Chemokine gene silencing in decidual stromal cells limits T cell access to the maternal-fetal interface

Abstract

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.

Fig. 1

The decidua resists infiltration by reactivated memory T cells. (A–D) B6CBAF1/J (H-2^b/k) females were given 3×10⁵ OVA-specific OT-I CD8⁺ T cells, in order to maximize potential T cell accumulation, and immunized with OVA protein 2–3 weeks prior to mating to C57BL/6 males. On E5.5, pregnant mice either received no additional treatment (A, C) or were intravenously injected with 0.5 mg OVA and adjuvant (CD40 antibodies plus poly(I:C)) (B, D). The mice were sacrificed on E8.5, and tissue immunostaining using CD3-specific antibodies (red) was performed on cross-sections of implantation sites (A, B) and inter-implantation sites (C, D). The two poles of the decidua (mesometrial and anti-mesometrial) are indicated. DAPI counterstain. (E, F) The decidual/myometrial border (E) and undecidualized endometrium (F) of a rechallenged mouse. Blood vessels (arrowheads) are identified by the presence of RBCs, which appear green as an artifact of the immunostaining protocol. myo, myometrium; dec, decidua; asterisk, uterine lumen. Data are representative of N=3 independent experiments (at least n=23 implantation sites each per group).

Fig. 2

The decidua produces low levels of Th1/Tc1-attracting chemokines in response to inflammation. (A–C) E8.5 pregnant (A, B) or pseudopregnant (C) B6CBAF1/J mice were either untreated (A) or injected with CD40 antibodies, poly(I:C), and OVA (B, C) 6 h prior to sacrifice. (A, B) CXCL9 immunostaining (red) of implantation site cross-sections. (C) Double CXCL9 (green)/CD45 (red) immunostaining of the endometrium of an inflamed pseudopregnant uterus. CXCL9 immunoreactivity appears largely confined to the endoplasmic reticulum, giving a punctate appearance. Data are representative of at least N=3 independent experiments. DAPI counterstain. (D) qRT-PCR analysis of cultured MSCs and DSCs. Cytokines were added for the last 6 h of a 24 h total culture period. Data show mean±SEM of N=4 independent experiments. n.s., not significant. (E) Migration of in vitro differentiated Th1 cells to supernatants collected from MSCs or DSCs treated as indicated over the entirety of a 24 h culture period. Data show mean±SEM of N=3 independent experiments. (F) Effect of CXCR3 desensitization (via pre-incubation of the Th1 cells with CXCL9) or CCL5 neutralization on Th1 cell migration to supernatants from TNFα+IFNγ-treated MSCs. Non-specific rat IgG antibodies served as the control for CCL5 antibodies. Data show mean±SEM of N=3 independent experiments.

Fig. 3

Chromatin configurations in uterine cells and tissue layers. (A) Ex vivo ChIP assays performed on cultured MSCs and DSCs. Cytokines were added for the last 6 h of a 24 h total culture period. Data show mean±SD of N=5 independent experiments. (B) In vivo ChIP assays performed on dissected uterine tissues and tissue layers. Deciduas were dissected free of embryos. Data show mean±SD of N=3 independent experiments.

Fig. 4

Effect of ectopic chemokine expression on effector T cell accumulation within the decidua. Mice were immunized with OVA 2–3 weeks prior to mating, rechallenged with OVA plus adjuvants on E4.5, injected with lentivirus on E5.5, and sacrificed on E7.5. Viral preparation included aliquots of EGFP reporter lentiviruses so that T cells within transduced areas could be identified on anti-CD3 and anti-GFP immunostained serial sections. (A) CD3⁺ cell densities in infected (GFP⁺) and uninfected (GFP⁻) decidual areas relative to myometrial CD3⁺ cell densities. Data show mean±SEM of at least N=3 independent experiments encompassing n=4 control (empty vector) virus-infected mice (75×10⁷ virus particles each), n=5 Cxcl9+Ccl5 virus-infected mice (37.5×10⁷ particles each), and n=3 each of mice infected with Cxcl9- or Ccl5-expressing viruses alone (75×10⁷ particles each). Myometrial CD3⁺ cell densities were not significantly different between the four groups (average: 0.037 cells/µm²). (B–K) GFP or CD3 immunostaining (as indicated, red) and DAPI counterstain (blue) of representative serial sections of decidualized uteri infected with Cxcl9+Ccl5 viruses (B–C, H–K) or control viruses (D–G). B and C: asterisks denote different areas of the decidual lumen, which frequently contained GFP⁺ cells, CD3⁺ cells, and granulocytes scattered among necrotic debris; the dashed line indicates the border between the myometrium and decidua. D–K: the dashed lines show the perimeters of infected areas used to calculate CD3⁺ cell densities; asterisks show areas excluded from analysis because of their necrotic appearance. Arrow, decidual lumen. Panels H–K are close-ups of B and C.