ORIGINAL ARTICLE
Maternal and Fetal Factors that Contribute to the Localization of T Regulatory Cells During Pregnancy Carrie M. Wambach1, Sonal N. Patel2, Daniel A. Kahn3 1
Division of Reproductive Endocrinology and Infertility, Department of OB/GYN, Cedars-Sinai Medical Center, Los Angeles, CA, USA; Division of Biology, California Institute of Technology, Pasadena, CA, USA; 3 Division of Maternal-Fetal Medicine, Department of OB/GYN, David Geffen School of Medicine, UCLA, Los Angeles, CA, USA 2
Keywords Allogeneic, cervix, interleukin-6, T regulatory, uteroplacental interface Correspondence Daniel A. Kahn, UCLA OB/GYN, Box 951740, 22-168 CHS, Los Angeles, CA 90095-1740, USA. E-mail: [email protected] Submission December 9, 2013; accepted February 5, 2014. Citation Wambach CM, Patel SN, Kahn DA. Maternal and fetal factors that contribute to the localization of T regulatory cells during pregnancy. Am J Reprod Immunol 2014; 71: 391–400 doi:10.1111/aji.12223
Problem To determine the interplay between fetal antigenicity and local maternal factors in determining reproductive tract T regulatory (Treg) cell accumulation during pregnancy. Method of study Examination of maternal Treg composition in the uterus, cervix, and uteroplacental interface (UPI) of murine syngeneic and allogeneic pregnancies and non-pregnant controls by flow cytometry. The impact of fetal antigenicity was defined by either fetal gender in syngeneic pregnancies or by allogeneic paternity. Impact of IL-6 on local Treg composition was determined using syngeneic pregnancies in IL-6/ females. Results An increased fraction of CD4+ T cells in the pregnant uterine lymphocytic infiltrate and draining pelvic lymph nodes are Tregs. Maternal IL-6 decreases Treg accumulation within the uterus and to a greater extent in the cervix in syngeneic pregnancy. Fetal antigenicity is matched by accumulation of Tregs to the UPI. Treg accumulation at the UPI of nonantigenic female fetuses is determined by the intrauterine position relative to male siblings. Conclusion Reproductive tract tissue Treg composition during pregnancy is influenced by maternal IL-6 and fetal antigenicity.
Introduction Failure of maternal immune tolerance of her semiallogeneic fetus results in poor pregnancy outcome. To protect her fetus from maternal immune attack, the activity of specialized CD4+ T cells that express the transcription factor FoxP3 (T regulatory cells) are normally invoked.1 T regulatory cells (Tregs) are essential for maintaining immune homeostasis, thus limiting autoimmunity.2 Pregnancy co-opts this fundamental mechanism to provide immune tolerance during pregnancy.3–6 Many groups have now shown American Journal of Reproductive Immunology 71 (2014) 391–400 ª 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
that interruption of Treg function during pregnancy leads to fetal antigen-specific poor pregnancy outcomes,3,6,7 thus highlighting the important role that these cells play in protecting the fetus from maternal immune attack. Our understanding of the conditions that govern the maternal Treg response during pregnancy is emerging. Experimental evidence points to Treg-mediated immune tolerance as a local phenomenon.8 Tregs have been shown to exert their suppressive effects by direct cell contact9 as well as through local soluble mediators such as IL-10.10 Thus, it is important 391
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to understand how Tregs accumulate at local sites where immune tolerance is needed, such as the maternal–fetal interface. Tregs are either thymically derived (natural Tregs) or are peripherally converted from na€ıve CD4+ T cells under special local conditions (peripheral Tregs).11 There are numerous differences between these two types of Tregs, although both exert suppressive effects.11 Specifically, it has been shown that na€ıve CD4+ T cells will become Tregs (express FoxP3) when exposed to T cell receptor stimulation in the context of local TGF-b.12 When inflammatory cytokines such as IL-6 or IL-1b are included in the local milieu, then na€ıve CD4+ T cells differentiate away from Tregs to become inflammatory TH17 cells.12 We, and others, have shown that pregnancy causes an expansion of peripheral Tregs in response to fetal antigens.3,5 As peripheral induction of na€ıve CD4+ T cells to become Tregs could occur within the target tissue, local tissue conditions [i.e., at the uteroplacental interface (UPI)] could play a role in determining local Treg accumulation. For example, an environment that is prone to inflammation could be less effective at fostering CD4+ T cell differentiation to Tregs.8,12 Thus, the development of maternal immune tolerance is hypothetically an interplay between local maternal conditions and the driving fetal antigens. Using a mouse-based model system, we sought to deconvolute the roles that local maternal