Association of immunosuppressive CD45⁺CD33⁺CD14⁻ CD10⁻HLA-DR^−/low neutrophils with poor prognosis in patients with lymphoma and their expansion and activation through STAT3/arginase-1 pathway in vitro

Abstract

Objective: This study aimed to explore the clinical significance of CD45+CD33+CD14−CD10−HLA-DR−/low neutrophils (Cluster of Differentiation 10 [CD10−] neutrophils) in B-cell non-Hodgkin’s lymphoma (B-NHL). An amplification system of CD10− neutrophils in vitro was constructed using cytokines, and the mechanisms underlying the cytokine-induced expansion and activation of the CD10− neutrophil subpopulation were investigated. Material and Methods: We identified a novel suppressive cell population known as CD10− neutrophils in the peripheral blood of patients with B-NHL in different statuses by flow cytometry and found it to be correlated with interleukin-6 levels, T cell counts, and plasma arginase-1 (Arg-1) levels. We then verified the effect of CD10− neutrophil expression on the prognosis of patients with B-NHL. Furthermore, we described a clinically compatible method for generating granulocyte populations rich in CD10− neutrophils using cultures of peripheral blood-isolated neutrophils supplemented with cytokines in vitro. Arg-1 expression was detected in neutrophils before and after induction by cytokines through reverse-transcription polymerase chain reaction, enzyme-linked immunosorbent assay, and flow cytometry. T-cell proliferation and apoptosis were measured by carboxyfluorescein succinimidyl ester assay and Annexin V-Propidium Iodide stains, and induced cells were exposed to Arg-1 inhibitor and ruxolitinib. signal transducer and activator of transcription 3 (STAT3)/Arg-1 signaling was studied mainly by western blot and chromatin immunoprecipitation experiments. Results: We established a correlation between high CD10− neutrophil levels and poorer survival outcomes in patients with B-NHL. Moreover, CD10− neutrophils were positively correlated with interleukin (IL)-6, T-reg cells, and plasma Arg-1 levels and negatively correlated with the absolute number of total T cells. Granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte colony-stimulating factor, and IL-6 could all induce the expansion of CD10− neutrophil phenotype cells in vitro, which exhibit typical immature cellular morphology, and the combination of IL-6 and GM-CSF was the most effective. We confirmed that the STAT3/Arg-1 signaling pathway could be a critical mechanism regulating CD10− neutrophil-mediated immunosuppression in vitro. Conclusion: CD10− neutrophils exhibited basic characteristics similar to conventional myeloid-derived suppressor cells. Our observations provide a promising STAT3 or Arg-1 targeting strategy for B-NHL and an important method for generating remarkably amounts of inhibitory granulocyte populations rich in CD10− neutrophils for immunotherapy.