSC^hi回溯门控和每周血细胞计数确认。结果：转录组学：TANs相较PBNs显示APC/共刺激程序的一致上调（CD83/CD86/CD40/ICOSLG；HLA-DR/DP；CD74/CALR/PSME2/HLA-DMA/DMB），与树突样、非细胞毒性的TAN状态一致。生存（双相）：早期抗Ly6G损害了结局，CT2A（n=9/组）中位17天对23天；HR 1.95（1.10-3.46），p=0.018。后期清除消除了这一不利影响，CT2A HR 1.23（0.68-2.22），p=0.49。流式（第8天）：早期清除的肿瘤含有更多Ly6C^hi单核细胞（占CD45+：29%对18%，p=0.006）、更少CD8+ T细胞（3.2%对6.1%，p=0.011），以及更低的CD8:Ly6C^hi比值（0.11对0.36，p=0.004）。方向性但无统计学意义的汇总指标：CD11b+髓系细胞（68%对55%，p=0.07）、CD3+ T细胞（9%对14%，p=0.09）、F4/80+ TAMs上MHC-II降低（p=0.08）、CD11c⁺MHC-II^hi APCs减少（q≈0.12）。结论：人类TANs采纳了APC/共刺激程序，且时机至关重要：植入前清除中性粒细胞造成髓系偏向、抗原贫乏、T细胞耗竭的环境，增强生存，而后期靶向则削弱此效应。这些观察结果支持时相特异性的TAN调控。选择性地维持或再教育早期TANs并抑制后期抑制性程序，以调控基于生物标志物的GBM免疫治疗。

查看英文原文 English abstract

Introduction: Tumor-associated neutrophils (TANs) are abundant in glioblastoma (GBM), yet their functions are phase-dependent. We investigated whether TANs are differentially transcribed from peripheral blood neutrophils (PBNs), if they acquire antigen-presenting functions in situ, and if neutrophil depletion timing influences survival and intratumoral immunity Methods: Transcriptomics: Integrated analysis of publicly deposited recent human GBM data sets (bulk RNA-seq and scRNA-seq; Seurat standard pipeline, stringent QC, integration/UMAP, Wilcoxon DE). Prespecified modules: co-stimulation (CD83/CD86/CD40/ICOSLG), MHC-II (HLA-DRB3/A, DPA1/DPB1), and antigen-processing/chaperones (CD74, CALR, PSME2, HLA-DMA/DMB). In vivo survival: Orthotopic CT2A and GL261; anti-Ly6G (1A8) or isotype on two schedules, (day −1 pre-implantation through ≥day 14) and delayed (start day +8). Kaplan-Meier/log-rank; Cox models (HR, 95% CI). Ex vivo flow (day 8): Bead-normalized spectral cytometry; CD45+ leukocytes; CD11b+ myeloids (Ly6G+ neutrophils; Ly6C^hi monocytes), F4/80+ TAMs with MHC-II, CD3+/CD8+ T cells. Two-sided Mann-Whitney; FDR where indicated. Ly6G epitope masking identified; depletion confirmed by CD11b+SSC^hi back-gating and weekly blood counts. Results: Transcriptomics: TANs vs PBNs showed coherent upregulation of APC/co-stimulatory programs (CD83/CD86/CD40/ICOSLG; HLA-DR/DP; CD74/CALR/PSME2/HLA-DMA/DMB), in line with dendritic-like, non-cytotoxic TAN states. Survival (biphasic): Previous anti-Ly6G impaired outcomes, CT2A (n=9/arm) median 17 vs 23 days; HR 1.95 (1.10-3.46), p=0.018. Subsequent depletion abrogated this penalty, CT2A HR 1.23 (0.68-2.22), p=0.49; Flow (day 8): Early-depleted tumors contained more Ly6C^hi monocytes (of CD45+: 29% vs 18%, p=0.006), fewer CD8+ T cells (3.2% vs 6.1%, p=0.011), and lower CD8:Ly6C^hi ratio (0.11 vs 0.36, p=0.004). Directional but non-significant aggregates: CD11b+ myeloids (68% vs 55%, p=0.07), CD3+T cells (9% vs 14%, p=0.09), reduced MHC-II on F4/80+ TAMs (p=0.08), reduced CD11c⁺MHC-II^hi APCs (q≈0.12). Conclusion: Human TANs take up APC/co-stimulatory programs, and timing is critical: pre-implantation neutrophil depletion imposes a myeloid-skewed, antigen-poor, T-cell-depleted setting, augments survival, while late targeting diminishes this. These observations favor phase-specific TAN modulation. maintenance or re-education of early TANs and suppression of late suppressive programs selectively, to regulate biomarker-based GBM immunotherapies.

利益披露 Disclosure

M. A. Abikenari, None.. J. Choi, None.. J. Liu, None.. A. Sjoholm, None.. G. Nageeb, None.. J. Poe, None.. A. Tran, None.. D. Bakalov, None.. R. Medikonda, None.. L. Kim, None.. C. Wu, None.. K. Cho, None.. M. Banu, None.. M. Lim, None.

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