Fig. 1.
Identification of CD4+CD25−LAG3+ regulatory T cells. (A) LAG-3 expression in the spleen, Peyer's patch (PP), mesenteric lymph node (MLN), and inguinal lymph node (ILN). (Top) LAG-3 and CD25 or CD45RB expression in splenocytes from C57BL/6 is shown for the gated on CD4+ (Upper Left) or CD4+CD25− (Upper Center and Upper Right) T cells, respectively. (Lower) LAG-3 and CD45RB expression of PP, MLN, and ILN gated on CD4+CD25− T cells. Representative FACS dot plots from at least three independent experiments are shown. (B) Proliferation of CD4+CD25−CD45RBhigh, CD4+CD25−, and CD4+CD25−LAG3+ splenocytes after 72 h stimulation with anti-CD3/anti-CD28. The results are the means of three independent experiments. (C) Cytokines in the culture supernatants of CD4+CD25−CD45RBhigh, CD4+CD25+, and CD4+CD25−LAG3+ T cells stimulated for 5 days with anti-CD3 mAb. Representative data from at least three independent experiments are shown. (D) Suppressive function of CD4+CD25−LAG3+ T cells. Naïve CD4+CD25−CD45RBhigh Thy1.1+ T cells were labeled with carboxyfluorescein diacetate succinimidyl ester (CFSE) and cocultured with the indicated Thy1.2+ T cells and irradiated whole splenocytes plus anti-CD3 mAb. Representative data from three independent experiments are shown. (E) Suppression of CD4+CD25−CD45RBhigh T-cell-mediated colitis in RAG-1−/− mice by CD4+CD25−LAG3+ T cells. Data represent body weight as a percentage of the initial weight of individual mice; n = 6 per group. (F) Representative photomicrographs of the colons stained with hematoxylin and eosin after transfer of the indicated cell populations. All error bars represent ±SD. (Scale bar, 100 μm.)