Systemic Reduction of Functionally Suppressive CD4^dimCD25^highFoxp3⁺ Tregs in Human Second Trimester Pregnancy Is Induced by Progesterone and 17β-Estradiol

Subjects

Thirty-eight healthy pregnant women with no signs of pregnancy complications at inclusion (pregnancy week 24–28) visiting the maternity outpatient care unit in Linköping (Kvinnohälsan), Sweden were asked to participate in the study. Obstetrical history of these women is given in Table I⇓. Twenty-five women were pregnant for the first time and 13 had experienced at least one previous pregnancy. Five women had previously given birth at least once. Excluding the women with previous pregnancy did not affect the statistical results. Seventy-one nonpregnant women (age 19–36 years, median age 26) who were blood donors or staff at Linköping University Hospital served as control subjects. There were no statistical age differences between the pregnant and nonpregnant women. In the functional in vitro assays, none of the nonpregnant women (n = 27) were using hormonal contraceptives. In the in vitro suppression assay, five of the women were in proliferative phase, six in secretory phase, and three women were on day 15 of their menstrual cycle according to questionnaires. No such information was available from the other nonpregnant women. Informed consent was obtained from all participants. The study was approved by the local ethics committee at Linköping University, Linköping, Sweden.

PBMC preparation

Whole blood was obtained in EDTA (for flow cytometry) or sodium-heparin (for functional assays) Vacutainer tubes. PBMC were separated within 1 h on Lymphoprep gradient (Axis-Shield) according to the manufacturer’s instructions, followed by washing in HBSS (Invitrogen). For direct culturing, cells were resuspended in T cell culture medium (TCM) consisting of IMDM (Invitrogen) supplemented with l-glutamine (292 mg/ml; Sigma-Aldrich), sodium bicarbonate (3,024 g/L; Sigma-Aldrich), penicillin (50 IE/ml), streptomycin (50 μg/ml) (Cambrex), 100× nonessential amino acids (10 ml/L; Invitrogen), and 5% heat inactivated FBS (Sigma-Aldrich). For flow cytometry, cells were resuspended in PBS (pH 7.4) (Medicago) supplemented with 0.1 or 2% heat-inactivated FBS (Sigma-Aldrich). For MACS separation, cells were resuspended in PBS with 2 mM EDTA (Sigma-Aldrich) and 2% FBS. Alternatively, PBMC were lysed in RNeasy RLT lysing buffer (Qiagen) and frozen at −80°C until RNA extraction.

RNA extraction and reverse transcriptase real-time PCR for quantification of Foxp3 mRNA expression

Expression of Foxp3 mRNA was analyzed in PBMC from pregnant (n = 13) and nonpregnant women (n = 25). Total RNA was extracted using the RNeasy mini kit (Qiagen) according to the manufacturer’s instructions. Approximately 200 ng of RNA was converted to cDNA in 20-μl reactions using the cDNA high-capacity archive kit (Applied Biosystems) with RNase inhibitor (Applied Biosystems) according to the manufacturer’s instructions. Reverse transcription was performed on a Mastercycler ep gradient S thermal cycler (Eppendorf). For real-time PCR, 1 μl of cDNA was mixed with TaqMan Universal Master Mix (Applied Biosystems) together with primers and probes for Foxp3 or 18S rRNA, respectively (Table II⇓). cDNA was amplified according to the TaqMan standard protocol as described by the manufacturer. Reactions were performed using the 7500 real-time PCR system (Applied Biosystems). Expression of 18S rRNA was used for normalization of RNA content in all samples. The absence of genomic DNA amplification was controlled by amplifying one sample of RNA. Data were analyzed with the 7300 system using SDS software version 1.3.1 (Applied Biosystems). Quantification was performed using the standard curve method. All samples were analyzed in duplicate and the variation limit between the duplicates was set to <15%.

