Foxp3cre/Dnmt1^−/− mice developed lethal autoimmunity by 3 to 4 weeks of age. (A) In WT Tregs, immunoprecipitation of Foxp3 led to co-precipitation of Dnmt1 and Dnmt3a (immunoglobulin-heavy chain, Igh, is also detected). (B) Male mice with Dnmt1^−/− Tregs became sick and runted, whereas WT littermate or female mice developed normally; (inset) representative Western blot showing the absence of Dnmt1 in the Tregs of targeted mice (developed by mating floxed Dnmt1 and Foxp3-Cre mice). (C) Kaplan-Meier survival plots showing death by 3 to 4 weeks of age of all mice with Dnmt1^−/− Tregs, whereas their adoptive transfer with WT Tregs at 2 to 4 days of life rescued mice in a dose-dependent manner; data are from 3 to 8 mice per group. (D) Dnmt1^−/− mice had small thymii but enlarged lymph nodes and spleens. (E) Dnmt1^−/− mice developed anemia and thrombocytopenia; data are from 5 mice per group. (F) Histologic examination mononuclear cell infiltrates of multiple organs, including extensive bronchovascular bundles of the lung, periductular areas of the pancreas, periportal areas of the liver, and submeningeal areas of the brain. (G) Sera of male Dnmt1^−/− mice contained autoantibodies directed against stratified squamous keratinizing epithelium in the skin (ear section, 1/40 to 1/80 titer), smooth muscle (eg, small bowel, titers of 1/40 to 1/80), striated muscle (titers of 1/320 to 1/640), nuclei of all tissues (nucleolar pattern, titers of 1/80 to 1/160, eg, in liver and pancreas in males vs unaffected nuclei in females), and antisperm antibodies (titers of 1/20 to 1/40 titers against mature sperm cells located in the central part of seminiferous tubules; arrows point to the tails of spermatozoa); sera from Dnmt1^−/− females (upper row) had no autoantibodies and were indistinguishable from the sera of healthy C57BL/6 mice (n = 3/group).