Supplementary Figure 2: Proliferation, survival and lineage stability of Treg cells from Foxp3CreAtg7fl/fl and Foxp3CreAtg7fl/flBcl2-TG mice. | Nature Immunology

Supplementary Figure 2: Proliferation, survival and lineage stability of Treg cells from Foxp3CreAtg7fl/fl and Foxp3CreAtg7fl/flBcl2-TG mice.

From: Autophagy enforces functional integrity of regulatory T cells by coupling environmental cues and metabolic homeostasis

Supplementary Figure 2

(a,b) Flow cytometry analyzing Ki67 expression in Treg cells from the spleen of Foxp3CreAtg7+/fl and Foxp3CreAtg7fl/fl mice (a) and Foxp3CreAtg7+/fl and Foxp3CreAtg7fl/fl chimeras (b). Numbers above bracketed lines indicate percent Ki67+ cells (a,b). (c) Flow cytometry analyzing CellTrace dilution in Treg cells stimulated with anti-CD3, anti-CD28, and IL-2 for 96 h. Numbers above bracketed lines indicate percent CellTracelo cells. (d) Foxp3CreAtg7+/fl or Foxp3CreAtg7fl/fl Treg cells were transferred into Rag1−/− mice, followed by flow cytometry analysis of CellTrace dilution in donor cells in the spleen and PLNs of recipients 7 days later. Numbers above bracketed lines indicate percent CellTracelo cells. (e,f) Flow cytometry analyzing the expression of active caspase-3 (e) and Bim (f) in CD45.2+ Treg cells in the spleen of Foxp3CreAtg7+/fl and Foxp3CreAtg7fl/fl chimeras. Numbers adjacent to outlined areas indicate percent caspase-3+ cells (e), and numbers above graph indicate MFI of Bim (f). (g) Flow cytometry analyzing the expression of IFN-γ and IL-17 (left), and frequency of IFN-γ+ cells and IL-17+ cells (right) in MC38 tumor-infiltrating Foxp3CreAtg7+/fl and Foxp3CreAtg7fl/fl Treg cells (n=8 mice per genotype). Numbers in quadrants indicate percent cells in each throughout (left). (h) Flow cytometry analyzing the expression of IFN-γ and IL-17 in Foxp3CreAtg7+/fl and Foxp3CreAtg7fl/fl Treg cells stimulated in vitro with anti-CD3, anti-CD28 and IL-2 for 96 h. (i) Flow cytometry analyzing active caspase-3 expression in Treg cells (from Foxp3CreAtg7+/fl, Foxp3CreAtg7fl/fl Foxp3CreAtg7+/flBcl2-TG and Foxp3CreAtg7fl/flBcl2-TG mice) stimulated with anti-CD3, anti-CD28, and IL-2 for overnight. Numbers above bracketed lines indicate percent caspase-3+ cells. (j,k) Flow cytometry analyzing the expression of Foxp3 in CD4+ T cells (j) and CD62L and CD44 in Foxp3−CD4+ T cells (k) in the spleen of Foxp3CreAtg7+/fl, Foxp3CreAtg7fl/fl Foxp3CreAtg7+/flBcl2-TG and Foxp3CreAtg7fl/flBcl2-TG mice. Numbers adjacent to outlined areas indicate percent Foxp3+ cells (j). (l) Flow cytometry analyzing the expression of IFN-γ and IL-17 in Treg cells in the spleen of Foxp3CreAtg7+/flBcl2-TG and Foxp3CreAtg7fl/flBcl2-TG mice. (m) Flow cytometry analyzing YFP-Foxp3 expression in divided Treg cells (from Foxp3CreAtg7+/flBcl2-TG and Foxp3CreAtg7fl/flBcl2-TG mice) stimulated in vitro with anti-CD3, anti-CD28 and IL-2 for 96 h. Numbers above graphs indicate MFI of YFP-Foxp3. NS, not significant (P > 0.05); * P < 0.05 (two-tail unpaired Student's t-test in g). Data are representative of four (a-f), two (h,m) or three (i-l) experiments, or pooled from two out of two (g) experiments (mean ± s.e.m in g).

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