Four-color flow cytometry

PBMC from pregnant (n = 14) and nonpregnant (n = 34) women were analyzed using four-color flow cytometry to determine the frequency of CD4⁺CD25^high cells and the expression of Foxp3, CTLA-4, and CD27. One million PBMC were incubated with mouse isotype controls (IgG1-FITC, IgG1-PE, IgG1-PerCP, and IgG1-allophycocyanin; all clone X40) (BD Biosciences) or mouse anti-human CD25-allophycocyanin (clone 2A3), CD4-PerCP (clone SK3), and CD27-FITC (clone M-T271) (BD Biosciences) for 30 min at 4°C in darkness. Cells were washed in PBS with 0.1% FBS by centrifugation at 500 × g for 5 min, followed by the addition of fixation/permeabilization buffer (eBioscience) and incubation as described above. Cells were washed twice in permeabilization buffer (eBioscience) by centrifugation at 500 × g for 5 min. Abs directed against intracellular CTLA-4 (clone BNI3) (BD Biosciences), Foxp3 (clone PCH101) (eBioscience), or isotype controls (BD Biosciences) were added and cells were incubated as above. Cells were washed once as described above and resuspended in PBS with 0.1% FBS. The absence of CTLA-4 surface expression was confirmed on two separate occasions and is in agreement with the findings of others (27). One hundred thousand lymphocytes were collected and analyzed using FACSCalibur and the CellQuest Pro software (BD Biosciences). The Foxp3 Ab clone PCH101 binds the amino terminus of the Foxp3 protein and has been shown to recognize both isoforms of the Foxp3 protein (7).

Six-color flow cytometry

PBMC from pregnant (n = 10) and nonpregnant (n = 10) women were analyzed by six-color flow cytometry to obtain a more detailed phenotype analysis. In addition, 17β-estradiol- and progesterone-stimulated PBMC (see below) were analyzed this way. Cells were labeled with isotype controls as described above or with mouse anti-human CD3-allophycocyanin-Cy7 (clone SK7), CD4-PerCP, CD25-allophycocyanin, CD45RA-FITC (clone L48), CD45R0-PE-Cy7 (clone UCHL1), CD127-PE (clone hIL-7R-M21), CD69-PE-Cy7 (clone FN50), and HLA-DR-FITC (clone L243). This was followed by permeabilization/fixation and staining of intracellular Foxp3 protein as described above. One hundred thousand lymphocytes were collected and analyzed using FACSCanto II (BD Biosciences) and the FACSDiva software (version 5.0.1; BD Biosciences). Absolute leukocyte (CD45), T lymphocyte (CD3), and Th cell counts (CD4) in 50 μl of EDTA whole blood were determined by using TruCount tubes (BD Biosciences) as described by the manufacturer.

Flow cytometric gating and analysis

All gating analysis was performed in a blinded manner, i.e., the evaluator did not know the origin of the sample. Cells were gated for the analysis of lymphocytes by side/forward scatter and, in six-color flow cytometry, gating for analysis of T cells was also based on CD3 expression. Gates for expression of CD25 in the CD4⁺ population (CD4⁺CD25⁺ or CD4⁺CD25⁻) were set according to isotype controls. The CD25^high gate was adjusted to contain CD4⁺ cells that expressed higher levels of CD25 than the discrete population of CD4⁻ cells that expressed CD25 (28, 29). This, and the development of other gating strategies, is further described in Results. To avoid the possible errors introduced when subjectively setting any gate for CD25^high expression, the 0.5% of CD4⁺ cells expressing the highest levels of CD25 were also evaluated. These cells are referred to as “0.5% CD4⁺CD25^highest” cells. Mean fluorescence intensity was evaluated by dividing the geometric mean fluorescence intensity for Foxp3⁺ cells with the geometric mean fluorescence intensity for Foxp3⁻ isotype controls to correct for the instrumental day-to-day variations in fluorescence intensity measurements.

