Real Time PCR primers used in this study.

<p>Real Time PCR primers used in this study.</p

Cytokine profiles of CD4+ and CD8+ T cells in the liver and spleen of IL-2Rα^−/− mice and IL-2Rα^+/− mice.

<p>Hepatic and splenic MNC were analyzed by flow cytometry. Flow cytometry profiles are shown in (A) and (C), and quantification of frequency of IFN-γ+ and IL-17A+ cells in the CD4+ and CD8+ T cell populations in (B) and (D). Data are representative with four mice in IL-2Rα^−/− group and five mice in IL-2Rα^+/− group. * P<0.05, ** P<0.01.</p

Comparison of CDR3 region of the TCR β family between dnTGFβRII and dnTGFβRII IL-6^−/− mice.

<p>The arrows indicate clonal expansion of specific Vβ. C57B6 mice were used as negative control. With this technique, if there is no detectable T cell expansion within a Vβ spectrum, a Gaussian distribution of CDR3 lengths is observed. In contrast, clonal expansions are observed as a perturbation of this Gaussian distribution.</p

Lymphoma-like T cell infiltrations were only found in homozygous dnTGFβRII p40^−/− and dnTGFβRII mice.

<p>A. Representative immunophenotype of hepatic lymphocytes (upper panels) and H&amp;E stained sections (lower panels) from inbred dnTGFβRII p40^−/− mice with lymphomatous lesions at age of 10 weeks. CD4 and CD8 double negative TCRβ⁺NK1.1⁺ cells are predominant in the liver of dnTGFβRII p40^−/−. Typical diffuse lymphomatous lesions were found in liver and spleen. B. Representative immunophenotype of liver infiltrating lymphocytes (upper panels) and H&amp;E stained sections from inbred dnTGFβRII mice with lymphomatous lesions at age of 12 weeks. C. Relative copy number of dnTGFβRII transgene detected by real-time PCR. D. The percentage of homozygous and hemizygous offspring from hemizygous TGFβRII parents.</p

Colitis severity.

<p>(A) Representative H&E staining of colon sections from IL-2Rα^+/−, IL-2Rα^−/−, IFN-γ^−/−IL-2Rα^−/− and IL-17A^−/−IL-2Rα^−/− mice. (B) Colitis score of IL-2Rα^+/− (n = 5), IL-2Rα^−/− (n = 8), IFN-γ^−/−IL-2Rα^−/− (n = 8) and IL-17A^−/−IL-2Rα^−/− (n = 9) mice.(C) Colon weight of IL-2Rα^+/− (n = 15), IL-2Rα^−/− (n = 15), IFN-γ^−/−IL-2Rα^−/− (n = 12) and IL-17A^−/−IL-2Rα^−/− (n = 24) mice. * P<0.05, ** P<0.01, *** P<0.001.</p

Histological features and immunophenotypes of lymphoma-like T cell infiltration.

<p>A. Flow cytometric analysis of HMNCs from dnTGFβRII and dnTGFβRII IL-6^−/− mice with and without lymphatomous lesion. The numbers above the plots indicate the frequency of TCRβ⁺NK1.1⁻ and TCRβ⁺NK1.1⁺ cells (left panels), the frequency of CD4 positive cells (middle panels) and the frequency of CD8 positive cells (right panels). Cells shown in the middle and right panels were gated on TCRβ⁺NK1.1⁻ or TCRβ⁺NK1.1⁺ populations as indicated in the left panels. B. The spleen weight of dnTGFβRII, dnTGFβRII IL-6^−/− and dnTGFβRII IL-6^−/− mice with a predominant NK1.1 positive or negative phenotype at age of 24–40 weeks. C. The total HMNC counts of dnTGFβRII, dnTGFβRII IL-6^−/− and dnTGFβRII IL-6^−/− mice with a predominant NK1.1 positive or negative phenotype at age of 24–40 weeks. D. Representative H&amp;E stained sections of tissue sample including liver (a–d), spleen (e–h), small intestine (i–l), colon (m–p) and lung (q–s) were prepared from dnTGFβRII and dnTGFβRII IL-6^−/− mice at age of 24–40 weeks (a–s,<b>×</b>200; t,<b>×</b>40). Typical diffuse lymphomatous lesions were found in liver (c) and spleen (g) of dnTGFβRII IL-6^−/− mice with a predominant NK1.1⁺ phenotype, while large focal lymphomatous lesions were found in liver (d,<b>×</b>200)(t,<b>×</b>40) and spleen (h) of dnTGFβRII IL-6^−/− mice with a predominant TCRβ⁺ NK1.1⁻ phenotype. No obvious lymphomatous lesions were found in lung, small intestine and colon in dnTGFβRII and dnTGFβRII IL-6^−/− mice.</p

