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The chemokine-mediated recruitment of effector T cells to sites of inflammation is a central feature of the immune response. The extent to which chemokine expression levels are limited by the intrinsic developmental characteristics of a tissue has remained unexplored. We show in mice that effector T cells cannot accumulate within the decidua, the specialized stromal tissue encapsulating the fetus and placenta. Impaired accumulation was in part attributable to the epigenetic silencing of key T cell-attracting inflammatory chemokine genes in decidual stromal cells, as evidenced by promoter accrual of repressive histone marks. These findings give insight into mechanisms of fetomaternal immune tolerance as well as reveal the epigenetic modification of tissue stromal cells as a modality for limiting effector T cell trafficking.
Besides being essential for reproductive success, the ability of the allogeneic fetus and placenta to avoid rejection by the maternal immune system during pregnancy (i.e. fetomaternal tolerance) has served as a paradigm for the study of organ-specific immune tolerance (1). Recent work on this problem has made use of the Act-mOVA mating system, in which wild-type female mice crossed with males hemizygous for the Act-mOVA transgene (2) generate concepti expressing a transmembrane form of the model antigen chicken egg ovalbumin (OVA) from the ubiquitously active β-actin promoter (3, 4). As early as embryonic day (E) 7.5, OVA is expressed at high levels by trophoblasts directly contacting the uterus (i.e. at the maternal/fetal interface) (3), thus exposing maternal tissue to a surrogate fetal/placental antigen that should in principle allow for T cell priming and render the conceptus susceptible to attack by antigen-specific cytotoxic T lymphocytes (CTLs).
This mating system has been used to show that fetal rejection is in part prevented by mechanisms that minimize the activation of naïve T cells with fetal/placental specificity (3, 5). However, antigen-specific fetal loss still does not occur when systemic anti-fetal/placental CTL activity is experimentally induced in late gestation (3). Thus, a fail-safe mechanism also exists to protect the conceptus from activated CTLs. To visualize this phenomenon more directly, we asked whether pregnant mice, immunized with soluble OVA prior to mating, would show Act-mOVA-specific fetal loss on E10.5 after being rechallenged with OVA plus adjuvant (agonistic CD40 antibodies plus poly(I:C)) on E5.5 (6). We studied this period of early gestation because the behavior of OVA-specific T cells would not be influenced by the systemic release of fetal/placental OVA, which starts on ~E10.5 (3). Strikingly, pregnant mice bore the expected Mendelian proportion of Act-mOVA+ concepti (17 of 26 total embryos from n=3 pregnant mice), even though 14–20% of splenic CD8 T cells were OVA-specific at the time of sacrifice (fig. S1). Thus, fetal rejection does not occur even when memory T cells with known fetal/placental specificity are reactivated in early pregnancy.
To find possible explanations for this observation, we evaluated the distribution of reactivated memory T cells in the uteri of C57BL/6-mated mice. Consistent with the ability of effector T cells to infiltrate peripheral tissues even in the absence of a localized antigen source (7, 8), E8.5 mice re-challenged with OVA plus adjuvant on E5.5 showed large numbers of CD3+ T cells distributed throughout the segments of myometrium (and associated submyometrial stroma) overlying each implantation site (Fig. 1, A and B), as well as throughout the myometrium and endometrium of the undecidualized uterine segments between implantation sites (i.e. inter-implantation sites; Fig. 1, C and D). In contrast, CD3+ cells in the decidua appeared sparse (Fig. 1B), with tissue densities remaining at levels similar to those seen throughout the uteri of mice that were not OVA-rechallenged. CD3+ cells in the decidua were most prominent within blood vessels; however, most were extravascular in the implantation site-associated myometrium and inter-implantation sites (Fig. 1E, F). Together, these results suggested that the decidua had a reduced capacity for T cell accumulation, possibly due an intrinsic inability to recruit T cells from the blood. Accordingly, reactivated memory T cells were also unable to infiltrate decidual tissue encapsulating OVA-expressing concepti (fig. S2), or in hormonally pseudopregnant females with oil-induced artificial deciduomas (see Fig. 4).