Stimulation of PBMC with 17β-estradiol and progesterone

Six-well plates (Costar) were coated with 0.005 μg/ml mouse anti-human CD3 Ab (clone UCHT1; AbD Serotec) for 2 h at 37°C followed by washing the wells with PBS. The chosen anti-CD3 Ab concentration was based on titration experiments where 0.005 μg/ml anti-CD3 Ab caused a low-grade activation of the CD4⁺ cells with slight elevation of CD69 and CD25 expression. PBMC, isolated from nonpregnant women (n = 13) at a final concentration of 10⁶ PBMC/ml, were cultured in uncoated or anti-CD3 Ab-coated wells with 10 nM, 100 nM, or 10 μM 17β-estradiol (water-soluble E; Sigma-Aldrich) and/or 200 nM, 2 μM, or 200 μM progesterone (water-soluble P; Sigma-Aldrich) in TCM for 3 days at 37°C with 5% CO₂ in a humidified atmosphere. After incubation, cells were stained for six-color flow cytometry analysis on FACSCanto II as described above.

Functional suppression assay: MACS-FACS-sorting and culturing conditions

The CD4⁺ T cell isolation kit II (Miltenyi Biotec) was used for negative selection of untouched CD4⁺ cells from PBMC separated from pregnant (n = 14) and nonpregnant women (n = 14). CD4⁺ selection was performed according to the manufacturer’s description using MS columns and a miniMACS separator (Miltenyi Biotec). The MACS-sorted CD4⁺ cells were then labeled with mouse anti-human CD4-FITC (clone MT466; Miltenyi Biotec) and mouse anti-human CD25-allophycocyanin (BD Biosciences). For analysis, a portion of cells was also labeled with mouse anti-human CD127-PE (BD Biosciences). Sorting of CD4⁺CD25⁻ responder cells and CD4^dimCD25^high Tregs (see below and Fig. 1⇓C for gating) was performed on a FACSAria cell sorter (BD Biosciences) equipped with a 100-μm nozzle. Sorted populations were collected in TCM and typically showed purities of >99% upon reanalysis. Ninety-six-well plates (BD Biosciences) were coated with 1 or 5 μg/ml anti-CD3 Ab (clone UCHT1; AbD Serotec) and 5 μg/ml rat anti-human CD28 (clone YTH913.12; AbD Serotec) for 24 h at 4°C, followed by washing in PBS.

• Download figure
• Open in new tab
• Download powerpoint

FIGURE 1.

Different gating strategies for the gating of Tregs (left column; pregnant, n = 14; nonpregnant, n = 34) and the expression of Treg-associated markers Foxp3, CD127^low, and HLA-DR within these gates (right column; pregnant, n = 10; nonpregnant, n = 10) in cells from pregnant and nonpregnant women. A, The classical CD4⁺CD25^high gate was adjusted to contain CD4⁺ cells that express higher levels of CD25 than CD4⁻ cells. B, Pregnant women show a distinctly scattered population of cells with high CD4 expression; the CD4^highCD25^high gate shows a low prevalence of Foxp3⁺, CD127^low, and HLA-DR⁺ cells. C, To avoid CD4^highCD25^high non-Tregs, a CD4^dimCD25^high gate was set to include the CD25^high cells with lower expression of CD4. D, With in the CD4^dimCD25^+/high gate there are distinctly scattered cells with lowered CD4 expression. E, The gates depict reference populations. The gate on the left depicts activated CD4⁺ cells, CD4⁺CD25^dim cells showing a non-Treg phenotype. The gate on the right depicts 0.5% CD4⁺CD25^highest cells showing a clear-cut Treg phenotype. See text for further explanation. All numbers and bars are given as means ± SD. ∗∗, p ≤ 0.01; ∗∗∗, p ≤ 0.001.

CD4⁺CD25⁻ responder cells were plated at 2.5 × 10⁴ cells/well alone or in coculture with CD4^dimCD25^high Tregs at ratios of 1:1, 2:1, or 4:1 in singlet or duplicate cultures and cultured for 91–93 h at 37°C and 5% CO₂ in a humidified atmosphere before harvesting the supernatants. The ability of CD4^dimCD25^high Tregs to suppress cytokine secretion from CD4⁺CD25⁻ responder cells was calculated as a suppressive index according to the formula: (1 − (secretion in coculture/secretion from CD4⁺CD25⁻ cells alone)) × 100.

