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

Inflammatory cytokine production in mice immunized with 2OA-BSA.

<p>(A) Serum levels of IL-17A, IFN-γ, TNF-α and IL-6 in WT, IL-17A^−/−, IL-17F^−/− and IL-22^−/− mice, respectively, at 2 weeks following 2OA-BSA immunization. (B) Production of IFN-γ in supernatant fluids of cultured splenic and hepatic MNCs (n = 4) with anti-CD3/CD28 mAbs at 3 days. (C) The level of inflammatory cytokines in extracted liver protein from WT, IL-17A^−/−, IL-17F^−/− and IL-22^−/− mice, respectively. Each group n = 8. *p&lt;0.05, **p&lt;0.01, ***p&lt;0.001.</p

IL-12p40 is required for 2OA-BSA-induced cholangitis.

<p>(A) H&amp;E staining of representative liver from WT, IL-12p40^−/− (p40^−/−), IL-23^−/− (p19^−/−) and IL-12p35^−/− (p35^−/−) mice 8 weeks after 2OA-BSA immunization. Portal inflammatory changes with interlobular bile duct damage (red arrow) were observed in the liver of WT, p19^−/− and p35^−/− mice but not p40^−/− mice; normal bile ducts in p40^−/− mice (blue arrow). (B) Portal inflammation and bile duct damage were examined in individual animals. The pathological score of portal inflammation and biliary cell damage were evaluated in WT (n = 17), p19^−/− (n = 14), p35^−/− (n = 3) and p40^−/− (n = 5) mice. *p&lt;0.05, **p&lt;0.01, ***p&lt;0.001. (C) Immunohistochemical analysis of the liver of WT mice and IL-23p19^−/− mice at 8 weeks after 2OA-BSA immunization using mAbs to CD4 and CD8.</p

Comparative levels of PDC-E2-specific autoantibodies in cytokine deficient mice induced by 2OA-BSA immunization; samples were collected at serial time points.

<p>The fold changes of AMA OD values of each group are compared to the data of WT mice (n = 8–16/group). AMA values of IL-17A^−/− and IFN-γ mice compared to other groups, *p&lt;0.05.</p

Pathological changes in liver of mice immunized with 2OA-BSA.

<p>(A) Representative H&amp;E staining profiles of liver from IL-17A^−/−, IL-17F^−/− and IL-22^−/− mice 8 weeks after immunization. Portal inflammatory changes with interlobular bile duct damage (red arrow) were observed. IL-17F^−/− demonstrating more epithelioid granulomas (red arrowhead) in liver compared to IL-17A^−/− and IL-22^−/− mice. (B) Scoring of portal inflammation and bile duct damage in liver from WT (n = 25), IL-17A^−/− (n = 19), IL-17F^−/− (n = 13) and IL-22^−/− (n = 14) mice.</p

Pathological changes in the liver of IFN-γ^−/− mice immunized with 2OA-BSA.

<p>(A) Representative H&amp;E stained of liver sections from IFN-γ^−/− mice compared with WT mice 8 weeks after 2OA-BSA immunization. Portal inflammatory changes with interlobular bile duct damage (red arrow) were observed in WT mice; normal bile ducts in IFN-γ^−/− mice (blue arrows). (B) Scoring of portal inflammation and bile duct damage in sections of liver from WT and IFN-γ^−/− mice (each group, n = 16).</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

Flow cytometric analysis of the lymphocytic infiltration in liver from IL-23p19^−/−, IL-12p35^−/− and control WT mice.

<p>HMNCs were isolated from liver samples at 8 weeks after the first immunization. The data are expressed as the cell number per gram of tissue. **p&lt;0.01, ***p&lt;0.001.</p

Inflammatory cytokine production in mice immunized with 2OA-BSA.

<p>(A) Serum levels of IFN-γ and IL-17A in B6 and IL-23p19^−/− mice 2 and 4 weeks after initial 2OA-BSA immunization. (B) Production of IFN-γ and IL-17A in supernatant fluids of cultured splenic and hepatic MNCs (n = 4) with anti-CD3/CD28 mAbs for 3 days (C) inflammatory cytokines in extracted liver protein from WT mice (n = 8) and IL-23p19^−/− (n = 8) mice. *p&lt;0.05, **p&lt;0.01, ***p&lt;0.001.</p