Fig. 5

Targeting sialoglycans and Siglec-9 on suppressive human CD33⁺ cells attenuates their function. A Experimental setup for generating suppressive myeloid cells in vitro. Fresh PBMCs were isolated from buffy coats of healthy donors and cocultured with the indicated cancer cell lines at a ratio of 100:1. On Day 7, CD33⁺ cells were isolated by magnetic positive selection, and their suppressive effect on autologous CD8⁺ T cells was assessed. Suppressive CD33⁺ cells were immediately used or were pretreated with sialidase or a Siglec-9 blocking antibody as indicated. CD8⁺ T cells were stained with CellTrace Violet (CTV) and stimulated by the addition of IL-2 and anti-CD3/28 microbeads. After 5 days, CD8⁺ T-cell proliferation was assessed by FACS. B Percentage of proliferating CD8⁺ cells upon coculture with the indicated suppressive CD33⁺ cells. Suppressive myeloid cells were generated from A459, A549 cells stably expressing sialidase (A549-sia) or A549 GNE KO cancer cells. N = 4–24 donors. A representative histogram for each condition is shown on the right. C Lectin staining was performed on suppressive CD33⁺ cells on Day 7 of the experiment to assess SNA and (D) MALII levels. Representative images of A549 (green) and A549-sia-MDSC-like cells (pink) are shown on the right. n = 7–10 donors in paired conditions. E Percentage of proliferating CD8⁺ cells upon coculture with suppressive CD33⁺ cells generated by A549 coculture. CD33⁺ cells were used immediately or were pretreated with sialidase or a Siglec-9 blocking antibody as indicated. N = 6–24 donors. F Assay setup to test the suppressive capacity of tumor-digested CD33⁺ cells. CD33⁺ cells were freshly isolated from the tumor homogenates of lung or colon cancer patients and used immediately or were pretreated with sialidase or a Siglec-9 blocking antibody. CD8⁺ cells were isolated from fresh PBMCs from the same donor and stained with CTV. The suppressive activity was assessed on Day 5 by flow cytometry. G Percentage of proliferating CD8⁺ cells of more than 3 generations upon coculture with tumor-derived CD33⁺ cells. MDSCs were left untreated and pretreated with sialidase or a Siglec-9 blocking antibody. Exemplary results for each condition are shown on the right. n = 5–8 per condition. The data are presented as the mean ± SD. A two-tailed paired t test was used. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001