Lymphoma-like T cell infiltration is transplantable into Rag1^−/− mice.

<p>A. Flow cytometric analysis of HMNCs from donor mouse showing a TCRβ⁺NK1.1⁺CD4⁻CD8⁻ phenotype. B. Representative flow cytometric analysis of splenic and hepatic MNCs from recipient mice 6 weeks post-transfer. C, Intracellular IFN-γ and IL-2 production. D. H&amp;E stained spleen and liver sections from Rag1^−/− recipient mice 6 weeks post-transfer of 2×10⁴ or 2×10⁵ HMNCs from inbred dnTGFβRII mice with lymphomatous lesions. E. Total HMNCs in Ly5.1Rag1^−/− recipient mice six weeks post-transfer. Ly5.1Rag1^−/− mice were adoptively transferred with 2×10⁴ or 2×10⁵ hepatic mononuclear cells from inbred dnTGFβRII mice with (n = 4) or without (n = 3) lymphomatous lesions, respectively.</p

Liver histopathology and mononuclear cell analysis.

<p>(A) Representative H&E staining of liver sections. (B) Score of liver portal inflammation, lobular inflammation and bile duct damage of IL-2Rα^−/− mice (n = 6), IFN-γ^−/−IL-2Rα^−/− mice (n = 6) and IL-17A^−/−IL-2Rα^−/− mice (n = 11). (C) Total number of mononuclear cells in liver from IL-2Rα^+/− (n = 8), IL-2Rα^−/− (n = 19), IFN-γ^−/−IL-2Rα^−/− (n = 7), IL-17A^−/−IL-2Rα^−/− mice (n = 17). * P<0.05, ** P<0.01.</p

Cytokine analysis.

<p>(A) Cytokines in serum samples of IL-2Rα^−/− (n = 6) IFN-γ^−/−IL-2Rα^−/− (n = 7) and IL-17A^−/−IL-2Rα^−/− mice (n = 7). (B) Relative levels of cytokine gene mRNA in colon and liver tissues of IL-2Rα^+/− (n = 8), IL-2Rα^−/− (n = 8), IFN-γ^−/−IL-2Rα^−/− (n = 8) and IL-17A^−/−IL-2Rα^−/− mice (n = 8). (C) Intracellular staining IFN-γ of liver CD4+ T cells and CD8+ T cells from IL-2Rα^−/− (n = 7) and IL-17A^−/−IL-2Rα^−/− mice (n = 11). * P<0.05, ** P<0.01, *** P<0.001.</p

CD4 and CD8 double negative T cells were detected in Ly5.1Rag1^−/− mice six weeks after adoptively transferred with one million hepatic CD8ab T cells from hemizygous dnTGFβRII mice (lymphomatous lesion-free).

<p>A. Flow cytometry analysis demonstrated the purity of hepatic CD8αβ T cells from hemizygous dnTGFβRII mice. The numbers in the plots indicate the percentage of cells. B. Flow cytometric analysis of splenic and hepatic mononuclear cells of recipient mice at 6 weeks post-transfer. Three Ly5.1Rag1^−/− mice were adoptively transferred with 1×10⁶ hepatic CD8αβ T cells from hemizygous dnTGFβRII mice. TCRβ staining was gated on CD45.2⁺ cells. The numbers in the plots indicate the percentage of cells. C. Weight and total MNC counts of spleen and liver from Ly5.1Rag1^−/− recipients 6 weeks post-transfer. D. H&amp;E staining sections of liver tissues from Ly5.1Rag1^−/− recipients six weeks post-transfer. R1, Recipient 1; R2, Recipient 2; R3, Recipient 3;</p