Prevention of graft rejection by Tregs.

<p><b>A,</b> Cardiac graft survival in mice treated with antigen-specific iTregs (CBA background) induced by the indirect pathway and direct pathway. Tregs (1×10⁶ cells) were injected intravenously one day before transplantation of the C57BL/6 heart into CBA recipients. BALB/c to CBA cardiac transplantation was performed after injection of indirect-iTregs (CBA) against the K^b peptide. (The Log-rank test was used; <i>P</i> values are compared between the indirect pathway and other pathways; no treatment, <i>P</i><0.001; anti-CD3 Ab/anti-CD28 Ab, <i>P</i><0.001; BALB/c vs. CBA, <i>P</i> = 0.0013; direct pathway, <i>P</i><0.001. Direct-iTreg treatment vs. no treatment, <i>P</i><0.001) <b>B,</b> HE staining of harvest cardiac grafts indicated days after transplantation. The bar of the right lower panels was 10 µm. Magnification; 200×.</p

Tregs features in the cardiac grafts on day 7 after transplantation(n = 3–5).

<p><b>A,</b> Proportion and cell number of Tregs in each graft. Mononuclear cells were separated from the graft by collagenase treatment and Percoll gradient centrifugation. After staining with CD3, CD4, CD25 and Foxp3 antibodies, cells were analyzed with FACS. In the lower panel, the number of infiltrated Tregs per graft is shown (n = 5). <b>B,</b> Mononuclear cells in the grafts were stained with anti-CTLA4 (left) and anti-IL-10 (left) antibodies after PMA, ionomycin, and brefeldin treatment, and analyzed with FACS (n = 3–5). The proportion of CTLA4- and IL-10-producing cells in the CD4⁺ T cell population in the grafts is shown. <b>C,</b> The cell number of IFN-γ and IL-6-producing cells. The mononuclear cells infiltrated into the grafts were stained with anti- IFN-γ and anti-IL-6 antibodies and analyzed with FACS (n = 3–5). (*** <i>P</i><0.01, ** <i>P</i><0.03, * <i>P</i><0.05, Tukey's Multiple Comparison Test).</p

CTLA4⁺CD8⁺ cells in filtrated cardiac grafts.

<p><b>A,</b> (upper panels) Fraction of CD4⁻CTLA4⁺ cells in mononuclear CD3⁺ cells in the grafts on day 7 and days 35–40 after transplantation (n = 3–5). *** <i>P</i><0.01 ** <i>P</i><0.03 (lower panels) FACS analysis of CTLA4⁺ cells in the graft of indirect-iTreg-treated mice on day 35 after transplantation. <b>B,</b> Histograms of CD3⁺CD8⁺ cells harvested from cardiac grafts 14 or 15 days after transplantation in mice treated with the indicated Tregs. <b>C,</b> Comparison of the expression of cell surface marker and cytokines between infiltrated CD8⁺ cells in indirect-iTreg-treated grafts on day 35, and effector CD8⁺ cells induced <i>in vitro</i>. Effector CD8⁺ cells were induced from naïve CD8⁺ T cells from CBA cultured with BMDCs from C57BL/6 mice (MLR). <b>D.</b> Induction of CD8⁺CTLA4⁺ cells. Using 96-well U-bottom plates, naïve CD8⁺ T-cells (2×10⁴/well, CBA) were cultured with 15 Gy-irradiated BMDCs (2×10⁴/well, C57BL/6) in either the presence or absence of direct-iTregs induced <i>in vitro</i> (4×10⁴/well (+) or 8×10⁴/well (++), CBA), as described above. After 5 days, the cells were analyzed using a FACSCanto. (* <i>P</i><0.05, Bonferroni's Multiple Comparison Test).</p

Expansion of Tregs by BMDCs <i>in vitro</i>.

<p><b>A, B, </b><i>In vitro</i> antigen-specific expansion of iTregs via the indirect pathway. Naïve CD4⁺ T cells from CBA mice were cultured with BMDCs (CBA) in the presence of the K^b peptide for 7 days at the indicated conditions. The Treg fraction was determined as CD3⁺CD4⁺ CD25⁺Foxp3⁺ (***<i>P</i><0.001, Bonferroni test) <b>C, D,</b> The-time course of Treg fraction (C) and number (D) in the <i>in vitro</i> culture with BMDCs in the presence of 10 µg/ml K^b peptide, 10 ng/ml IL-2, 2 ng/ml TGF-β, 5 µg/ml anti- IFN-γ and anti-IL-4 antibodies, and 20 nM ATRA. <b>E,</b> The cell surface marker and purity of the Tregs before and after cell sorting, on day 8 after culture via the indirect and direct pathway. Tregs induced via the direct pathway were cultured with C57BL/6 BMDCs and 10 ng/ml IL-2, 2 ng/ml TGF-β, 10 µg/ml anti- IFN-γ and 5 µg/ml anti-IL-4 antibodies, and 20 nM ATRA. The cells were stained with the indicated antibodies and analyzed with FACS.</p

Analysis of DCs in the grafts.

<p><b>A,</b> Gr-1 and CD11c staining in cells filtrated in cardiac grafts 7 days after transplantation in mice with the indicated treatment. <b>B,</b> Population of CD11c⁺H2b⁺ cells in the filtrated cardiac graft cells. The graft undergoing no treatment was harvested 14 days after transplantation, and other 2 grafts were harvested 35–40 days after transplantation. <b>C,</b> Cell-surface marker expression of CD11c⁺ cells filtrated in the cardiac grafts 14 or 15 days after transplantation in mice tread with the indicated Tregs.</p

The effect of nTregs expanded by the indirect pathways.

