Composition and plasticity of triple-negative breast carcinoma-infiltrating regulatory T cells

Low Foxp3⁺ regulatory T-cell (Treg) presence in the tumor-infiltrating lymphocytes (TILs) is considered favorable in breast cancer, and numerous CD25-targeting agents have been applied in the attempt to remove Foxp3⁺ Treg cells, which typically present CD4⁺ CD25^+/hi surface phenotype. However, CD25 is not Treg-exclusive and can be upregulated by effector T cells. Hence, CD25 depletion may cause the elimination of activated T cells that are responding to tumor-specific antigens. In this study, the composition and function of CD4⁺ CD25⁺ cells inside the microenvironment of triple-negative breast carcinoma (TNBC) were investigated. Directly ex vivo, the Foxp3⁺ Treg cells represented a minor subset in total CD4⁺ CD25⁺ TILs. Significant differences were observed in the expression of Treg-associated molecules between CD4⁺ CD25⁺ Foxp3⁺ TILs and CD4⁺ CD25⁺ Foxp3^- TILs. While both the CD4⁺ CD25⁺ Foxp3⁺ and the CD4⁺ CD25⁺ Foxp3^- TILs could express CTLA-4 and LAG-3, the expression levels were significantly higher in CD4⁺ CD25⁺ Foxp3⁺ TILs than in CD4⁺ CD25⁺ Foxp3^- TILs. Upon TCR stimulation, the expression of TGF-beta was significantly higher in CD4⁺ CD25⁺ Foxp3⁺ TILs, while the expression of IL-10 was significantly higher in CD4⁺ CD25⁺ Foxp3^- TILs. These differences were conserved in the blood counterparts of these cells. Interestingly, the level of CD25⁺ Foxp3⁺ cells in circulating CD4⁺ T cells was positively correlated with the level of CD25⁺ Foxp3⁺ cells in CD4⁺ TILs, but the level of CD25⁺ Foxp3^- cells in circulating CD4⁺ T cells was not associated with the level of CD25⁺ Foxp3^- cells in CD4⁺ TILs. Th17-polarizing medium could readily remodel CD4⁺ CD25⁺ Foxp3^- , but not CD4⁺ CD25⁺ Foxp3⁺ , T cells into RORgammat and IL-17-expressing T cells, demonstrating stronger plasticity of the former subset. Together, these data demonstrated that the CD4⁺ CD25⁺ TILs were composed of distinctive Foxp3^- and Foxp3⁺ cells, with the former representing the major subset. The antigen specificity and effector molecule expression of the CD4⁺ CD25⁺ Foxp3^- thus require further analyses.