Figure 2. Flow cytometry, gene expression and functional analysis of effector and Treg cells in TCRmini-Foxp3GFP mice bearing B16 melanoma tumors expressing NP-Ep63K.
(A) Cells from control, draining lymph nodes and tumor infiltrates were isolated and single cell suspensions were stained with the relevant antibodies. Expression of Foxp3GFP (left column) and CD25 (second column) on CD4+ T cells are shown. Expression of activation markers CD44 and CD62L is shown on gated effector CD4+ Foxp3− cells (third column) and Foxp3 and CD25 expression are shown on gated CD4+ T cells (right column). Gates used to define Foxp3GFP- cells (left column, continuous line) and subsets of naive CD44−CD62L+ (third column, continuous line) and activated CD44+CD62L− (third column, dotted line) cells as well as Foxp3GFPlo (left column, broken line) and Foxp3GFPhi (left column, dotted line) Treg cells for TCR repertoire studies are shown as rectangles. Naive cells were absent in tumors. Figure shows representative data of at least five mice analyzed. (B) Expression of GITR (left panels) and CTLA-4 (right panels) in CD4+Foxp3GFP- (upper panels) and CD4+Foxp3GFP+ (lower panels) cells isolated from tumor draining lymph nodes and tumors. Two individual mice were analyzed. (C) Analysis of Foxp3, IL-10 and TGF-β expression in Foxp3GFP- and Foxp3GFP+ CD4+ T cells isolated from tumors. Sorted cells were lysed directly and gene expression was detected by RT-PCR. Samples were normalized for β-actin expression. Two individual mice were analyzed. (D) Foxp3GFP+CD4+ T cells isolated from the draining lymph nodes (DrLN) or tumors (TILs) suppress proliferation of effector CD4+ T cells. One of two experiments is shown.