Figure 2

Immunosuppressive properties of tumor-induced CD8⁺ Treg cells. (A) Flow cytometric histoplot represented TGFβ (first panel), IL10 (second panel), and IFNγ (third panel) positivity within CD8⁺FOXP3⁺ Treg cells (blue) and CD8⁺FOXP3⁻ cells (pink). Percentages of the respective population were represented graphically in right panels. (B) The relative transcript levels of TGFβ, IL10, and IFNγ were analyzed by RT-PCR (upper panel) and quantified by qPCR (lower panel). The gel bands were cropped, whole gel pictures were given in Supplementary Fig. S2A. (C) CD4⁺ responder T cell proliferation was measured by CFSE-dilution assay in presence and absence of isolated tumor-CD8⁺ Treg cells (CD8⁺CD25⁺CTLA4⁺ T cells). Responder cells were stimulated with anti-CD3/-CD28 antibodies and rIL2 for 96 h. Numbers in outlined areas indicated the percent of dividing cells in each histo-plot and presented statistically (right panel). (D) CD8⁺ cytotoxic cells (Tc) were primed with CD8⁺ Treg cells. Control and Treg-primed Tcs were then co-incubated separately with breast tumor cells for a 48 h and apoptotic index of the tumor cells i.e. CD8-negative population, were analyzed flow cytometrically by Annexin-V/PI-positivity and represented statistically (right panel). (E) CD4⁺ T cell apoptosis was determined in presence and absence of tumor-CD8⁺ Treg cells and analyzed statistically. (F) Percent suppression of CD4⁺ responder cells was determined by CFSE dilution assay in presence or absence of CD4⁺ and CD8⁺ Treg cells alone or a combination. Isotype-matched control antibodies were used for all flow cytometric experiments. Data are representative as the mean ± SEM and are the cumulative results of five independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001. GAPDH was used as internal loading control in RT-PCR and qPCR.