Multiplex bead array analysis of IL-2, IL-4, IL-10, TNF-α, and IFN-γ

Supernatants were analyzed by a LINCOplex human cytokine kit according to the manufacturer’s instructions (Linco Research) using the Luminex 100 instrument (Luminex). STarStation software (version 2.3; Applied Cytometry Systems) was used for acquisition and analysis of data. The range of the standard curves was 0.13–10 000 pg/ml with a dilution factor of 5. The lowest (detection limit) and highest standard concentrations used for each cytokine were adjusted according to the standard curve fitting of the standard concentrations after mathematical interpolation. Values below the detection limit were given half the value of the detection limit.

Statistics

The statistical guidance resource at Linköping University was consulted for the statistical analyzes. Due to multiple comparisons and the risk of mass significances, the significance level was set to 1%, i.e., p ≤ 0.01 (sometimes depicted as ∗∗) was considered statistically significant and p ≤ 0.05 (sometimes depicted as ∗) was regarded as a statistical tendency. Results from the flow cytometric analyzes were analyzed using Student’s unpaired t test and presented as mean ± SD. Data on cytokines did not follow Gaussian distribution and were therefore analyzed using a Wilcoxon signed rank test or a Mann-Whitney U test and presented as medians and the interquartile range (25th and 75th percentile values). Data on cytokines were also logarithmically transformed to obtain Gaussian distribution and analyzed using parametrical statistical methods. Because this did not affect the results, data were kept in linear mode and analyzed nonparametrically as described above. The coefficient of variation was expressed as percentage by calculating (SD/mean) × 100. All statistical analyzes were performed using the GraphPad Prism version 4 software (GraphPad Software).

Determination of an optimal gating strategy for CD4⁺CD25^high cells during pregnancy; the CD25^high cells with low CD4 expression (CD4^dimCD25^high) show the most pronounced Treg phenotype

The optimal flow cytometric gating strategy for CD25^high cells was investigated using four-color flow cytometry in conjunction with six-color flow cytometry for analysis of the Treg markers Foxp3, CD127^low, and HLA-DR. CD4⁺CD25^high cells were first gated according to the classical CD4⁺CD25^high gate, i.e., adjusted to contain CD4⁺ cells that expressed higher levels of CD25 than the discrete population of CD4⁻ cells that express CD25 (Fig. 1⇑A) as described previously by others (28, 29). Using this gating strategy, pregnant women displayed an increased frequency of CD4⁺CD25^high cells of CD4⁺ cells (Fig. 1⇑A). However, it was noted that within the classical CD4⁺CD25^high gate, the pregnant women showed a population of distinctly scattered cells with high CD4 expression (CD4^high). This CD4^highCD25^high population was expanded in pregnant women (Fig. 1⇑B) whereas it was almost absent in nonpregnant women (<0.5% of CD4⁺ in 35 of total 44 controls). Importantly, detailed subpopulation analysis showed that the CD4^highCD25^high population contained few cells expressing the Treg markers Foxp3 and CD127^low as well as HLA-DR (Fig. 1⇑B, right panel). Thus, the CD4^highCD25^high population was more similar to activated CD4⁺CD25^dim cells than the 0.5% CD4⁺CD25^highest cells in regard to the expressions of Foxp3, CD127, and HLA-DR (Fig. 1⇑, B and E, right panels) and also with regard to high cytokine secretion and the lack of suppressive activity (see further below). To avoid inclusion of these apparent non-Tregs, a gate was set to include CD25^high cells with a lower expression of CD4; i.e., CD4^dimCD25^high (Fig. 1⇑C). This CD4^dimCD25^high population showed a high resemblance to the 0.5% CD4⁺CD25^highest cells, with high prevalence of Foxp3⁺, CD127^low, and HLA-DR⁺ cells (Fig. 1⇑, C and E, right panels). Therefore, CD4^dimCD25^high was considered the optimal definition of Tregs. To our surprise, the CD4^dimCD25^high population was reduced in size (in the percentage of CD4⁺ cells) in pregnant as compared with nonpregnant women (Fig. 1⇑C). We also considered a distinctly scattered population of CD25^+/high cells with low expression of CD4 (CD4^dimCD25^+/high; Fig. 1⇑D). This population, which formed a discrete cloud of cells in a CD4/CD25 plot (Fig. 1⇑D), comprised fewer Foxp3⁺, CD127^low, and HLA-DR⁺ cells and it was considered less appropriate than the CD4^dimCD25^high gate (Fig. 1⇑, C and D, right panels) for analysis of Tregs. Thus, taken together, the CD4^dimCD25^high population was considered the optimal definition of Tregs, and this gating strategy was applied in all of the following investigations of Treg frequency, phenotype, and function.

