Coordinate regulation of tissue macrophage and dendritic cell population dynamics by CSF-1

CSF-1 drives the homeostatic expansion of macrophages within the growing myometrium of pregnant mice by stimulating in situ proliferation and inducing monocyte precursor recruitment from the blood

Cis-acting pathways selectively enforce the non-immunogenicity of shed placental antigen for maternal CD8 T cells.

Maternal immune tolerance towards the fetus and placenta is thought to be established in part by pathways that attenuate T cell priming to antigens released from the placenta into maternal blood. These pathways remain largely undefined and their existence, at face value, seems incompatible with a mother's need to maintain a functional immune system during pregnancy. A particular conundrum is evident if we consider that maternal antigen presenting cells, activated in order to prime T cells to pathogen-derived antigens, would also have the capacity to prime T cells to co-ingested placental antigens. Here, we address this paradox using a transgenic system in which placental membranes are tagged with a strong surrogate antigen (ovalbumin). We find that although a remarkably large quantity of acellular ovalbumin-containing placental material is released into maternal blood, splenic CD8 T cells in pregnant mice bearing unmanipulated T cell repertoires are not primed to ovalbumin even if the mice are intravenously injected with adjuvants. This failure was largely independent of regulatory T cells, and instead was linked to the intrinsic characteristics of the released material that rendered it selectively non-immunogenic, potentially by sequestering it from CD8α(+) dendritic cells. The release of ovalbumin-containing placental material into maternal blood thus had no discernable impact on CD8 T cell priming to soluble ovalbumin injected intravenously during pregnancy, nor did it induce long-term tolerance to ovalbumin. Together, these results outline a major pathway governing the maternal immune response to the placenta, and suggest how tolerance to placental antigens can be maintained systemically without being detrimental to host defense

Non-immunogenic responses of maternal CD8 T cells to shed placental mOVA.

<p>(A) Effects of mating, pregnancy, adjuvant (anti-CD40 Abs +poly(I:C)), sOVA, and mOVA on the expansion of OVA-specific CD8 T cells. Adjuvant±sOVA was injected 6 days prior to sacrifice. Representative dot plots and mean±SEM of the percentage of CD44^hi APC-conjugated K^b/OVAp-tetramer⁺ cells of total splenic CD8 T cells. Pregnant mice were killed on E17.5 to 1 day after delivery. Mated mice that failed to become pregnant were killed on what would have been E17.5-21.5. Aside from group 3 (one experiment), data are from at least 4 independent experiments per group. In addition (*), <i>n</i> = 7 group 2 and <i>n</i> = 4 group 3 mice showed no difference compared to <i>n</i> = 9 virgin, untreated (group 1) mice when analyzed with PE-conjugated K^b/OVAp-tetramers (2-8 experiments). a, <i>P</i><0.02 vs. group 4; b, <i>P</i> = 0.38 vs. group 5; c, <i>P</i> = 0.01 vs. group 5; d, <i>P</i> = 0.01 vs. group 1; e, <i>P</i> = 0.53 vs. group 7. (B) Effects of pregnancy and mOVA shedding from the placenta on the induction of IFNγ-expressing splenic CD8 T cells by intravenous sOVA+adjuvant injection. Experimental groups are a subset of those shown in panel A. Representative dot plots and mean±SEM of the percentage of CD44^hi IFNγ+ cells of total splenic CD8 T cells. Splenocytes were treated <i>ex vivo</i> with OVAp prior to analysis. Data are from two independent experiments. a, <i>P</i> = 0.005 vs. group 5; b, <i>P</i> = 0.04 vs. group 8. (C) Effect of PC61-mediated Treg depletion on OVA-specific CD8 T cell expansion in Act-mOVA-mated pregnant mice treated with anti-CD40 Abs+poly(I:C). Top row: representative dot plots and mean±SEM of the percentage of CD44^hi PE-conjugated K^b/OVAp-tetramer⁺ cells of total splenic CD8 T cells. a, <i>P</i><0.001 vs. virgin, untreated. Of note, due to their increased brightness, the PE-conjugated K^b/OVAp tetramers used in this experiment detected about twice as many cells as the APC-conjugated K^b/OVAp tetramers used in the experiment shown in Fig. 3A. Bottom row: representative plots showing loss of FOXP3⁺ CD25^hi cells at the time of sacrifice. Data are from 2–8 independent experiments.</p

Pregnancy does not induce DC activation in the spleen, nor does it inhibit adjuvant-induced DC activation.