factors and fetal antigenicity play in determining maternal Treg accumulation at the UPI. The localization of Tregs to the reproductive tract during pregnancy is complex, but using defined fetal antigens and genetically modified mice we were able to delineate the impact of local maternal inflammation and variable fetal antigenicity on local Treg accumulation. These observations provide a framework for understanding some of the driving factors behind the peripheral conversion of na€ıve CD4+ T cells to Tregs during pregnancy. Materials and methods Mice The colony was housed in a barrier facility used for transgenic mice generation. The female mice used were C57BL/6 (6–8 weeks old), C57BL/6-FoxP3GFP,13 and C57BL/6-IL-6/ (6–8 weeks old). The male mice used were C57BL/6 (8–12 weeks old) and Balb/c (8–12 weeks old). All mice were obtained from Jackson Laboratories (Bar Harbor, 392
ME, USA). Mice were subjected to syngeneic or allogeneic matings. Syngeneic matings: female C57BL/ 6 9 male C57BL/6, female C57BL/6-FoxP3eGFP 9 male C57BL/6-FoxP3-eGFP, or female C57BL/6IL-6/ 9 male C57BL/6. Allogeneic mating: female C57BL/6 or C57BL/6-FoxP3-eGFP 9 male Balb/c. Timed matings were defined from the day of vaginal plug observation and were denoted as days post-coitus (d.p.c.) = 0.5. Virginal (no mating) C57BL/6 female mice (6–8 weeks old) were used as controls. Mice were housed and handled in accordance with National Institutes of Health guidelines under Institutional Animal Care and Use Committeeapproved protocols (UCLA 2010-118-11). Tissue Lymphocyte Isolation Virgin or pregnant mice were euthanized at indicated gestational ages (d.p.c. = 12.5 or 18.5). Uteri were removed, and the number of fetuses was identified. Each fetal-placenta unit was isolated. Sharp dissection was used to isolate the maternal aspect of the decidua from each placenta. The cervix was also identified and carefully dissected from the uterine horns. To isolate lymphocytes, tissues from each placenta were individually processed by mechanical dissociation and enzymatic digestion. First, they were placed in a digestion solution [Hanks Balanced Salt Solution, 1.3 lM ethylenediaminetetraacetic acid (EDTA), 25 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) 100 U/mL collagenase, 0.1 mg/mL DNase I and 5 lM b-mercaptoethanol] and manually disrupted using a tissue homogenizer. Homogenized samples were incubated in the same buffer for 60 min at 37°C while shaking at 200 r.p.m. in an orbital incubator shaker (New Brunswick Scientific, Edison, NJ, USA). Samples were then strained to separate the lymphocytes from undigested tissue, and the strained lymphocytes were washed and re-suspended in a 44% Percoll solution. Percoll density gradient separation was then performed by layering cells over a 67% Percoll solution and centrifuging at 800 9 g at 20°C for 20 min. Purified lymphocytes were washed twice with phosphate-buffered saline (PBS), and cell pellets were re-suspended in 1% bovine serum albumin (BSA) in PBS in preparation for cell staining and flow cytometric analysis. The same protocol was applied to cervical and uterine tissues in non-pregnant controls. Inguinal and pelvic/periaortic lymph nodes were collected and single-cell suspensions were prepared by passing the tissue through a 70-lm polypropylene American Journal of Reproductive Immunology 71 (2014) 391–400 ª 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
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mesh cell strainer (BD) in PBS on ice. Spleens were harvested, and single-cell suspensions were prepared by passing the tissue through a metal screen (300 lm). Red blood cell (RBCs) were lysed by room temperature incubation in a hypotonic solution [0.83% NH4Cl, 0.02 M Tris (pH 7.6)]. Flow Cytometric Analysis Gating strategy was based on lymphoid tissues processed in parallel. To identify Tregs in C57BL/6 and C57BL/6-IL-6/ mice, fluorescently labeled monoclonal antibodies to CD4 [fluorescein isothiocyanate (FITC)] and FoxP3 (APC) (Biolegend, San Diego, CA, USA) and a FoxP3 staining kit (eBioscience, San Diego, CA, USA) were used. In C57BL/6-FoxP3-eGFP mice, Tregs were identified as a percentage of the CD4-APC-positive population. Extracellular and intracellular staining was done according to the manufacturer’s instructions. Analysis was performed on a BD Accuri C6 with post-acquisition analysis performed with Flowjo (Treestar, Palo Alto, CA, USA). Fetal Gender Identification Fetal tissue was placed in 75 lL Base Solution (0.025 M NaOH, 0.2 mM EDTA, pH 12) and incubated at 95°C for 30 min. To this was added 75 lL of Neutralization Solution (0.04 M Tris–HCl, pH 5.0). The supernatant was used as a DNA template in a polymerase chain reaction (PCR) reaction (Platinum Amplitaq; Invitrogen, Grand Island, NY, USA) with primers for murine SRY 5 0 -TTGTCTAGAGAGCAT GGAGGGCCATGTCAA-3 