Analyses of immune cell populations present in the uterus of NP or pregnant mice, and in placenta (16.5 d<i>pc</i>).

<p>Only viable cells excluding propidium iodide were analysed. Enriched leukocytes from NP (A,B) or pregnant (16.5 d<i>pc</i>) uterus (C,D) and placenta (E,F) were analysed by flow cytometry. R2-gated cells (left, cf. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107267#pone-0107267-g002" target="_blank">Figure 2</a>) were analysed on the basis of the following cell surface markers: TCRβ⁺/CD4⁺ (CD4 T cells); TCRβ⁺/CD8⁺ (CD8 T cells); NK1.1⁺/TCRβ⁻ (NK cells); TCRβ⁺/NK1.1⁺ (NKT cells); CD19⁺/B220⁺ (B cells); Gr1⁺/CD11b⁺ (myeloid Gr1+ cells including monocytes); Gr1^−/CD11c⁺/CD11b^Hi/low (myeloid CD11c+ cells including DCs). R1-gated cells (right, cf. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107267#pone-0107267-g002" target="_blank">Figure 2</a>) were analysed on the basis of the following cell surface markers: Gr1⁺/CD11b⁺ (Granulocytes); CD11b^Hi/Gr1⁺/Gr1^+/−/CD11c^+/− (Highly granulosity cells or HGC). The results are representative from a typical experiment of a pool of 7 mice (A, B) or from a single mouse (C, D, E, F). The experiment was repeated at least twice with 3–6 animals per assay.</p

Quantification of uterine and placental cytokines and chemokines in syngeneic pregnancy (day 16.5 <i>pc</i>).

<p>Uterine and placental tissues were prepared as described in Materials and Methods : (A) Uteri from NP mice, (B) Uteri from <b>syngeneic</b> pregnancy (day 16.5 d<i>pc</i>), (C) Placenta from <b>syngeneic</b> pregnancy (same animals). Samples were analyzed simultaneously for the following 22 cytokines: IL-1α, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-7, IL-9, IL-10, IL-12(p70), IL-13, IL-15, IL-17, CSF2 (GM-CSF), CSF3 (G-CSF), IFNγ, CXCL10 (IP-10), CXCL1 (KC), CCL2 (MCP-1), CCL3 (MIP-1α), CCL5 (RANTES), TNF-α. Only IL-9, IL-10, GM-CSF (CSF2), G-CSF (CSF3), CXCL10 (IP-10), IL-1α, CCL3 (MIP-1α), CXCL1 (KC), CCL2 (MCP-1) yielded reproducibly significant measurements and have been presented. Striped bars: whole organ, black bars: enriched leukocytes from the same organ. Data are from 4 to 5 different samples. Statistically significant differences: (*) p≤0.05, (***) p≤0.001.</p

Cytokine and chemokine expression levels in uterus from NP mice versus syngeneic or allogeneic pregnancies (day 16.5 <i>pc</i>).

<p>The same protocol as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107267#pone-0107267-g007" target="_blank">Figure 7</a> was followed. Proteins were measured from whole uterus (A), or from enriched uterine leukocytes (B) from NP uterus, or syngeneic or allogeneic pregnancies (note the important scale variations). Striped bars: NP uterus, grey bars: syngeneic pregnancy at 16.5 d<i>pc</i>, black bars: allogeneic pregnancy at 16.5 d<i>pc</i>. Data are from 4 to 5 different samples. (*) p≤0.05, (**) p≤0.005, (***) p≤0.001.</p

Drastic changes in the scatter distribution of leukocytes from NP, or pregnant uterus, and placenta at various stages of a syngeneic pregnancy.

<p>The same protocol as <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107267#pone-0107267-g001" target="_blank">Figure 1</a> was followed. Viable cells excluding propidium iodide were gated on the basis of forward (FSC) and side scatter (SSC) criteria, from uterus (A) or placenta (D). Mean percentages (B, E) and total cell numbers (C, F) in « granular » (R1 gate, white bars) or « lymphoïd/monocytoïd » (R2 gate, black bars) are presented. NP uteri were pooled from 4 to 15 mice in all phases of œstrus cycle and the experiment was repeated 6 times. At each stage of pregnancy or post-partum, the data were collected from 4 to 14 mice assayed in 2 to 9 separate experiments. (*) p≤0.05, (**) p≤0.005, (***) p≤0.001.</p

High granulosity cells in non pregnant uterus are primarily eosinophils.

<p>Viable R1- or R2- gated CD45.2+ cells from NP uterus were analysed by FACS stained for APC and granulocytes markers (CD11b, CD24, CD11b, F4/80, Ly6C, Ly6G, MHC class II and CCR2) (A, B and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107267#pone-0107267-t001" target="_blank">Table 1</a>) and for NK and T cell markers (CD11b, NK1.1, CD3ε, CD8α and CD4) (B and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107267#pone-0107267-t001" target="_blank">Table 1</a>). The experiment was repeated twice with 6 females each time (A, B). Viable CD45.2+, CD11b+, F4/80+ R1 (C) or R2 (D) cells were sorted by flow cytometry and spun onto Super + glass microscope slides using a Cytospin cytofuge. Cells were fixed with methanol and stained with Wright-Giemsa. Pictures were taken on a Nikon H600L microscope equipped with a DS-Fi2-Nikon camera (C, D). The experiment was performed twice with at least 5 females each time.</p

Purity of leukocytes and presence of fetal cells after enrichment from placenta and uterus (16.5 d<i>pc</i>).