<p>(A) Induction of CD86 expression by splenic DC subsets in virgin and pregnant mice by anti-CD40 Abs or poly(I:C). Data show <i>n</i> = 4–12 mice per group (mean±SD), from at least 2 independent experiments per group. *, <i>P</i><0.001. (B) Representative histograms. For clarity, untreated pregnant mice are not shown.</p

Incomplete deletion and a lack of tolerance induction in maternal CD8 T cells exposed to mOVA.

<p>(A) OVA-specific CD8 T cell expansion in postpartum Act-mOVA-mated females. Mice were challenged with sOVA+adjuvants either as virgins or 7–10 days postpartum following pregnancies sired as indicated. Six days later, the percentage of CD44^hi PE-conjugated K^b/OVAp-tetramer⁺ cells of total splenic CD8 T cells was determined. (B) Splenocytes from subsets of the same mice were treated <i>ex vivo</i> with OVAp and then stained intracellularly with IFNγ Abs. n.s., not significant. (C) Mice were given 10,000 CD45.1 OT-I T cells on E5.5-8.5, then challenged as in (A) 8–9 days postpartum. Groups of virgin mice were treated and analyzed in parallel. The OT-I cells were identified as being CD45.1⁺; endogenous OVAp-specific cells were identified with PE-conjugated K^b/OVAp-tetramers. All XAct-mOVA postpartum females analyzed are shown; all other groups show four representative mice from <i>n</i> = 4–5 mice per group. (D) Mice were given 5×10⁵ CD45.1 OT-I T cells either as virgins or on E12.5-13.5. Some mice were then injected on the same day with sOVA+adjuvants, or with adjuvants alone. The percentage of OT-I cells was determined 6 days later. All data are from at least 2 independent experiments.</p

Shedding of mOVA into maternal blood.

<p>OVA immunoprecipitation followed by anti-OVA western blotting was performed on plasma samples. For comparison, 10 and 50 ng of sOVA were directly loaded into the last two lanes of each gel. (A) Plasma from B6- and Act-mOVA-mated pregnant mice was pooled (<i>n</i> = 4 mice per pool) and purified by differential centrifugation. The amount of material loaded into each lane was derived from the same initial amount of plasma (25 µl). (B) sOVA clearance from mouse plasma. Mice were uninjected (-) or injected with 100 µg sOVA. Plasma was obtained at the indicated times post-injection. Both blots are representative of two independent experiments.</p

Selectively impaired mOVA presentation to CD8 T cells in <i>Ccr7^-/-</i> mice.

<p>Representative CFSE dilution profiles and the percentage of cells having undergone at least one division cycle for all mice in the group (mean±SEM). All data are from 2–5 independent experiments. (A) Response of OT-I T cells transferred into virgin females injected with 100 µg sOVA on the day of T cell transfer. (B) Response of OT-I and OT-II T cells transferred into virgin or E12.5-17.5 pregnant females mated with the indicated partners. * <i>P</i> = 0.001, ** <i>P</i><0.0001 compared to corresponding Act-mOVA-mated B6 controls.</p

Cis-Acting Pathways Selectively Enforce the Non-Immunogenicity of Shed Placental Antigen for Maternal CD8 T Cells

Maternal immune tolerance towards the fetus and placenta is thought to be established in part by pathways that attenuate T cell priming to antigens released from the placenta into maternal blood. These pathways remain largely undefined and their existence, at face value, seems incompatible with a mother's need to maintain a functional immune system during pregnancy. A particular conundrum is evident if we consider that maternal antigen presenting cells, activated in order to prime T cells to pathogen-derived antigens, would also have the capacity to prime T cells to co-ingested placental antigens. Here, we address this paradox using a transgenic system in which placental membranes are tagged with a strong surrogate antigen (ovalbumin). We find that although a remarkably large quantity of acellular ovalbumin-containing placental material is released into maternal blood, splenic CD8 T cells in pregnant mice bearing unmanipulated T cell repertoires are not primed to ovalbumin even if the mice are intravenously injected with adjuvants. This failure was largely independent of regulatory T cells, and instead was linked to the intrinsic characteristics of the released material that rendered it selectively non-immunogenic, potentially by sequestering it from CD8α(+) dendritic cells. The release of ovalbumin-containing placental material into maternal blood thus had no discernable impact on CD8 T cell priming to soluble ovalbumin injected intravenously during pregnancy, nor did it induce long-term tolerance to ovalbumin. Together, these results outline a major pathway governing the maternal immune response to the placenta, and suggest how tolerance to placental antigens can be maintained systemically without being detrimental to host defense