Over the last decade numerous studies have led to the realization that suppressive function of Treg cells extends far beyond autoimmunity, the originally suggested sphere of their activity. Treg cells have been implicated in control of acute and chronic infections, tissue homeostasis at barrier sites populated by commensal microbiota, allergy, injury response and tissue repair, metabolic syndrome, and cancer (Josefowicz et al., 2012
). In this study, we find that the Foxp3
intronic enhancer CNS1, essential for extrathymic differentiation of Treg cells, is present only in eutherian mammals, but not in marsupials or monotremes and that pTreg cell paucity in CNS1-deficient females mated to MHC-mismatched males results in increased spontaneous abortion of embryos. An implication of these observations is that generation of Treg cells in the thymus does not afford adequate protection of the fetus expressing allogeneic MHC alleles from immune mediated attack by maternal T cells. This latter notion is also supported by the comparable extent of embryo resorption associated with acute depletion of pTreg and pan-Treg ablation, i.e. elimination of both pTreg and tTreg cells. This finding implies that extrathymically generated Treg cells serve as the predominant subset mitigating maternal-fetal allogeneic conflict. We would propose that once in place, extrathymic generation of Treg cells, primarily driven by the pressure to enforce maternal fetal tolerance, likely assumed additional functions including control of responses to non-self antigens leading to allergy and asthma and to commensal organisms in the gut (Lathrop et al., 2011
; Josefowicz et al., 2012
Although pTreg cell deficiency in CNS1-deficient mice resulted in significantly increased resorption of fetuses during allogeneic pregnancy, its penetrance was incomplete. This was a rather expected result likely due to multiple mechanisms that operate during pregnancy to limit encounter of maternal alloreactive T cells with, or their response to, fetal alloantigens. These mechanisms include, but are not limited to, inactivation of immune cells by tryptophan deprivation by indoleamine 2,3-dioxygenases (Munn et al., 1998
), Fas-Fas ligand mediated apoptosis of activated alloreactive T cells (Hunt et al., 1997
), expression of immunosuppressive mediators such as TGF-ß and galectin-1 (Simpson et al., 2002
; Blois et al., 2007
), entrapment of endometrial dendritic cells (Collins et al., 2009
), limited expression of MHC molecules on trophoblasts (Erlebacher et al., 2007
), and increased expression of inhibitory B7 family members (PD-L1, B7H3, B7H4) (reviewed in Petroff and Perchellet, 2010
). It seems reasonable to suggest that pTreg cell mediated suppression enforces maternal-fetal tolerance not single-handedly, but jointly with other numerous immunomodulatory mechanisms.
Additional factors, which could influence the degree of immune mediated resorption associated with pTreg cell or pan-Treg cell deficiency include the genetic background, microbial status, and stress exposure. It is likely that in the absence of pTreg cells infection may result in a more severe pregnancy disruption. It must be also noted that the three week-long gestation period in mice is relatively short; extrathymic generation of Treg cells may play a more pronounced role in maternal-fetal tolerance in mammals with longer gestation times where there would be higher probability of the encounter of alloreactive T cells of the mother with paternally encoded alloantigens and for the immune response to develop.
The aforementioned possible influences affecting severity of pregnancy disruption and differences in experimental design might account for a varying degree of embryo resorption observed in our experiments and in previous reports employing adoptive T cell transfers and CD25 antibody mediated Treg cell depletion in lymphopenic or lymphoreplete mice (Aluvihare et al., 2004
; Shima et al., 2010
). However, we have not encountered pervasive fetal death observed upon continuous DT-mediated ablation of Treg cells starting at mid-gestation daily which was likely due to secondary effects of the poor health condition of the mother (Rowe et al., 2011
Our demonstration of a key role of extrathymic generation of Treg cells in maternal-fetal tolerance substantially adds to previous studies demonstrating the general importance of Treg cells in control of maternal immune responses to the allogeneic fetus in mice. In humans, Treg cells are present in increased numbers during pregnancy in the blood and the decidua (Heikkinen et al., 2004
; Somerset et al., 2004
). Decreases in Treg cells have been associated with frequent human pregnancy disorders including preeclampsia and repeated spontaneous abortions (Arruvito et al., 2009
; Darmochwal-Kolarz et al., 2012
). The histological features of allogeneic pregnancy in pTreg cell-deficient females were redolent of abnormal spiral artery remodeling associated with pre-eclampsia and other complications of pregnancy in humans and accompanying increased local inflammation (Redman and Sargent, 2005
; Avagliano et al., 2011
; Renaud et al., 2011
). These observations raise an intriguing possibility of a link between these conditions and impaired pTreg generation or function. Our study suggests that the reduced Treg accumulation and resulting pathology may be partially due to defective peripheral induction of Treg cells to paternal antigens and potential therapies could be developed to address this defect.