The CD4^dimCD25^high population is reduced in size during pregnancy

As shown both by four-color and six-color flow cytometry, the percentage of circulating CD4^dimCD25^high cells of CD4⁺ cells was in fact decreased in pregnancy (four-color: 1.2 ± 0.35% vs 1.9 ± 0.6%, p = 0.0005, Fig. 1⇑C; six-color: 1.5 ± 0.3% vs 2.8 ± 1.1%, p = 0.002). In addition, TruCount analysis of the absolute cell count confirmed these results, showing a decrease of the total CD4^dimCD25^high count per microliter of blood in pregnant compared with nonpregnant women (14 ± 4 cells/μl vs 21 ± 8 cells/μl; p = 0.01).

The CD4^dimCD25^high population shows a less regulatory and a more activated phenotype

Alongside the reduction of the CD4^dimCD25^high compartment in pregnancy, the CD4^dimCD25^high Tregs as well as the entire CD4⁺ population contained a lower proportion of cells expressing Foxp3 during pregnancy (Fig. 2⇓, A and B). Similar results were obtained in the CD4⁺CD25⁺ population as well as in the 0.5% CD4⁺CD25^highest population (p = 0.006 and p ≤ 0.0001, respectively; data not shown). Also, the Foxp3 expression intensity, expressed as geometric mean fluorescence intensity, was significantly reduced in the CD4⁺ as well as in the CD4^dimCD25^high population when comparing pregnant and nonpregnant women (p = 0.003 and p = 0.018, respectively; Fig. 2⇓D). In accordance with this, PBMC isolated from pregnant women showed a tendency toward lower Foxp3 mRNA expression than PBMC from nonpregnant women (relative values: 2.4 ± 0.7 vs 2.9 ± 0.8; p = 0.05) (Fig. 2⇓C).

• Download figure
• Open in new tab
• Download powerpoint

FIGURE 2.

Expression of intracellular Foxp3 protein and mRNA in PBMC from pregnant and nonpregnant women. A, Expression of Foxp3 protein in CD4⁺ cells (pregnant, n = 14; nonpregnant, n = 32). B, Expression of Foxp3 protein in CD4^dimCD25^high (pregnant, n = 14; nonpregnant, n = 32). C, Expression of Foxp3 mRNA in total PBMC (pregnant, n = 13; nonpregnant, n = 25) D, Foxp3 fluorescence intensity (pregnant, n = 14; nonpregnant, n = 33) where the peaks on the left are Foxp3⁻ lymphocytes (isotype controls) and the peaks on the right are Foxp3⁺CD4^dimCD25^high. This histogram overlay was constructed using FlowJo software (Tree Star) and the y-axis scale (percentage of maximum) is used to normalize for the number of events in the samples displayed. All bars represent means.

No changes in CTLA-4 expression were seen within the CD4^dimCD25^high population (data not shown). Because CD27, which has been suggested to be a marker for Tregs in inflamed tissues (30), was highly expressed throughout the entire CD4⁺ population and showed no specificity for Foxp3⁺ or CD4^dimCD25^high cells, it was excluded from further analysis (data not shown).