<p>A. Enriched leukocyte preparations from non-pregnant (NP) uterus, pregnant (16.5 d<i>pc</i>) uterus (P) and placenta were analyzed by flow cytometry. Viable cells excluding propidium iodide were gated on the basis of forward (FSC) and side scatter (SSC) criteria. The average leukocyte purity assessed by the presence of the CD45.2 marker was 89.3% for NP uterus, 97% for pregnant uterus, and 95% for placenta, compared to 98% for the spleen (positive control for the assessment of purity) from the same mice. Analyses were performed on at least 3 animals per group B. CD45.2+ B6 females were crossed with CD45.1+ B6 males. CD45+ leukocyte populations from the uterus and placenta were analyzed by flow cytometry on day 16.5 <i>pc</i>. The detection of CD45.1+2+ cells revealed the presence of a small percentage (<5%) of fetal leukocytes. The experiment has been repeated 3 times.</p

Cytokine and chemokine expression levels in placentae from syngeneic or allogeneic pregnancies (day 16.5 <i>pc</i>).

<p>Analyses were performed on the placentas from the same pregnant mice as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107267#pone-0107267-g008" target="_blank">Figure 8</a>. Proteins were measured from whole placenta (A), or from enriched placental leukocytes (B) from syngeneic or allogeneic pregnancies (note the important scale variations). Grey bars: syngeneic pregnancy, black bars: allogeneic pregnancy. Data are from 4 to 5 different samples. (*) p≤0.05, (**) p≤0.005, (***) p≤0.001.</p

Antibody and dilutions used for flow cytometry staining.

<p>NA: not applicable.</p><p>Antibody and dilutions used for flow cytometry staining.</p

Loss of P2X7 Receptor Plasma Membrane Expression and Function in Pathogenic B220⁺ Double-Negative T Lymphocytes of Autoimmune MRL/<em>lpr</em> Mice

<div><p>Lupus is a chronic inflammatory autoimmune disease influenced by multiple genetic loci including <em>Fas Ligand</em> (FasL) and <em>P2X7 receptor</em> (P2X7R). The Fas/Fas Ligand apoptotic pathway is critical for immune homeostasis and peripheral tolerance. Normal effector T lymphocytes up-regulate the transmembrane tyrosine phosphatase B220 before undergoing apoptosis. Fas-deficient MRL/<em>lpr</em> mice (<em>lpr</em> mutation) exhibit lupus and lymphoproliferative syndromes due to the massive accumulation of B220⁺ CD4^–CD8^– (DN) T lymphocytes. The precise ontogeny of B220⁺ DN T cells is unknown. B220⁺ DN T lymphocytes could be derived from effector CD4⁺ and CD8⁺ T lymphocytes, which have not undergone activation-induced cell death due to inactivation of Fas, or from a special cell lineage. P2X7R is an extracellular ATP-gated cell membrane receptor involved in the release of proinflammatory cytokines and TNFR1/Fas-independent cell death. P2X7R also regulate early signaling events involved in T-cell activation. We show herein that MRL/<em>lpr</em> mice carry a <em>P2X7R</em> allele, which confers a high sensitivity to ATP. However, during aging, the MRL/<em>lpr</em> T-cell population exhibits a drastically reduced sensitivity to ATP- or NAD-mediated stimulation of P2X7R, which parallels the increase in B220⁺ DN T-cell numbers in lymphoid organs. Importantly, we found that this B220⁺ DN T-cell subpopulation has a defect in P2X7R-mediated responses. The few B220⁺ T cells observed in normal MRL^+/+ and C57BL/6 mice are also resistant to ATP or NAD treatment. Unexpectedly, while P2X7R mRNA and proteins are present inside of B220⁺ T cells, P2X7R are undetectable on the plasma membrane of these T cells. Our results prompt the conclusion that cell surface expression of B220 strongly correlates with the negative regulation of the P2X7R pathway in T cells.</p> </div

Dose–response experiments for ATP-induced CD62L shedding on T lymphocytes.

<p>Spleen cells from 3 to 4-mo-old MRL<i>^+/+</i> (<i>solid line</i>), MRL/<i>lpr</i> (<i>dashed line</i>) and P2X7-deficient B6<i>^P2X7R−/−</i> (<i>dotted line</i>) mice (n = 3 mice/group) were treated for 45 min at 37°C with doses of ATP ranging from 100 to 5000 µM. Spleen cells were then triple-stained with anti-CD90, anti-CD19 and anti-CD62L mAb to assess by flow cytometry: (A) the percentage of CD62L-expressing CD19^–CD90⁺ T cells and (B) MFI of CD62L on CD19^–CD90⁺ T cells. Results are expressed as the mean percentage of initial expression ± SE.</p