Rapid advances in DNA synthesis techniques have made it possible to engineer viruses, biochemical pathways and assemble bacterial genomes. Here, we report the synthesis of a functional 272,871 bp designer eukaryotic chromosome, synIII, which is based on the 316,617 bp native Saccharomyces cerevisiae chromosome III. Changes to synIII include TAG/TAA stop-codon replacements, deletion of subtelomeric regions, introns, tRNAs, transposons and silent mating loci as well as insertion of loxPsym sites to enable genome scrambling. SynIII is functional in S. cerevisiae. Scrambling of the chromosome in a heterozygous diploid reveals a large increase in “a mater” derivatives resulting from loss of the MATα allele on synIII. The total synthesis of synIII represents the first complete design and synthesis of a eukaryotic chromosome, establishing S. cerevisiae as the basis for designer eukaryotic genome biology.

Foxp3 + regulatory T cells in peripheral tissues (pT regs ) are instrumental in limiting inflammatory responses to non-self antigens. Within the intestine, pT regs are located primarily in the lamina propria, while intraepithelial CD4 + T cells (CD4 IELs ), which also exhibit anti-inflammatory properties and depend on similar environmental cues, reside in the epithelium. Using intravital microscopy, we show distinct cell dynamics of intestinal T regs and CD4 IELs . Upon migration to the epithelium, T regs lose Foxp3 and convert to CD4 IELs in a microbiota-dependent fashion, an effect attributed to the loss of the transcription factor ThPOK. Finally, we demonstrate that pT regs and CD4 IELs perform complementary roles in the regulation of intestinal inflammation. These results reveal intra-tissue specialization of anti-inflammatory T cells shaped by discrete niches of the intestine.The gut mucosa is exposed to large amounts of both harmless and potentially pathogenic stimuli on a daily basis, hence diverse immune regulatory mechanisms must operate to avoid inflammatory diseases (1). Peripheral Foxp3-expressing regulatory T cells (pT regs ) mediate suppression of a variety of immune cells and actively prevent inflammatory bowel diseases and food allergies (2-7). Similar to pT regs , Foxp3 − CD8αα + CD4 + intraepithelial lymphocytes (CD4 IELs ) depend on retinoic acid (RA) and transforming growth factor (TGF)-β signaling for their development and also have anti-inflammatory properties (4,(8)(9)(10)(11)(12)(13). However, while CD4 IELs accumulate in the intestinal epithelium, very few total T regs (including pT regs or thymically-derived T regs ) can be found at this site ( fig. S1A and B fig. S2A). Because previous studies have demonstrated that the majority of "ex-Foxp3" cells in the steady state were derived from uncommitted precursors that transiently upregulated Foxp3 (18), we also performed fate mapping after pulse-labeling iFoxp3 Tomato mice with tamoxifen (14), a strategy more likely to target bona fide T regs (19). Nevertheless, while stable Foxp3 expression was again observed in several peripheral tissues examined, over 50% of Tomato + CD4 + T cells that accumulated in the small intestine and almost 10% in the large intestine epithelium isolated from iFoxp3 Tomato mice no longer expressed Foxp3 fives weeks post tamoxifen administration ( fig. S2B and C). The contribution of Tomato + cells to the CD8αα + and CD8αβ + CD4 IEL pools was roughly 10% and 25%, respectively ( fig. S2D). Consistent with a ThPOK-dependent process, ex-T regs that underwent IEL differentiation showed low levels of ThPOK ( fig. S2E). These results indicate that a substantial proportion of intestinal T regs physiologically convert to CD4 IELs .Commensal bacteria play a major role in the induction of lamina propria pT regs in the large intestine (3,(5)(6)(7)20). In contrast, we observed an increased frequency of pT regs (Neuropilin-1 − Foxp3 + ) in the small intestinal epithelium isolated from germ-free (GF) mice when compared to...

Oral tolerance prevents pathological inflammatory responses towards innocuous foreign antigens via peripheral regulatory T cells (pTreg cells). However, whether a particular subset of antigen-presenting cells (APCs) is required during dietary antigen exposure to instruct naïve CD4+ T cells to differentiate into pTreg cells has not been defined. Using myeloid lineage-specific APC depletion in mice, we found that monocyte-derived APCs are dispensable, while classical dendritic cells (cDCs) are critical for pTreg cell induction and oral tolerance. CD11b− cDCs from the gut-draining lymph nodes efficiently induced pTreg cells, and conversely, loss of IRF8-dependent CD11b− cDCs impaired their polarization, although oral tolerance remained intact. These data reveal the hierarchy of cDC subsets in pTreg cell induction and their redundancy during oral tolerance development.

SummaryCD4+ T cells differentiate into phenotypically distinct T helper cells upon antigenic stimulation. Regulation of plasticity between these CD4+ T-cell lineages is critical for immune homeostasis and prevention of autoimmune disease. However, the factors that regulate lineage stability are largely unknown. Here we investigate a role for retinoic acid (RA) in the regulation of lineage stability using T helper 1 (Th1) cells, traditionally considered the most phenotypically stable Th subset. We found that RA, through its receptor RARα, sustains stable expression of Th1 lineage specifying genes, as well as repressing genes that instruct Th17-cell fate. RA signaling is essential for limiting Th1-cell conversion into Th17 effectors and for preventing pathogenic Th17 responses in vivo. Our study identifies RA-RARα as a key component of the regulatory network governing maintenance and plasticity of Th1-cell fate and defines an additional pathway for the development of Th17 cells.

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