Further characterization by six-color flow cytometry showed that the Treg markers CD127^low (Fig. 3⇓A) and HLA-DR⁺ (Fig. 3⇓B) in the CD4^dimCD25^high Treg population were similarly expressed in pregnant and nonpregnant women. However, the frequency of HLA-DR⁺ cells was significantly lower in pregnant women in the entire CD4⁺ population (mean 2.6 ± 0.9 vs 4.1 ± 1.3%; p = 0.007) and in the 0.5% CD4⁺CD25^highest population (mean 53.1 ± 9.8 vs 67.8 ± 8.6%; p = 0.002). When looking at the activation markers CD45R0 (effector/memory) and CD45RA (naive), pregnant women showed an activated Treg phenotype with increased frequency of CD45R0⁺ and decreased frequency of CD45RA⁺ cells within the CD4^dimCD25^high population (Fig. 3⇓, C and D). No changes in the very early activation marker CD69 could be seen between pregnant and nonpregnant women, and the expression of this marker was generally very low throughout the entire CD4⁺ population (∼1%; data not shown).

• Download figure
• Open in new tab
• Download powerpoint

FIGURE 3.

Expression of CD127 (A), HLA-DR (B), CD45R0 (C), and CD45RA (D) in CD4^dimCD25^high cells from pregnant (n = 10) and nonpregnant (n = 10) women. All bars represent means.

The pregnancy-related changes in CD4^dimCD25^high Treg frequency and phenotype can be induced in vitro by 17β-estradiol and progesterone

PBMC from nonpregnant women were treated with 10 μM 17β-estradiol and/or 200 μM progesterone, which significantly lowered the frequency of CD4^dimCD25^high cells and Foxp3⁺ cells compared with untreated cells (Fig. 4⇓A). This reduction of the CD4^dimCD25^high population, most pronounced for progesterone treatment, was observed in both anti-CD3 stimulated and unstimulated (data not shown) cultures. However, in contrast to results from pregnant women, neither progesterone nor 17β-estradiol induced an expansion of the classical CD4⁺CD25^high nor the CD4^highCD25^high population (data not shown). Within the CD4^dimCD25^high population, 17β-estradiol and progesterone, alone or in combination, lowered the frequencies of Foxp3⁺, CTLA-4⁺, and HLA-DR⁺ cells (Fig. 4⇓B), whereas the expression of CD127 remained unchanged (data not shown). Again, these changes were prominently induced by progesterone and also seen in non-anti-CD3 stimulated cultures (data not shown). Furthermore, the effects of hormone treatments were similar in the secretory and proliferative phases of the menstrual cycle. The lower concentrations of hormones (10 nM and 100 nM for 17β-estradiol and 200 nM and 2 μM for progesterone) caused changes that followed the effects of the highest concentrations (10 μM 17β-estradiol and 200 μM progesterone) but were much less pronounced (not all changes were statistically significant; data not shown). Thus, only data from the highest concentrations are shown (Fig. 4⇓).

• Download figure
• Open in new tab
• Download powerpoint

FIGURE 4.

PBMC from nonpregnant women (n = 7) were stimulated with 10 μM 17β-estradiol (E) and 200 μM progesterone (P) in the presence of plate-bound 0.005 μg/ml anti-CD3 Ab for 3 days. A, Proportion of CD4^dimCD25^high and Foxp3⁺ cells in the CD4⁺ population. B, Proportion of Foxp3⁺, CTLA-4⁺, and HLA-DR⁺ cells within the CD4^dimCD25^high population. Bars represent means and SD. Significance-markers (∗) indicate differences between hormone stimulated and unstimulated cells. ∗, p ≤ 0.05; ∗∗, p ≤ 0.01; ∗∗∗, p ≤ 0.001.

CD4^dimCD25^high Tregs suppress secretion of IL-2, TNF-α, and IFN-γ but not IL-4 and IL-10, whereas CD4^highCD25^high cells are completely nonsuppressive

The functional effects of CD4^dimCD25^high Tregs from pregnant and nonpregnant women were evaluated in a system with plate-bound anti-CD28 (5 μg/ml) and anti-CD3 Abs at two different concentrations (suboptimal: 1 μg/ml; optimal: 5 μg/ml). Both concentrations generated results according to the same pattern, but because the optimal anti-CD3 concentrations gave higher and more reliable cytokine responses without abrogation of Treg-suppressive function, data from this stimulation are shown.

