K16-19R, Foxp3 K17R or K18R, or along with one million native Treg cells isolated from C57BL/6 mice. On day 7 post-transfer, we enumerated cells from the lymph nodes (LN) and spleen of each mouse. The co-transfer of CD4+ T cells transduced with WT Foxp3, or native Tregs, led to substantially decreased homeostatic T cell proliferation of Thy1.1+ CD4+CD252 cells compared with the results of transfer of Thy1.1+ CD4+CD252 alone, or cotransfer of Thy1.1 CD4+CD252 cells transduced with EV (Fig. 5A). However, adoptive transfer of Thy1.1+ CD4+CD252 cells, plus CD4+ T cells transduced with mutant Foxp3 K17R or Foxp3 K18R, led to impaired suppression of homeostatic T cell proliferation as compared with cotransfer of Thy1.1+ CD4+CD252 cells plus CD4+ T cells transduced with WT Foxp3 (Fig. 5A). We also evaluated the percentage of Thy1.1+ CD4+CD252 cells among these groups (Fig. 5B). Transfer of Thy1.1+ CD4+CD252 alone exhibited the highest percentage of Thy1.1+ cells, followed by cotransfer with EV, and then Foxp3 K18R. However, there was no statistically significant difference between the other groups, including cotransfer with WT Foxp3, B6 Treg, Foxp3 K16-19R or Foxp K17R. Thy1.1+ CD4+ T cells cotransfered with CD4+ T cells transduced with Foxp3 K18R displayed significantly higher cell numbers than those of Foxp3- K17R, consistent with our finding that Foxp3 K18R has lower DNA binding ability (Fig. 3). These data showed that the 2 lysine residues are important for Treg in vivo suppression of lymphopenia- Lysine Acetylation and Foxp3 Function induced expansion of CD4+ T cells. They also suggest that there are differences between the 2 Foxp3 mutants that might be reflected by differences in gene expression in corresponding Treg cells. FKH lysine mutations affect Treg gene expression profiles To assess the possible effects of Foxp3 K17 and K18 mutations on Treg gene expression, global gene expression profiling was performed using Affymetrix 430 2.0 mouse oligonucleotide gene expression arrays. Normalized gene expression values of the CD4+ T cells transduced with EV were used as the reference for comparison with data from CD4+ T cells transduced with WT, K17R or K18R Foxp3. Compared with EV transduced CD4+ T cells, CD4+ T cells transduced with WT Foxp3 had 147 up- regulated genes, whereas CD4+ T cells transduced with Foxp3 K17R or K18R had only 14 or 33 up-regulated genes, respectively (Log2 .1). Among the up-regulated genes, 7 or 16, respectively, overlapped with WT Foxp3 (Fig. 6A). Conversely, CD4+ T cells transduced with WT Foxp3 had 293 down-regulated genes, Foxp3 K17R had 101 down-regulated genes, and Foxp3 K18R had 103 down-regulated genes (Log2,21). Among the down-regulated genes, 70 or 73, respectively, were shared with WT Foxp3 (Fig. 6A). These data showed that a single lysine mutation (K17R or K18R) within the Foxp3 FKH domain markedly affects Foxp3- dependent gene expression, suggesting an important role of each lysine in controlling Foxp3+ Treg functions. Figure 5. Foxp3 mutants impair Treg function in vivo. (A) 16106 Thy1.1+ CD4+CD252 T cells were co-transferred with 16106 Thy1.2+ CD4+ T cells transduced with WT Foxp3, K16-19R, K17R, K18R or EV, or with purified normal B6 Treg cells, into Rag12/2 mice. At 7 d post-transfer, single-cell suspensions from lymph node or spleen samples were stained for FACS analyses; the numbers (A) or percentages (B) of CD4+ Thy1.1+ cells are shown. Results are representative of 2 independent experiments, and *p,0.05 compared to WT Foxp3. doi:10.1371/journal.pone.0029035.g005 PLoS ONE | www.plosone.org 7 January 2012 | Volume 7 | Issue 1 | e29035