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. Author manuscript; available in PMC: 2016 Sep 1.
Published in final edited form as: Nat Immunol. 2016 Feb 1;17(3):286–296. doi: 10.1038/ni.3352

Figure 2.

Figure 2

Cpdm−/− mice exhibit impaired generation of Treg cells. (a) Flow cytometry of cells from the thymus (Thy), spleen (SP), lymph nodes (LN), mesenteric lymph nodes (MLN), colon, small intestine (SI) and lungs of 4-week-old Cpdm+/− and Cpdm−/− mice (n = 7–12 per group), analyzing the expression of Foxp3 and CD4 (left), and frequency of Foxp3+CD4+ Treg cells in those tissues (right). Numbers adjacent to outlined areas (left) indicate percent Foxp3+CD4+ (Treg) cells. (b) Total Foxp3+CD4+ cells in the thymus, spleen, lymph nodes and mesenteric lymph nodes of Cpdm+/− and Cpdm−/− mice (n = 8 per group). (c) Flow cytometry (as in a) of pregated CD45.1+ or CD45.2+ cells from the thymus and spleen of host mice (CD45.1+) (n = 6–9 per group) reconstituted with Cpdm+/− (CD45.2+) BM cells or Cpdm−/− (CD45.2+) BM cells (BMC) or with a mixture of wild-type (CD45.1+) BM cells (WT) and Cpdm−/− (CD45.2+) BM cells (Mixed) (above), and frequency of Foxp3+CD4+ Treg cells in those tissues (below). (d,e) Flow cytometry analyzing Foxp3 expression in naive CD4+CD62L+CD44−CD25 T cells obtained from Cpdm+/− and Cpdm−/− mice and stimulated with various concentrations (above plots) of anti-CD3 (0.5, 1 or 3 µg/ml) plus anti-CD28 (1 µg/ml), and TGF-β (1 ng/ml) (d), or with anti-CD3 (2 µg/ml) plus anti-CD28 (1 µg/ml) and various concentrations (above plots) of TGF-β (0.5, 1 or 2.5 µg/ml). Numbers above bracketed lines indicate percent Foxp3+ cells. (f) Flow cytometry of cells from the spleen and lymph nodes of congenic B6 (CD45.1+) host mice (n = 4 per group) that received adoptively transferred naive CD4+CD62L+CD44−CD25 T cells from Cpdm−/− and Cpdm−/− (CD45.2+) OT-II mice (which have transgenic expression of a TCR specific for ovalbumin peptide), followed by immunization of the host mice with that ovalbumin peptide (10 µg), analyzing the expression of Foxp3 and CD45.2 (left), and frequency of CD45.2+ Treg cells in those tissues (right). Numbers adjacent to outlined areas (left) indicate percent Foxp3+ (Treg) CD45.2+ (donor) cells. Each symbol (a,f) represents an individual mouse; small horizontal lines indicate the mean (± s.d.). *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001 (two-tailed unpaired t-test). Data are pooled from or representative of two to four independent experiments (mean and s.d. in b).