0 and 30 -AGCCTCCCGAT TTCACAGTGTCTCCTCACC-50 . In the same reaction, control primers for Fabpi 50 -CCTCCGGAGAGCAGCGATTAAAAGTGT-30 and 30 -TAGAGCTTTGCCACATC ACAGGTCATTCA-50 were included. The amplicons of SRY, 268 bp, and Fabpi, 455 bp were evaluated by agarose gel electrophoresis. Statistical Analysis Normality was determined using D’Agostino and Pearson omnibus test. Differences in normally distributed populations were statistically analyzed using unpaired Student’s t test or analysis of variance (ANOVA) with Bonferroni/Dunn post-test. Nonparametric analysis was done with a Mann–Whitney test. Analysis was accomplished with PRISM (Graphpad, La Jolla, CA, USA). American Journal of Reproductive Immunology 71 (2014) 391–400 ª 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Results Analysis of lymphocyte populations isolated from the reproductive tract required mechanical and enzymatic disruption. Identification of Tregs in uterine tissue as CD4+ FoxP3+ cells was done as shown in Fig. 1a. We found T lymphocytes to be an overall low frequency occurrence at the UPI in accordance with other published observations.14 Thus, we applied strict gating criteria based on isotype controls to the samples. In addition, the same gating strategy also identified CD4+ FoxP3+ cells more readily in lymphoid tissues (Fig. 1b). We did not find an effect of allogeneic pregnancy on the Treg composition of the CD4+ population in inguinal lymph nodes or spleen (Fig. 1c left and middle) in accordance with our previous observations in syngeneic pregnancies.6 In contrast to syngeneic pregnancies, we found a significant increase in the Treg composition in the uterine draining pelvic lymph nodes (Fig. 1c right) in allogeneic pregnancies. Local Treg Composition of the Uterocervical Lymphocyte Population Changes with Pregnancy Compared with non-pregnant controls, we found that the reproductive tract lymphocyte non-T cell population increases with pregnancy (data not shown) consistent with previously published observations.14,15 When examining the T-cell composition of the uterocervical lymphocyte populations, we found no significant change in the percentage of CD4+ T cells (Fig. 2a,b) as a consequence of pregnancy. When examined more closely, we found that the composition of the CD4+ population shifted within the uterine environment to significantly increase the percentage of cells that expressed FoxP3 (Fig. 2c), consistent with the notion of local immune suppression during pregnancy. Conversely, we found that within the lymphocyte population of the pregnant cervix, we found a significant decrease in the Treg composition (Fig. 2d). Local Maternal Inflammatory Response Drives Decreased Cervical Treg Composition The finding that the local intracervical Treg population decreases with pregnancy led us to consider local factors that could disrupt tissue differentiation of na€ıve CD4+ T cells into Tregs. Many lines of investigation have found that the cervix and uterus 393
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are subjected to ascending inflammatory stimuli from the vagina.16,17 It has been shown that na€ıve CD4+ T cells can differentiate into FoxP3-expressing Tregs under the local influence of TGF-b.12 Conversely, when the same na€ıve CD4+ T cells are exposed to TGF-b and IL-6, the result is differentiation away from Tregs towards the highly inflammatory TH17 cell.12 To address the hypothesis that normally occurring local maternal cervical inflammation (presumably in response to vaginal microflora) could include IL-6 production that would inhibit Treg accumulation, we evaluated uterocervical lymphocyte populations from wild type (IL-6+/+) and deficient (IL-6/) mice during syngeneic pregnancy. As we had seen previously, in wild-type mice there was a decrease in cervical Treg composition with wild-type pregnancy (Fig. 2e right). Consistent with our hypothesis, we found that the overall uterocervical Treg population increases in the 394
Fig. 1 Flow cytometric gating scheme for the identification of T regulatory cells in murine uterine tissues. (a) Uterine tissues were isolated by dissection from the underlying placental tissue from mice (C57BL/ 6) that had undergone timed allogeneic matings. Lymphocytes were isolated by collagenase digest followed by Percoll density centrifugation. Flow cytometric gating strategy involved identification of lymphocyte population by size/granularity, single cell discrimination; CD4 (fluorescein isothiocyanate)- and FoxP3 (APC)-positive lymphocyte population was identified by isotype controls. (b, c) Application of gating strategy to lymphoid organs from virgin (n = 8) C57BL/6 females or pregnant (n = 10) C57BL/6 females (d.p.c. = 18.5) that had undergone an allogeneic mating with a Balb/c male. Statistical difference determined by two tailed Mann–Whitney (N.S. non – significant) test.
