This study showed remarkable effects on the immune system caused by the over-expression of Nur77 in T cells. In the only previous study of these mice in a disease model, the incidence and severity of collagen-induced arthritis was significantly decreased in Nur77-transgenic mice versus WT controls 
. Attenuation of the disease was associated with apoptosis of transgenic T cells and decreased production of collagen type II-specific IgG2a antibodies, consistent with impaired capacity for normal T cell proliferation and provision of help for T cell-dependent B cell functions. In contrast, we now show significant therapeutic consequences of the differential susceptibility of Treg versus non-Treg cells to undergo Nur77-induced apoptosis.
As expected, given its pro-apoptotic role during thymocyte development, Nur77 over-expression led to a marked reduction in the numbers of mature T cells in the periphery. However, the residual peripheral T cells themselves showed markedly increased rates of apoptosis upon activation in vitro or in vivo, indicating that Nur77 is an important regulator of mature T cell apoptosis. Despite their decreased numbers and increased rates of apoptosis following cell activation, in the absence of Tregs, T cells of Nur77Tg mice were able to promote rejection of allografts at a similar pace to that exerted by WT T cells. By contrast, CD4+CD25+ naturally occurring Tregs were more resistant to apoptosis induced by Nur77 over-expression, such that secondary lymphoid tissues of Nur77Tg mice had a markedly expanded population of Foxp3+ Tregs. The spontaneous long-term acceptance of fully MHC-mismatched cardiac allografts in this strong-responder strain combination highlights the extent to which the Treg to T effector cell ratio can dampen host immune responses.
Allografts in Nur77Tg recipients at both peri-transplant and later periods were infiltrated by Foxp3+ Tregs, and had increased expression of several genes recently implicated in Treg biology, including HDAC9, TIP60 and Foxp1 
. They also showed decreased levels, compared to allografts in WT recipients, of pro-inflammatory cytokines and chemokine receptors important to allograft rejection in this model 
. The finding that Nur77 could be co-immunoprecipitated with HDAC7, extends to Foxp3+ Tregs the known relationship of these two molecules in T cell development, especially within the thymus 
. Further characterization of the roles of HDAC7, HDAC9 and related proteins in Tregs is underway in our laboratory 
Despite the presence of relatively large numbers of Tregs in Nur77Tg mice, adoptive transfer of small numbers of WT T effector cells led to prompt rejection of cardiac allografts, while transferring even large numbers of Nur77Tg T effector cells, so as to increase the T effector to Treg cell ratio, did not abrogate spontaneous allograft acceptance, pointing to the important role of T effector cell apoptosis in achieving transplant tolerance. Hence, efforts to induce allograft acceptance as a result of Nur77 over-expression will likely require enhanced expression in both CD4 and CD8 T cells whereas, at the least, associated enhancement of Nur77 expression in Treg cells does not appear to have negative consequences.
Previous studies showed using Foxp3 transgenic mice that increased expression of Foxp3 led to increased apoptosis of CD4+CD25− T cells following TCR activation, along with upregulation of pro-apoptotic genes such as FasL, TRAIL. TNF-α and Bad 
. The Fas-FasL pathway was also implicated in the control of Treg apoptosis in studies of FasL mutant (gld) mice; these contain markedly increased numbers of CD4+CD25+ Tregs within the CD4 population 
. Recent data suggest that adult human Foxp3+ Tregs are comprised of a naive (CD4+CD25+CD45RAhi
) CD95− subset, as well as a more frequent memory (CD4+CD25+CD45RO) CD95+ subset, and that these two subsets differ in their susceptibility to CD95L-induced apoptosis 
. Thus, while the memory subset is prone to CD95L-mediated death, naive Tregs are resistant. Tregs are known to be more resistant than conventional T cells to activation-induced cell death 
, and the current data indicate that Tregs are also more resistant than conventional Tregs to Nur77-induced cell death.
In addition to transgenic approaches, Nur77 expression can be modulated by physiologic and chemical agents. Nur77 was first identified as an immediate-early gene induced by serum (9) or TCR signaling in T cell hybridomas and thymocytes 
. Natural and synthetic retinoic acids affect Nur77 activity through its interaction with RAR and RXR; all-trans or 9-cis-retinoic acids, which block activation-induced cell death, inhibit the transcriptional activity of Nur77 (and expression of FasL) in T cells 
. By contrast, CD437, a RARγ-specific agonist, induces Nur77 expression through an unknown mechanism and enhances Nur77-mediated activity through a Nur77-binding response element 
With regard to effects of pharmacologic agents on Nur77 expression and function, thapsigargin inhibits endoplasmic reticulum-dependent Ca2+
-ATPase, increases cytosolic Ca2+
levels, and induces Nur77 expression and causes apoptosis in T cells 
. Likewise, Nur77 transcription triggered by TCR signaling can be inhibited by cyclosporin A, suggesting that calcineurin plays an important role in its regulation. Cyclosporin A blocks apoptosis by inhibiting the DNA binding activity of the transcription factor Nur77 through the N-terminal region of the protein 
. Lastly, Nur77 was shown to be rapidly induced in vitro and in vivo in thymocytes exposed to DBTC, and antisense oligonucleotide inhibition of Nur77 expression prevented apoptosis induced by DBTC, suggesting that DBTC caused thymocyte apoptosis by inducing Nur77 expression 
Given our initial data, DBTC may be a suitable agent to test the effects of pharmacologic induction of Nur77 in T cells, including in experimental allograft models. Future studies from our group will involve testing of this compound in allograft models, as well as assessment of the effects of engineering of a dominant-negative form of Nur77 on T cell and Treg function. Collectively, the current data, along with the proposed studies, should provide a basis for assessing whether manipulation of Nur77 levels by pharmacologic or genetic means warrants further development and testing in pre-clinical disease models.