Natural and in vitro-generated regulatory T-cell subsets differentially modulate phenotype and drug response in K562...

Regulatory T cells (Tregs) are key mediators of immune suppression in cancer and are frequently elevated in hematologic malignancies, including acute and chronic leukemias, however, their role in modulating the leukemic cell phenotype and therapy response remains unclear. This study investigated whether natural Tregs (nTregs), in vitro-generated CD4⁺CD25⁺Foxp3⁺ Tregs (iTregs), and type 1 regulatory T (Tr1) cells directly modulate the phenotype, viability and drug response of the K562 chronic myeloid leukemia cell line in vitro. K562 cells were cultured either in direct co-culture with Treg subsets or in their culture supernatants, followed by flow cytometric assessment of stemness-associated and progression/drug-resistance-related markers. Anti-TGF-β1 or anti-IL-10 blocking antibodies were used to assess cytokine-dependent mechanisms. Functional effects were evaluated using an MTT-based assay under drug-free conditions or after treatment with daunorubicin or gemcitabine. Natural Tregs increased the proportion of CD34⁺, PDGFRβ⁺, ABCG2₊ and VEGFR1⁺ K562 cells. Neutralization of TGF-β1 in the co-culture system partially attenuated the nTreg-associated upregulation of PDGFRβ and ABCG2. iTregs increased ABCG2 expression, which was also reduced upon TGF-β1 blockade. Tr1 cells increased the proportion of CD24⁺ cells, upregulated PDGFRβ and ABCG2, and increased K562 cell metabolic activity/viability under drug-free conditions. All Treg subsets attenuated daunorubicin cytotoxicity, whereas reduced sensitivity to gemcitabine was observed only in iTreg- and Tr1-containing cultures. These findings suggest that Treg cell subsets may directly promote stemness-associated and drug-tolerance-related features in K562 cells in vitro, supporting a tumor-promoting role within the leukemia microenvironment and warranting further validation in primary leukemia samples and in vivo models.