Because anti-CD40 antibodies plus poly(I:C) induce a type 1-polarized T cell response (9), we tested whether impaired decidual T cell infiltration was due to low expression of T helper 1(Th1)/T cytotoxic 1 (Tc1) chemoattractants. Strikingly, 6 h after the intravenous injection of anti-CD40 antibodies, poly(I:C), and endotoxin-contaminated OVA, a regimen expected to increase blood levels of the pro-inflammatory cytokines tumor necrosis factor-α (TNFα) and interferon-γ (IFNγ) (10, 11), high levels of the key Th1/Tc1-attracting chemokine CXCL9 (a CXCR3 ligand) (12) were apparent in the segments of myometrium overlying each E8.5 implantation site (Fig. 2, A and B). In contrast, much lower CXCL9 expression was apparent in the decidua. High CXCL9 expression was also induced in both the endometrium and myometrium of the undecidualized uteri of pseudopregnant females (fig. S3), with the vast majority of the expressing cells being CD45− stromal cells (Fig. 2C). Because endometrial stromal cells (ESCs) are the precursors of decidual stromal cells (DSCs), the major cell type of the decidua, these results suggested that the developmental process of decidualization reduced the cells’ capacity to produce T cell chemoattractants under inflammatory conditions.
We next independently prepared highly enriched stromal cells from E7.5 artificial deciduomas and overlying myometrium (fig. S4) and evaluated their inflammatory response in vitro (Fig. 2D). As with other cell types (13, 14), myometrial stromal cells (MSCs) treated with a combination of TNFα and IFNγ showed synergistic mRNA induction of both Cxcl9 and Cxcl10, which encodes a second CXCR3 ligand. Furthermore, Ccl5, whose product CCL5 (RANTES) has also been implicated in Th1/Tc1 recruitment to inflamed tissues (12), was upregulated ~15-fold in TNFα-treated MSCs, with expression further augmented by IFNγ. In contrast, Ccl5 and Cxcl9 transcript levels in DSCs remained unchanged after cytokine treatment, whereas TNFα+IFNγ mildly induced Cxcl10 expression to levels that barely exceeded those of MSCs at baseline. The expression pattern of Cxcl11, which encodes the third known CXCR3 ligand, was similar to that of Cxcl9 and Cxcl10 (fig. S5). The inability of DSCs to produce Th1 chemoattractants was functionally confirmed using transwell migration assays, which furthermore showed that MSCs attract Th1 cells through the induction of CXCR3 ligands and CCL5 (Fig. 2E, F). Together, these results suggested that the inability of DSCs to produce T cell-attracting chemokines under inflammatory conditions in vivo was due to a cell-intrinsic defect in their inflammatory cytokine response.
The inability of DSCs to produce CXCR3 ligands and CCL5 was not explained by decreased activation of NF-κB or STAT1, the major transcription factors mediating TNFα and IFNγ signaling (fig. S6A, B). Moreover, we could find examples of NF-κB and STAT1 target genes that were induced in DSCs in a relatively robust fashion (fig. S6C). These results suggested that the DSC chemokine expression defects were gene-specific and independent of inflammatory signaling per se. We therefore evaluated gene-specific chromatin configurations using chromatin immunoprecipitation (ChIP) assays. Elevated basal levels of the repressive histone H3 trimethyl lysine 27 (H3K27me3) mark (15) were present on the Cxcl9 and Cxcl10 promoters in DSCs as compared to MSCs (Fig. 3A, top row). Conversely, TNFα+IFNγ treatment increased Cxcl9/10 promoter levels of acetylated histone H4 (H4Ac), a mark of active gene transcription, in MSCs but not DSCs (Fig. 3A, bottom row). Both cell populations showed the inverse patterns of H3K27me3 and H4Ac occupancies on the Gapdh and Cd8a promoters expected from these genes' respectively high and low constitutive expression levels. Thus, low cytokine inducibility of Cxcl9/10 in DSCs was associated with the gene-specific presence of the repressive H3K27me3 histone mark.
In vivo ChIP assays performed directly on dissected E7.5 uterine tissue layers also revealed high levels of the H3K27me3 mark on the Cxcl9/10 promoters in whole decidua as compared to overlying myometrium, thus demonstrating that Cxcl9/10 silencing was also a feature of true, pregnancy-associated decidua in vivo (Fig. 3B). Recognizing that undecidualized uteri are comprised equally in volume by endometrium and myometrium (5), this result also meant that in vivo ChIP assays could be used to infer chromatin configurations in undecidualized ESCs, as these cells could not be sufficiently purified for ex vivo assays. Accordingly, Cxcl9/10 H3K27me3 promoter occupancies in whole non-pregnant uteri and E7.5 inter-implantation sites were similar to those in segments of implantation site-associated myometrium, and clearly not intermediate between the myometrium and decidua (Fig. 3B). This result strongly suggested that the H3K27me3 modification of the Cxcl9/10 promoters appears upon transformation of ESCs into DSCs.
