Systems Biologic Analysis Of Regulatory T Cells Genetic Pathways And Therapeutic Trial In Murine Primary Biliary Cirrhosis

Primary biliary cirrhosis (PBC) is a progressive autoimmune biliary disease whose pathogenesis involves most immune cell subsets, but the specific role of each cell subset remains unclear. CD4+Foxp3+regulatory T cells (Tregs) play a non-redundant role in control of excessive immune responses, and defects in Tregs have been shown both in patients and murine models of primary biliary cirrhosis (PBC). Herein, we took advantage of a murine model of PBC, the dominant negative transforming growth factor β receptor Ⅱ (dnTGFβRⅡ) mice, to seek for the answers to three simple and fundamental questions:First, what are the Tregs genetic defects and their functional effects in murine PBC; Second, Tregs in murine PBC model play positive role or negative role in the pathogenesis; Third, whether exchanging dnTGFβRⅡ CD4lineage with wild type CD4+T cells is sufficient to reverse the cholangitis.By using high-resolution microarrays with verification by PCR and protein expression, we found profound and wide-ranging differences between dnTGFβRⅡ and normal, wild type Tregs. Critical transcription factors were down-regulated including Eos, Ahr, Klf2, Foxpl in dnTGFβRⅡ Tregs. Functionally, dnTGFβRⅡ Tregs expressed an activated, pro-inflammatory phenotype with upregulation of Ccl5, Granzyme B and IFN-y. Genetic pathway analysis suggested that the primary effect of loss of TGF-β pathway signaling was to down regulate immune regulatory processes, with a secondary upregulation of inflammatory processes. These findings provide new insights into T regulatory genetic defects; aberrations of the identified genes or genetic pathways should be investigated in human PBC Tregs. This approach which takes advantage of biologic pathway analysis illustrates the ability to identify genes/pathways that are affected both independently and dependent on abnormalities in TGFβ signaling.Then, we developed dnTGFβRⅡ;CD4-/-mice lacking any CD4cell lineage including Tregs; these mice displayed more severe cholangitis than unmodified dnTGFβRⅡ mice. In the absence of CD4+cells, the total numbers of intrahepatic, effector CD8+T cells producing IFN-y increased significantly.Moreover, the biliary disease is alleviated in mixed bone marrow chimeric mice constructed from dnTGFPRII;CD4-/-and B6;CD8-/-mice, and the number of terminally differentiated CD8+T effector cells decreased in the presence of WT CD4cells in these mice. Similar results were seen in dnTGFβRⅡ;CD4-/-mice parabiosised with B6mice. These results provide further evidence that PBC requires abnormalities in several immune cell subsets. Correcting CD4+T cell subset deficiencies eliminated disease despite the presence of pathogenic CD8cells. Thus targeting therapy to the CD4+T cell subset may be sufficient to treat the disease.