<p><b>A,</b> Expansion of alloantigen-specific nTregs <i>in vitro</i>. nTregs (5×10⁴) from CBA were cultured with 5×10⁴ allo-BMDCs in the presence of 10 µg/ml K^b peptide, 100 ng/ml IL-2, 2 ng/ml TGF-β, 5 µg/ml anti- IFN-γ and anti-IL-4 antibodies, and 20 nM ATRA, and the number (left) and fraction (right) of Tregs were estimated. <b>B,</b> Comparison of cell surface markers of iTregs and nTregs expanded by the indirect pathway for 8 days before cell sorting. <b>C,</b> Effect of iTregs and nTregs on graft survival in cardiac transplantation models. <b>D,</b> HE staining of harvested cardiac grafts.</p

Characterization of <i>Foxp3</i>^Cre-YFP-<i>TRAF6</i>^f/f (cKO) mice.

<p>(A) Representative appearance of a 22-week-old cKO mouse. (B) Incidence of dermatitis in cKO mice (<i>n</i> = 20). (C) Representative macroscopic observations of the LN from WT mice and age (about 20 weeks old)-matched TRAF6 cKO mice. (D) Representative histopathologies of the skin (H&E staining) and joint (Safranin-O staining) lesion of representative cKO mice. (E) Serum titers of immunoglobulin subclasses and anti-dsDNA antibodies from WT and cKO mice measured by ELISA. Each symbol indicates an individual host mouse (^*<i>p</i> < 0.05). (n=7) (F) Spontaneous splenic germinal center formation in cKO mice. Sections of spleen were stained with anti-IgE and anti-CD3 antibodies. Representative data form three independent mice are shown.</p

Reduced stability of Foxp3 in <i>TRAF6</i>-deficient Treg cells

<p>(A) Foxp3 expression in freshly isolated CD4⁺ T cells from the spleen, mesenteric lymph node (LN) and thymus of the indicated mice. Foxp3 levels were measured using YFP-fluorescence. Data are representative of five independent experiments with similar results. The numbers of YFP⁺ Tregs in each organ are shown in right graphs (n=5). (B) Expression of KLRG1 was measured in CD4⁺Foxp3(YFP)⁺ gated cells. (C) <i>In </i><i>vitro</i> suppression assay. FACS purified 5×10⁴ CD4⁺CD25^-CD62L⁺CD44^- naïve T cells from WT mice were stimulated for 72 hours with 10⁵ T-cell depleted spleen cells and anti-CD3 (2C11) (1µg/ml), in the absence or presence of CD3⁺CD4⁺Foxp3(YFP) + Treg cells from WT (diamond) or cKO (square) mice as indicated concentrations. Cells were pulsed with 0.5 µCi of ³H-thymidine for the final 16 hours before being harvested. Error bars indicate SD; n=3. (D) <i>In </i><i>vivo</i> suppression assay. 3 x 10⁵ naïve T cells from the LN of CD45.1⁺ WT mice together with or without 1.5 x 10⁵ Tregs (CD4⁺YFP ⁺ RFP⁺) from the LN of CD45.2 Foxp3 ^YFP-CreROSA26^RFP TRAF6^+/+ (WT-Tregs) or CD45.2 Foxp3 ^YFP-CreROSA26^RFP TRAF6^fl/fl (cKO-Tregs) were transferred into Rag2^-/- mice. Body weight changes were measured daily. Data are representative from four independent experiments. *<i>p</i><0.05 **<i>p</i><0.01; Representative histology of the colon is shown in right panels. Magnification x200 (E) Flow cytometric analysis of Foxp3-YFP and CD45.1 expression on CD3⁺CD4⁺ T cells from the LN in Rag2^-/- mice. Data are representative from four independent experiments. Quantification of the data of the fraction of YFP ⁺ CD45.1^- cells (remaining Foxp3 ⁺ Tregs) is shown in the right panel. **<i>p</i>< 0.01.</p

Activation of T cells in Treg-specific TRAF6-cKO mice.

<p>(A) Flow cytometry for activated/memory phenotypes of freshly isolated CD4⁺ T cells from thymus, spleen and mesenteric lymph nodes (MLNs) in the indicated mice at 22 weeks of age. Representative data from three independent mice are shown. (B) Cytokine production from splenic T cells. Freshly isolated splencytes (1x10⁶) from WT and cKO mice were stimulated with anti-CD3 antibody for 2 days. Cytokine levels in the culture supernatant was measured using ELISA (n=3).</p

Fate mapping study of <i>TRAF6</i>-deficeint Tregs.

<p>(A) FACS profiles of CD4+ T cells in the spleen of WT (Foxp3 ^YFP-CreROSA26^RFP TRAF6^+/+) or cKO (Foxp3 ^YFP-CreROSA26^RFP TRAF6^fl/fl) mice. A representative profile of WT and cKO mice (16-20 weeks old) with or without severe inflammation. (B) RFP ⁺ YFP^-/RFP ⁺ YFP⁺ ratio in the spleen of WT and cKO mice at 12 to 22 weeks of age (n=5). (C) mRNA levels in each CD4⁺T cell fraction. After sorting using FACS with YFP and RFP, mRNA was isolated from each fraction, and the levels of indicated genes were measured using quantitative real time RT-PCR. The mean ± SD of three independent experiments are shown. All data were analyzed using a Student’s t test. *<i>p</i><0.05, **<i>p</i><0.01vs. WT.</p