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Bone marrow-derived DCs from Tgfbr2fspKO and Tgfbr2floxE2/floxE2 littermate control mice were generated as described above. DCs were treated overnight with 1 μg/ml lipopolysaccharide (LPS), 500 U/ml tumour necrosis factor α (TNFα) or 20 μg/ml zymosan to induce maturation. A total of 1106 treated and untreated DCs from each group underwent FACS as described above for major histocompatibility complex (MHC) class II, CD11c, CD86 (BD Pharmingen) and 7-AAD. The percentage of mature DCs was determined for each group and treatment as defined by cells simultaneously staining positive for CD11c and highly positive for CD86 and MHC class II after gating out dead (7-AAD-positive) cells. Experiments were conducted in triplicate with results reported as mean percentage mature DCs (SD), and one-way analysis of variance (ANOVA) was employed to compare Tgfbr2fspKO and Tgfbr2floxE2/floxE2 DCs using SPSS 16.
Since TGFβ is known to regulate DC function and control of DC maturation is needed to maintain immune tolerance, we hypothesised that Tgfbr2fspKO DCs lack normal maturational regulation, resulting in the development of autoimmunity. To study DC maturation, bone marrow-derived DCs from Tgfbr2fspKO and Tgfbr2floxE2/floxE2 control mice were treated in vitro with molecules that induce DC maturation, including LPS, TNFα and zymosan. After 24 h, DCs were harvested and upregulation of the maturation markers MHC class II and CD86 was compared using FACS. While the number of mature DCs in untreated Tgfbr2floxE2/floxE2 and Tgfbr2fspKO cultures was similar, LPS induced maturation of Tgfbr2fspKO DC cultures more than threefold compared with wild type DC cultures (fig 8C). Similar results were seen with TNFα and zymosan treatment (data not shown).
Although TGFβ is known to modulate DC maturity,18 our studies implicate disruption of TGFβ signalling in DCs as a critical event in the development of autoimmunity. DCs normally maintain immune tolerance by remaining immature during the presentation of self-antigens to T cells, resulting in T cell anergy. Since Tgfbr2fspKO DCs have increased maturation in response to activation, we hypothesise that autoimmunity occurs in the Tgfbr2fspKO mouse due to precocious DC maturation in response to self-antigen, resulting in the activation of self-reactive T cells. Studies using targeted disruption of Tgfbr2 in helper T cells to induce autoimmunity are consistent with the idea that TGFβ affects autoimmunity by modulating interactions between DCs and T cells. By expressing a dominant-negative form of Tgfbr2 under direction of the CD4 promoter, murine models of human primary biliary cirrhosis19 and inflammatory bowel disease20 associated with autoantibody production have been generated. In the model of inflammatory bowel disease, T cells were shown to evade immune tolerance by undergoing DC-independent activation. 041b061a72