CD4^dimCD25^high Tregs from both pregnant and nonpregnant women suppressed the secretion of IL-2, TNF-α, and IFN-γ from CD4⁺CD25⁻ cells (Fig. 5⇓). Although the suppression appeared to be more pronounced with increasing numbers of CD4^dimCD25^high Tregs, no statistical differences were observed between the different Treg doses. Confirming their role as suppressors, CD4^dimCD25^high Tregs alone secreted less IL-2, TNF-α, and IFN-γ compared with CD4⁺CD25⁻ alone or when cocultured with CD4⁺CD25⁻ cells (Fig. 6⇓, A–C). Despite the reduced expression of Foxp3 in CD4^dimCD25^high Tregs in pregnancy, the ability of the Tregs to suppress IL-2, TNF-α, and IFN-γ secretion was similar in pregnant and nonpregnant women, both when analyzed as cytokine concentrations and as suppressive indexes.

• Download figure
• Open in new tab
• Download powerpoint

FIGURE 5.

The suppressive capacity of FACSAria-sorted CD4^dimCD25^high Tregs in coculture with autologous CD4⁺CD25⁻ responder cells from pregnant (n = 7–13) and nonpregnant (n = 14) women expressed as suppressive index (1 − (secretion in coculture/secretion from CD4⁺CD25⁻ cells alone)) × 100. Solid lines indicate median values and dotted lines indicate the estimated intra-assay variation (30%).

• Download figure
• Open in new tab
• Download powerpoint

FIGURE 6.

The secretion of IL-2 (A), TNF-α (B), IFN-γ (C), IL-4 (D), and IL-10 (E) from CD4⁺CD25⁻ cells alone, CD4^dimCD25^high Tregs alone, or in 1:1 combination. Dark gray filled bars show data from pregnant women (n = 13) and light gray filled bars show data from nonpregnant women (n = 14). Bars represent medians and the 75th percentile.

Tregs from pregnant and nonpregnant women did not suppress the secretion of IL-4 or IL-10 (Fig. 6⇑, D and E). In fact, supernatants from CD4⁺CD25⁻/Treg cocultures and Tregs alone contained significantly more IL-4 than supernatants from CD25⁻ cells cultured alone (Fig. 6⇑D). CD4⁺CD25⁻/Treg cocultures from pregnant women tended to produce more IL-4 than cocultures from nonpregnant women (p = 0.06). As for IL-4, CD4^dimCD25^high Tregs from pregnant, but not from nonpregnant women, tended to secrete more IL-10 than CD4⁺CD25⁻ cells alone (Fig. 6⇑E).

CD4^highCD25^high cells (Fig. 1⇑B) from pregnant women, defined as having higher CD4 expression than CD4^dimCD25^high Tregs (Fig. 1⇑C) yet falling into the classical CD4⁺CD25^high gate (Fig. 1⇑A), did not suppress the secretion of IL-2, TNF-α, and IFN-γ (Fig. 7⇓). Rather, CD4^highCD25^high cells secreted high levels of IL-2, TNF-α, and IFN-γ (Fig. 7⇓) as well as IL-4 (median, 1147 pg/ml; minimum, 87 pg/ml; maximum, 2413 pg/ml) and IL-10 (median, 2294 pg/ml; minimum, 15 pg/ml; maximum, 2574 pg/ml), hence contributing to an increase in the bulk amount of these cytokines in coculture with CD4⁺CD25⁻ cells. Thus, this confirmed the phenotypic data and further strengthened the notion that CD4^highCD25^high cells are not suppressive Tregs.

• Download figure
• Open in new tab
• Download powerpoint

FIGURE 7.

Lack of suppressive capacity in the pregnancy-associated population CD4^highCD25^high (n = 3; i.e., data is shown as minimum, maximum, and median values). The secretion of IL-2, TNF-α, and IFN-γ from CD4⁺CD25⁻ cells alone (far left) is suppressed by Tregs (CD4^dimCD25^high cells; second from left) but not by CD4^highCD25^high cells (second from right) in 2:1 (responder:suppressor) combination. The secretion from CD4^highCD25^high cells (25,000 cells, n = 1; or 6 250 cells, n = 2) alone are shown on the far right.