Impact of innate lymphoid cell type 2 in chronic lymphocytic leukemia on the function of treg and CD8+ T cells through IL-9.

Abstract

Objective: This study investigated the impact of innate lymphoid cell type 2 (ILC2s) on the function of regulatory T cells (Treg) and CD8⁺ T cells in chronic lymphocytic leukemia (CLL) through IL-9.

Methods: Peripheral blood samples were collected from CLL patients (n = 52) and healthy controls (n = 30). ILC2 proportions and IL-9 levels were assessed using flow cytometry and ELISA. Immunofluorescence staining was performed to stain GATA3, CRTH2, and IL-9 in cervical lymph nodes from CLL patients (n = 10) and control subjects with reactive lymphadenitis (n = 10). Correlation analysis between ILC2s and IL-9 was conducted using the Spearman test. ILC2s were sorted and cultured from CLL patients, followed by co-culture experiments with PBMCs of healthy controls and MEC-1 cells, with or without anti-IL-9 antibody intervention. Flow cytometry was used to measure the proportions of ILC2s, Treg cells, PD-1⁺/TIGIT⁺/CTLA-4⁺ Treg subsets, and granzyme B⁺/perforin⁺ CD8⁺ T cells, along with MEC-1 cell apoptosis.

Results: The proportions of ILC2s and Treg, along with serum IL-9 levels, were significantly elevated in CLL patients (P < 0.05). Peripheral blood ILC2s were positively correlated with IL-9 (r = 0.609, P < 0.001). The average fluorescence intensity of GATA3, CRTH2, and IL-9 in the cervical lymph nodes of CLL patients increased significantly (P < 0.001), and IL-9 showed colocalization with GATA3 and CRTH2. In vitro, IL-9 levels in the supernatant of sorted ILC2s from CLL patients increased. Treatment with anti-IL-9 antibody significantly reduced the PD-1⁺ Treg and TIGIT⁺ Treg cells while increasing granzyme B⁺ CD8⁺ T cells (P < 0.05). However, there was no significant effect on Treg, CTLA-4⁺ Treg, and perforin⁺ CD8⁺ T cells (P > 0.05). Additionally, anti-IL-9 antibody significantly increased early apoptosis (P < 0.05).

Conclusion: ILC2s affect CD8⁺ T cells and Treg cells through IL-9, weakening the anti-tumor effects of CD8⁺ T cells and enhancing the immunosuppressive effects of Treg cells, thereby contributing to CLL pathogenesis.