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

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

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

Antibody and dilutions used for flow cytometry staining.

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

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

Image_1.tif

<p>A previous report has shown that regulatory T cells (Treg) were markedly more sensitive to adenosine-5′-triphosphate (ATP) than conventional T cells (Tconv). Another one has shown that Tregs and CD45RB^low Tconvs, but not CD45RB^high Tconvs, displayed similar high sensitivity to ATP. We have previously reported that CD45RB^low Tconvs expressing B220/CD45RABC molecules in a pre-apoptotic stage are resistant to ATP stimulation due to the loss of P2X7 receptor (P2X7R) membrane expression. To gain a clearer picture on T-cell sensitivity to ATP, we have quantified four different cellular activities triggered by ATP in mouse T cells at different stages of activation/differentiation, in correlation with levels of P2X7R membrane expression. P2X7R expression significantly increases on Tconvs during differentiation from naive CD45RB^highCD44^low to effector/memory CD45RB^lowCD44^high stage. Maximum levels of upregulation are reached on recently activated CD69⁺ naive and memory Tconvs. Ectonucleotidases CD39 and CD73 expression levels increase in parallel with those of P2X7R. Recently activated CD69⁺ CD45RB^highCD44^low Tconvs, although expressing high levels of P2X7R, fail to cleave homing receptor CD62L after ATP treatment, but efficiently form pores and externalize phosphatidylserine (PS). In contrast, naive CD45RB^highCD44^low Tconvs cleave CD62L with high efficiency although they express a lower level of P2X7, thus suggesting that P2X7R levels are not a limiting factor for signaling ATP-induced cellular responses. Contrary to common assumption, P2X7R-mediated cellular activities in mouse Tconvs are not triggered in an all-or-none manner, but depend on their stage of activation/differentiation. Compared to CD45RB^low Tconvs, CD45RB^lowFoxp3⁺ Tregs show significantly higher levels of P2X7R membrane expression and of sensitivity to ATP as evidenced by their high levels of CD62L shedding, pore formation and PS externalization observed after ATP treatment. In summary, the different abilities of ATP-treated Tconvs to form pore or cleave CD62L depending on their activation and differentiation state suggests that P2X7R signaling varies according to the physiological role of T convs during antigen activation in secondary lymphoid organs or trafficking to inflammatory sites.</p

Image_4.tif

<p>A previous report has shown that regulatory T cells (Treg) were markedly more sensitive to adenosine-5′-triphosphate (ATP) than conventional T cells (Tconv). Another one has shown that Tregs and CD45RB^low Tconvs, but not CD45RB^high Tconvs, displayed similar high sensitivity to ATP. We have previously reported that CD45RB^low Tconvs expressing B220/CD45RABC molecules in a pre-apoptotic stage are resistant to ATP stimulation due to the loss of P2X7 receptor (P2X7R) membrane expression. To gain a clearer picture on T-cell sensitivity to ATP, we have quantified four different cellular activities triggered by ATP in mouse T cells at different stages of activation/differentiation, in correlation with levels of P2X7R membrane expression. P2X7R expression significantly increases on Tconvs during differentiation from naive CD45RB^highCD44^low to effector/memory CD45RB^lowCD44^high stage. Maximum levels of upregulation are reached on recently activated CD69⁺ naive and memory Tconvs. Ectonucleotidases CD39 and CD73 expression levels increase in parallel with those of P2X7R. Recently activated CD69⁺ CD45RB^highCD44^low Tconvs, although expressing high levels of P2X7R, fail to cleave homing receptor CD62L after ATP treatment, but efficiently form pores and externalize phosphatidylserine (PS). In contrast, naive CD45RB^highCD44^low Tconvs cleave CD62L with high efficiency although they express a lower level of P2X7, thus suggesting that P2X7R levels are not a limiting factor for signaling ATP-induced cellular responses. Contrary to common assumption, P2X7R-mediated cellular activities in mouse Tconvs are not triggered in an all-or-none manner, but depend on their stage of activation/differentiation. Compared to CD45RB^low Tconvs, CD45RB^lowFoxp3⁺ Tregs show significantly higher levels of P2X7R membrane expression and of sensitivity to ATP as evidenced by their high levels of CD62L shedding, pore formation and PS externalization observed after ATP treatment. In summary, the different abilities of ATP-treated Tconvs to form pore or cleave CD62L depending on their activation and differentiation state suggests that P2X7R signaling varies according to the physiological role of T convs during antigen activation in secondary lymphoid organs or trafficking to inflammatory sites.</p