Background: Galectin-9 (Gal-9), a crucial immune checkpoint ligand, plays an essential role in human tumors, but its clinical significance in chronic lymphocytic leukemia (CLL) is still unclear. In the current study, we took a closer look at Gal-9 expression as a part of our quest to identify novel prognostic biomarkers of CLL.

Methods: In this study, 126 untreated CLL patients and 40 age and gender-matched controls were analyzed. Plasma Gal-9 levels were quantified using enzyme-linked immunosorbent assay (ELISA), while Gal-9 expression on immune cells was assessed by multicolor flow cytometry. Confocal microscopy was used to visualize Gal-9 localization on B cell membranes. Epstein-Barr virus (EBV)-miR-BamH1 fragment H rightward facing 1-1 (BHRF1-1) expression was detected via reverse transcription quantitative polymerase chain reaction (RT-qPCR), and digital PCR was used to quantify Gal-9 transcripts in purified CD19⁺ B cells. The presence of CLL-associated cytogenetic abnormalities was determined by fluorescence in situ hybridization (FISH). The study also identified and characterized B regulatory cell (Breg)-like CLL cells and myeloid-derived suppressor cell (MDSC) subsets (monocytic (M-MDSC), polymorphonuclear (PMN-MDSC), and early-stage (eMDSC)) expressing Gal-9. Survival analyses, including Kaplan-Meier and Cox regression models, were performed to evaluate the prognostic significance of Gal-9 expression.

Results: Our results confirmed an increased expression of Gal-9 mRNA (p < 0.0001) as well as an elevated percentage of B-cells with membrane Gal-9 expression (p = 0.04) in CLL patients compared to healthy volunteers. Elevated Gal-9 levels in plasma and increased Gal-9 expression on B cells were associated with poor prognostic markers. Notably, the percentage of Gal-9-positive B-cells was found to be an independent prognostic factor of time to first treatment in CLL patients (p = 0.006). Plasma levels of Gal-9 were closely linked to the percentages of CD19⁺/Gal-9⁺ (p < 0.001), eMDSC/Gal-9⁺ (p < 0.001), and M-MDSC/Gal-9⁺ (p = 0.001) cells. Moreover, it was noted that Gal-9 expression was significantly higher (p < 0.0001) within the CD19⁺CD5⁺CD24^highCD38^high fraction in comparison to the non-Breg CLL cells. Finally, higher levels of Gal-9 expression were linked to EBV positivity and the expression of EBV-miR-BHRF1-1.

Conclusions: Gal-9 expression in leukemic B cells and plasma may serve as a marker of disease activity in CLL. Moreover, an increase in malignant B cell production, along with a boost in MDSC cells, appears to coincide with a significant increase in Gal-9 concentration. Breg-like CLL cells show distinct Gal-9 expression compared to non-Breg CLL cells. Gal-9 may enhance the immunosuppressive potential of Breg. Moreover, EBV-driven Gal-9 upregulation may worsen prognosis in CLL.