The analysis of CNS1 sequence conservation suggests that this enhancer was gained during evolution of eutherian mammals. CNS1 contains binding sites for transcription factors downstream of three major signaling pathway required for pTreg generation and its deletion results in a selective impairment of this differentiation process (Zheng et al., 2010
). Thus, the introduction of this several hundred base pair-long DNA sequence into the Foxp3
locus could have been sufficient to enable the differentiation of pTreg cells. This line of reasoning implies that the increased interaction between the mother and fetus during gestation necessitated a mechanism of acquired active tolerance afforded by peripheral generation of regulatory T cells. Consistent with this idea, diminished litter size observed in allogeneic pregnancy in CNS1-deficient vs. –sufficient females suggested that pTreg generation afforded a reproductive advantage.
The process of insertion of CNS1 sequence into the first intron of the Foxp3
locus appears to have occurred via a MIR family retrotransposon activity during the Mesozoic era at a time overlapping with the evolution of placental mammals. We found that in the mouse genome MIR elements resembling CNS1 are enriched for SMAD and RXR binding sites (data not shown) suggesting that these elements may have endowed TGF-β and retinoic acid response capacity to Foxp3 and other genes in agreement with the idea of exaptation where portions of transposable elements acquire a function that serves their host (Brosius and Gould, 1992
). These elements can then confer novel signaling pathway responsiveness to existing genes, thereby augmenting their function. A mechanism of retrotransposon-mediated exaptation affecting the structure or regulation of pre-existing genes upon introduction of novel exons or enhancers has been previously reported (Bejerano et al., 2006
; Mikkelsen et al., 2007
). We suggest that acquisition of CNS1 supporting extrathymic Treg cell generation in eutherian mammals represents a novel example of retrotransposition-mediated innovation in regulation of gene expression where a distinct biological purpose and novel functionality associated with the CNS1 enhancer is implicit of the potential evolutionary pressure underlying its conservation.
It is noteworthy in this regard that the emergence of chorioallantoic placenta, which allowed for viviparity in therian mammals, was assisted by appropriation of several retroviral genes including retrotransposon-derived Peg10 and Peg11/Rtl1 and syncitin-A and –B originating from the envelope protein of a defective retrovirus. These genes are essential for normal function of placenta and trophoblast fusion, respectively (Mi et al., 2000
; Ono et al., 2006
; Sekita et al., 2008
; Dupressoir et al., 2011
). Taken together, these results and our findings suggest that in addition to facilitating placentation during mammalian evolution retrotransposon-mediated innovation helped to alleviate immune conflict associated with this acquisition.
In conclusion, we suggest that the mechanism of extrathymic differentiation of Treg cells may have been gained during evolution to reinforce tolerance to paternal alloantigens presented by the fetus during the increasingly long gestation period in placental mammals. This adaptation was realized with the aid of the Foxp3 CNS1 enhancer responsible for induction of Foxp3 in peripheral CD4+ Foxp3- T cells, which likely emerged upon capture of a MIR retrotransposon containing TGF-β and retinoic acid receptor response elements. A role of extrathymically generated Treg cells in maternal-fetal tolerance may provide an important insight into potential clinical complications of human pregnancies.