Induction of in vitro Treg cells was most easily accomplished with anti-CD3 mAb mitogen-based stimulation. Therefore, to control for the use of mitogen-based stimulation, it was necessary to confirm that n-butyrate anergized mitogen-stimulated CD4+ T cells similarly to antigen-stimulated CD4+ T cells. Primary cultures of isolated C57BL/6 CD4+ T cells were stimulated with plate-bound anti-CD3 mAb and soluble

anti-CD28 mAb for 7 days in the presence or absence of n-butyrate. As seen in Fig. 1A, n-butyrate reduced proliferation of CD4+ T cells by approximately 95% in mitogen-stimulated primary cultures. To test whether n-butyrate induced unresponsiveness was retained after the removal of the HDAC inhibitor, the CD4+ T cells from the primary culture were re-stimulated in secondary cultures that did not contain n-butyrate. As shown in Fig. 1B, control CD4+ T cells find more from the

primary cultures proliferated vigorously when re-stimulated in secondary cultures. In contrast, CD4+ T cells from the n-butyrate-treated primary cultures proliferated 83–91% less than untreated CD4+ T cells. The retention of proliferative unresponsiveness in the secondary cultures demonstrated that the CD4+ T cells from the n-butyrate-treated mitogen-stimulated primary cultures were anergic. Anergy in CD4+ T cells usually involves an inability to generate IL-2 in association with proliferative unresponsiveness. Consequently, IL-2 secretion DNA Damage inhibitor by the CD4+ T cells was also examined to confirm the onset of anergy (Fig. 1C). CD4+ T cells from control primary cultures secreted IL-2 in secondary cultures stimulated with anti-CD3 mAb. In contrast, IL-2

secretion second was inhibited in CD4+ T cells from the n-butyrate-treated primary cultures. The anergic CD4+ T cells did not generate any additional IL-2 beyond the detected background levels in response to anti-CD3 mAb stimulation in the secondary cultures. The decreased IL-2 concentration within the anergic CD4+ T cell culture supernatants had no bearing upon proliferation in the n-butyrate-treated CD4+ T cells as seen in Fig. 1B. Taken together, the results in Fig. 1 revealed that n-butyrate induced anergy within mitogen-stimulated CD4+ T cells as determined through significant reduction of proliferation and IL-2 secretion. To determine if n-butyrate increased the percentage of FoxP3+ Treg cells in primary or secondary cultures, CD4+ T cells from transgenic FoxP3EGFP C57BL/6 mice were stimulated in primary cultures with or without n-butyrate. Natural Treg cells as determined by the presence of FoxP3EGFP comprised approximately 8% of isolated lymphoid CD4+ T cells (data not shown). TGF-β was added to additional primary cultures to generate FoxP3+ T cells as a positive control [21]. Percentages of FoxP3+ T cells were quantified daily over the course of 5 days (Fig. 2A). The percentage of CD4+FoxP3+ T cells increased only in the primary cultures stimulated in the presence of TGF-β, as shown on Day 4 in Fig.