The Ccl5 promoter also showed increased H3K27me3 occupancy in whole decidua as compared to myometrium and undecidualized uterus (Fig. 3B), suggesting a shared pathway for minimizing decidual chemokine expression. Interestingly, this increase was not readily apparent ex vivo, while H3K27me3 levels on the Cxcl9/10 promoters appeared somewhat reduced upon TNFα+IFNγ treatment (Fig. 3A). These results suggest some level of reversibility of the H3K27me3 mark at the locations we assessed by ChIP, possibly as a result of just isolating and culturing the cells. Since H4Ac levels on the Cxcl9/10 and Ccl5 promoters were nonetheless unchanged in TNFα+IFNγ-treated DSCs (Fig. 3A), it is likely that the continued repressed status of these genes after 24 h culture also involves either the presence of the H3K27me3 mark in other regions of their respective loci, or the presence of other repressive modifications.
We next determined the effect of ectopic chemokine expression within the decidua by injecting artificial deciduomas in OVA-rechallenged mice with control, Cxcl9, or Ccl5-expressing lentiviruses mixed with EGFP reporter lentiviruses. For each mouse, decidual CD3+ T cell densities were normalized to myometrial CD3+ cell densities in order to account for systemic differences in the magnitude of the anti-OVA T cell response. CD3+ cell densities in the GFP+ decidual areas of control virus-injected mice were elevated as compared to uninfected, GFP− decidual areas, thus revealing a non-specific effect of viral infection per se. However, these densities were not further altered in mice injected with Cxcl9- or Ccl5-expressing viruses. In contrast, CD3+ cell densities in decidual areas infected with mixtures of Cxcl9- and Ccl5-expressing viruses were significantly elevated compared to control virus-infected areas (Fig. 4). The synergistic effect of dual CXCL9/CCL5 expression thus apparent is consistent with recent observations in a malignant melanoma model (16). Furthermore, since CXCL9 expression in Cxcl9+Ccl5-infected decidual areas was undetectable by tissue immunostaining (fig. S7), it was unlikely that T cell infiltration into these areas was due to supra-physiologic chemokine expression. Together, these results suggested that inadequate endogenous expression of CXCR3 ligands and CCL5 was limiting for decidual T cell accumulation.
Provocatively, T cells have been reported to be relatively scarce in the human decidua (17, 18), implicating the developmental program of decidual chemokine silencing described here as a potentially conserved mechanism of fetomaternal tolerance. Consistent with this possibility, Cxcl10 is expressed only focally in the human decidua, in association with peri-glandular leukocyte aggregates (19). As the presence of even low numbers of activated T cells at the maternal/fetal interface might disturb placental development or function, dysregulation of this pathway might also contribute to a variety of pregnancy complications. Conversely, altered chemokine silencing may influence the susceptibility of the decidua to infection. More generally, however, our results demonstrate that genes encoding Th1/Tc1 attracting chemokines are subject to epigenetic regulation in tissue stromal cells, and that such regulation can significantly influence a tissue’s capacity for T cell accumulation. This demonstration raises questions regarding how the repressive H3K27me3 histone mark is targeted to select chemokine genes, and whether related pathways control T cell access to the stroma of infected, autoimmunity-afflicted, or cancer-bearing tissues.
We thank S. K. Dey and K. Johnson for advice, and A. Frey and J. Ernst for comments on the manuscript. The Histopathology and Vaccine & Cell Therapy core facilities of the NYU Cancer Institute provided histology services and tetramer reagents, and were supported by NIH–NCI (P30CA016087). P.N., E.T. and C.-S. T. performed experiments, P.A. and D.E.L. provided critical expertise and reagents, P.N. and A.E. analyzed data, P.N. and A.E. designed experiments and wrote the manuscript. The data reported in the manuscript are tabulated in the main paper and in the supplementary materials. Supported by grants from the NIH (RO1AI062980) and the American Cancer Society to A.E. (RSG-10-158-01-LIB).
Supporting Online Material
Materials and Methods
Figs. S1 to S7