absence of maternal IL-6 and that the difference between uterine and cervical Treg composition was abolished (Fig. 2e left). Fetal Antigenicity and Length of Gestation Influence Local Treg Accumulation at the Uteroplacental Interface The ‘strength’ of a particular tissue antigen has been ascribed to the time to which significant graft rejection occurs in a transplant.18 The maternal immune system during syngeneic pregnancies only has to cope with the presence of male fetuses due to the expression of a minor transplant antigen; H-Y.6 We have previously shown that during syngeneic pregnancies the mother develops a protective Treg response to H-Y.6 Similarly, others have shown that allogeneic pregnancies lead to protective Treg-mediated responses to those fetal antigens.3,5 As alloantigens American Journal of Reproductive Immunology 71 (2014) 391–400 ª 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
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Fig. 2 Helper T cell composition within the uterus and cervix changes with pregnancy. Uterine (n = 8) or cervical (n = 4) tissue was isolated from euthanized virgin C57BL/6 female mice (6–8 weeks old), from uterine tissue not in direct placental contact (n = 8), or from cervical tissue (n = 4) from pregnant C57BL/6 that had undergone a timed syngeneic mating (day 18.5 post observed vaginal plug). Lymphocytes were isolated by mechanical disruption of the tissue, followed by collagen digest and Percoll density centrifugation, and lastly stained for CD4 and FoxP3. Flow cytometric analysis was carried out for CD4 (a, b) or CD4/FoxP3 (c, d) with composition displayed as average percentage S.E.M. Statistical difference was determined by two tailed Mann–Whitney (N.S. non-significant) test. Maternal IL-6 production influences local distribution of Tregs within uterocervical tissues during pregnancy. Uterine or cervical tissue was isolated from euthanized pregnant (e) wildtype C57BL/6-IL-6+/+ female mice (6–8 weeks old; n = 8) and C57BL/6 – IL-6/ female mice (6–8 weeks old; n = 8) that had undergone a timed syngeneic mating (day 18.5 postobserved vaginal plug). Lymphocytes were isolated by mechanical disruption of the tissue, followed by collagenase digest and Percoll density centrifugation, and lastly stained for CD4 and FoxP3. Flow cytometric analysis was carried out for CD4/FoxP3 with composition displayed as average percentage S.E.M. Statistical difference determined one-way ANOVA with Bonferroni– Dunn post-test (*,†P < 0.01, N.S. nonsignificant).
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are more provocative in terms of tissue rejection than H-Y antigen, we hypothesized that more local Tregs would be found in allogeneic pregnancies than in synAmerican Journal of Reproductive Immunology 71 (2014) 391–400 ª 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
geneic pregnancies in the maternal uterine tissues in contact with the fetus. When we examined the lymphocytes isolated from the UPI, we found a significant 395
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increase in the percentage of Tregs in allogeneic pregnancies as compared with syngeneic pregnancies (Fig. 3). We were surprised to find that over the course of a syngeneic pregnancy there was a peak in uteroplacental Treg composition at mid-gestation (Fig. 3b). In contrast, highly antigenic (allogeneic) pregnancies had a sustained accumulation of Tregs evident from d.p.c. 12.5 onwards (Fig. 3b).
