Suppression of early IL-4 production underlies the failure of CD4 T cells activated by TLR-stimulated dendritic cells to differentiate into Th2 cells

Dendritic cells (DCs) activated through TLRs provide a potent negative signal for Th2 cell development that is independent of positive signals for Th1 cell development such as IL-12 and IFN-gamma. In this study we demonstrate that the ability of TLR-activated DCs to suppress Th2 cell development is Ag dose-independent and unique to DCs that have been activated through TLRs vs by cytokines. We show that TLR-activated DCs inhibit early IL-4 production by CD4 T cells and thus inhibit their ability to subsequently increase GATA-3 expression and commit to the Th2 lineage. This occurs independently of expression of the GATA-3 antagonist T-bet. Although CD4 T cells activated by TLR-activated DCs make IL-2, they are not capable of phosphorylating STAT5 in response to this cytokine. This inhibition of responsiveness to IL-2 appears to underlie the failure to make early IL-4. Our findings suggest that DCs provide instructional signals for T cell differentiation before cytokine-mediated Th cell selection and outgrowth.     

TGF-beta 1 uses distinct mechanisms to inhibit IFN-gamma expression in CD4+ T cells at priming and at recall: differential involvement of Stat4 and T-bet

TGF-beta1 plays a critical role in restraining pathogenic Th1 autoimmune responses in vivo, but the mechanisms that mediate TGF-beta1's suppressive effects on CD4(+) T cell expression of IFN-gamma expression remain incompletely understood. To evaluate mechanisms by which TGF-beta1 inhibits IFN-gamma expression in CD4(+) T cells, we primed naive wild-type murine BALB/c CD4(+) T cells in vitro under Th1 development conditions in the presence or the absence of added TGF-beta1. We found that the presence of TGF-beta1 during priming of CD4(+) T cells suppressed both IFN-gamma expression during priming as well as the development of Th1 effector cells expressing IFN-gamma at a recall stimulation. TGF-beta1 inhibited the development of IFN-gamma-expressing cells in a dose-dependent fashion and in the absence of APC, indicating that TGF-beta1 can inhibit Th1 development by acting directly on the CD4(+) T cell. During priming, TGF-beta1 strongly inhibited the expression of both T-bet (T box expressed in T cells) and Stat4. We evaluated the importance of these two molecules in the suppression of IFN-gamma expression at the two phases of Th1 responses. Enforced expression of T-bet by retrovirus prevented TGF-beta1's inhibition of Th1 development, but did not prevent TGF-beta1's inhibition of IFN-gamma expression at priming. Conversely, enforced expression of Stat4 partly prevented TGF-beta1's inhibition of IFN-gamma expression during priming, but did not prevent TGF-beta1's inhibition of Th1 development. These data show that TGF-beta1 uses distinct mechanisms to inhibit IFN-gamma expression in CD4(+) T cells at priming and at recall.     

Roles of autocrine TGF-beta receptor and Smad signaling in adipocyte differentiation

TGF-beta inhibits adipocyte differentiation, yet is expressed by adipocytes. The function of TGF-beta in adipogenesis, and its mechanism of action, is unknown. To address the role of TGF-beta signaling in adipocyte differentiation, we characterized the expression of the TGF-beta receptors, and the Smads which transmit or inhibit TGF-beta signals, during adipogenesis in 3T3-F442A cells. We found that the cell-surface availability of TGF-beta receptors strongly decreased as adipogenesis proceeds. Whereas mRNA levels for Smads 2, 3, and 4 were unchanged during differentiation, mRNA levels for Smads 6 and 7, which are known to inhibit TGF-beta responses, decreased severely. Dominant negative interference with TGF-beta receptor signaling, by stably expressing a truncated type II TGF-beta receptor, enhanced differentiation and decreased growth. Stable overexpression of Smad2 or Smad3 inhibited differentiation and dominant negative inhibition of Smad3 function, but not Smad2 function, enhanced adipogenesis. Increased Smad6 and Smad7 levels blocked differentiation and enhanced TGF-beta-induced responses. The inhibitory effect of Smad7 on adipocyte differentiation and its cooperation with TGF-beta was associated with the C-domain of Smad7. Our results indicate that endogenous TGF-beta signaling regulates the rate of adipogenesis, and that Smad2 and Smad3 have distinct functions in this endogenous control of differentiation. Smad6 and Smad7 act as negative regulators of adipogenesis and, even though known to inhibit TGF-beta responses, enhance the effects of TGF-beta on these cells.     

An intrinsic mechanism predisposes Foxp3-expressing regulatory T cells to Th2 conversion in vivo

Naturally occurring regulatory T (nTreg) cells express Foxp3 and were originally discovered as immune suppressors critical for self-tolerance and immune homeostasis. Through yet-to-be-defined mechanisms, nTreg cells were recently shown to convert into proinflammatory cells. Particularly, attenuation of Foxp3 expression led to Th2 conversion of nTreg cells in vivo. In this paper, we demonstrated an nTreg-specific mechanism controlling their Th2 conversion. We found that wild-type nTreg cells expressing reduced levels of Foxp3 but not those expressing no Foxp3 produced the Th2 cytokine IL-4. Intriguingly, IL-4 production by converted nTreg cells is required for Th2 differentiation of coexisting naive CD4 T cells in vivo, suggesting that Th2 conversion of nTreg cells might be critical for directing Th2 immune responses. Th2 conversion of nTreg cells was not due to their inability to become Th1 cells, because IFN-γ was produced by Foxp3-low-expressing cells when IL-4/STAT-6 signaling was abrogated. Surprisingly, however, unlike naive CD4 T cells whose IL-4 production is dependent on STAT-6, Foxp3-low-expressing cells generated IL-4 independent of STAT-6, indicating an intrinsic mechanism that favors nTreg-to-Th2 differentiation. Indeed, compared with naive CD4 T cells, nTreg expressed elevated levels of GATA-3 independent of STAT-6. And GATA-3 was required for nTreg-to-Th2 conversion. Foxp3 may account for this GATA-3 upregulation in nTreg cells, because ectopic expression of Foxp3 preferentially promoted GATA-3 but not T-bet expression. Thus, we have identified an intrinsic mechanism that imposes a Th2/Th1 imbalance and predisposes Foxp3-expressing cells to IL-4 production independent of STAT-6 signaling.