Gémes Nikolett, Rónaszéki Benedek, Modok Szabolcs, Borbényi Zita, Földesi Imre, Trucza Éva, Godza Blanka, László Zsuzsanna, Csernus Balázs, Krenács László, Bagdi Enikő, Szabó Enikő, Puskás László G, Bertagnolo Valeria, Szebeni Gábor J
Laboratory of Functional Genomics, Core Facility, HUN-REN Biological Research Center, Szeged, Hungary.
Department of Internal Medicine, Hematology Center, Faculty of Medicine, University of Szeged, Szeged, Hungary.
Front Immunol. 2025 Apr 17;16:1563386. doi: 10.3389/fimmu.2025.1563386. eCollection 2025.
Understanding leukemia-associated immunophenotypes (LAIP) could assist in the design of therapies to ameliorate patient benefits in acute myeloid leukemia (AML). In our study, focusing on single-cell heterogeneity in therapeutic resistance, flow cytometric immunophenotyping of the peripheral blood of therapy-naive and follow-up AML patients versus age and sex-matched healthy controls (HCs) was performed.
The FACS panel consisted of Viobility 405/520 Fixable Dye, Anti-human CD45, CD19, CD3, CD7, CD33, CD34, CD38, CD64, CD117, CD135, HLA-DR antibodies. Unsupervised clustering algorithms such as Uniform Manifold Approximation and Projection for Dimension Reduction (UMAP) and Flow cytometry data that builds Self-Organizing Maps (FlowSOM) were used to reveal the LAIP. The measurable residual disease (MRD) was monitored by our proposed manual gating. To complement the characterization of peripheral immune cells, Luminex MAGPIX was used to measure the concentration of 31 soluble immune-oncology mediators from the plasma of AML patients and HC.
Both manual gating, UMAP and FlowSOM showed normalization of LAIP similar to the HC immune landscape following therapy. Eleven metaclusters (MCs) were associated with AML before therapy. The follow-up of AML samples revealed four MCs of therapy sensitive cells, and one MC composed of therapeutic resistant cells (MC12: CD3-CD7-CD33-CD38- CD64- HLA-DR- CD117- CD135-) identified by the FlowSOM analysis. The initial AML blasts in the MRD gate (CD19-, CD45+, CD3-, CD38+/CD34±, CD7+/CD117+, CD117+/CD135+) were detectable at the lowest frequency in our current study at 22 cells per 100,000 (0.022%) CD45+CD3- living singlet parental population. In the plasma of AML patients the levels of BAFF, B7-H2, B7-H4, CD25, MICA, and Siglec-7 were increased versus HCs.
This study focused on understanding the LAIP in AML before and after therapeutic intervention. The study highlights the potential of using single-cell LAIP profiling and immune mediator measurements to monitor therapy response and identify measurable residual disease and therapy resistant cell populations in AML.
了解白血病相关免疫表型（LAIP）有助于设计改善急性髓系白血病（AML）患者获益的治疗方案。在我们的研究中，聚焦于治疗耐药中的单细胞异质性，对初治及随访的AML患者与年龄和性别匹配的健康对照（HC）的外周血进行了流式细胞术免疫表型分析。
流式细胞术检测板由Viobility 405/520固定染料、抗人CD45、CD19、CD3、CD7、CD33、CD34、CD38、CD64、CD117、CD135、HLA - DR抗体组成。使用无监督聚类算法，如用于降维的均匀流形近似和投影（UMAP）以及构建自组织映射的流式细胞术数据（FlowSOM）来揭示LAIP。通过我们提出的手动设门法监测可测量残留病（MRD）。为补充外周免疫细胞的特征描述，使用Luminex MAGPIX检测AML患者和HC血浆中31种可溶性免疫肿瘤介质的浓度。
手动设门法、UMAP和FlowSOM均显示治疗后LAIP正常化，类似于HC的免疫格局。治疗前有11个元簇（MC）与AML相关。对AML样本的随访发现了4个治疗敏感细胞的MC，以及通过FlowSOM分析鉴定出的1个由治疗耐药细胞组成的MC（MC12：CD3 - CD7 - CD33 - CD38 - CD64 - HLA - DR - CD117 - CD135 -）。在我们当前的研究中，MRD门（CD19 -，CD45 +，CD3 -，CD38 + / CD34±，CD7 + / CD117 +，CD117 + / CD135 +）中的初始AML原始细胞在每100,000个（0.022%）CD45 + CD3 - 活单细胞亲本群体中以最低频率22个细胞可检测到。与HC相比，AML患者血浆中BAFF、B7 - H2、B7 - H4、CD25、MICA和Siglec - 7的水平升高。
本研究聚焦于了解治疗干预前后AML中的LAIP。该研究强调了使用单细胞LAIP分析和免疫介质测量来监测治疗反应以及识别AML中可测量残留病和治疗耐药细胞群体的潜力。