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Neighboring Antigenic Fetuses can Influence Local Treg Populations at the Uteroplacental Interface We found that allogeneic pregnancies have a greater Treg composition at the UPI than syngeneic pregnancies (Fig. 3a). On closer examination, we found that a small, but significant, increase in uteroplacental Tregs was found in syngeneic pregnancies involving the male fetuses, compared to female fetuses (Fig. 4a). This finding was consistent with previous observations regarding maternal Treg immune response to H-Y antigen during syngeneic pregnancies.6 Occasionally, we noticed increased Treg accumulation in the UPI of female fetuses in syngeneic matings. This finding led us to the hypothesis that Treg populations at male fetuses were spreading to neighboring female fetuses possibly through diffusion. In fact, when we considered the gender of flanking fetuses, we found that female uteroplacental lymphocyte populations with increasing numbers of neighboring males had increasing Treg percentages (Fig. 4b). In contrast, the degree to which male fetuses were isolated from neighboring males did not impact the local Treg population (Fig. 4c). Discussion In this study, we provide additional insight into the complexity of Treg accumulation at the UPI. We have found that the lymphocytic infiltrate characteristic of pregnancy changes in terms of the CD4+ T cell composition to include a greater percentage of Tregs. We find that maternal IL-6 plays a role in determining local Treg composition. Lastly, we find that the degree of fetal antigenicity plays a role in accumulation of Tregs to the UPI, and that local fetal antigenicity can influence bystander accumulation. Histologic evaluation of the UPI has found that a characteristic lymphocytic infiltrate, which includes a large number of uterine natural killer (NK) cells, occurs during pregnancy.15 CD4+ T cell subsets play many diverse roles in guiding the ‘theme’ of a local 396
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Fig. 3 Treg accumulation at the uteroplacental interface is influenced by fetal antigenicity. (a) Lymphocytes were isolated from the uterus directly opposite the placenta by careful dissection; tissues were from pregnant C57BL/6-FoxP3-eGFP females that had undergone a timed syngeneic (n = 31 uterine samples) mating with male C57BL/6 mice, or allogeneic (n = 10 uterine samples) mating with male Balb/c mice. Pregnant females were euthanized 18.5 days post-coitus (d.p.c.). (b) Treg composition of the uterine lymphocytes was determined from pregnant C57BL/6-FoxP3-eGFP mice that had undergone a timed syngeneic or allogeneic (Balb/c male) mating, and evaluated either as virgin (n = 8), d.p.c. 12.5 (n = 12), or d.p.c. 18.5 (n = 12). Lymphocytes were recovered from the maternal tissue by mechanical disruption, followed by collagenase digestion and Percoll density centrifugation, and lastly stained for CD4. Flow cytometric analysis was carried out for CD4/FoxP3 with composition displayed as average percentage S.E.M. Statistical difference determined by two-tailed unpaired Student’s t test.
immune response.8 Thus, we are not surprised to find that the overall CD4+ T cell percentage of the lymphocytic infiltrate does not change with pregnancy, but rather that the composition of the CD4+ population shifts to include a greater percentage of Tregs to meet the challenge of requisite immune tolerance. While not addressed in this study, the large increase in uterine NK cells during American Journal of Reproductive Immunology 71 (2014) 391–400 ª 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
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pregnancy may be a target of Treg-mediated local immune control. Recent evidence has suggested that Tregs can directly control NK cell activity.19 As uterine NK cells are phenotypically distinct from those found in the periphery, it is not clear if those findings are applicable.20 It is clear that uterine NK cells are necessary to support pregnancy, but retain the potential to cause pathology.20 Perhaps local Tregs modulate NK cell activity to favor a permissive environment for pregnancy. Experimental evidence supports the possibility that pregnancy invokes peripherally induced Tregs.3,21,22 We find that within the reproductive tract a variation in Treg composition exists, with fewer Tregs in the cervix as compared to the more distal uterus during pregnancy. As the vagina is heavily colonized under normal conditions, and the intrauterine environment is sterile, the microbial interface is at the
cervix. Furthermore, the cervix is an important site for priming of the maternal immune response to paternal (e.g., seminal fluid) antigens.23,24 Contained within this polymicrobial flora are inflammatory stimuli that play a role in modulating the normal maternal local immune response.16,17 One of the consequences of the ascending inflammatory stimuli could be the production of IL-6. We hypothesized that the observed decrease in Treg accumulation within the cervix compared with the uterus could be a result of local maternal IL-6 preventing local na€ıve CD4+ T cells from becoming Tregs. Our observation that a deficiency of maternal IL-6 abolishes the difference in cervical versus distal uterine Treg composition suggests that local maternal IL-6 is a determinant of Treg composition. Furthermore, we found that there was a significant increase in the uterine Treg composition in the IL-6/ mouse,
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Fig. 4 Local fetal antigen influences regional accumulation of Tregs at the uteroplacental interface (UPI). Treg composition of the UPI lymphocytes was determined from pregnant C57BL/6-FoxP3-eGFP mice that had undergone a timed syngeneic mating and evaluated at d.p.c. 18.5. Fetal gender was determined by PCR. Lymphocytes were recovered from the maternal tissue by mechanical disruption, followed by collagenase digestion and Percoll density centrifugation, and lastly stained for CD4. (a) Comparison of Treg accumulation at female UPI (n = 20) vs male UPI (n = 25). (b) Determination of influence of local males on Treg accumulation at non-antigenic (female) UPI, with zero males flanking (n = 2), one flanking male (n = 7), or two flanking males (n = 3). (c) Determination of influence of local males on Treg accumulation at antigenic (male) UPI, with zero males flanking (n = 2), one flanking male (n = 4), or two flanking males (n = 3). Flow cytometric analysis was carried out for CD4/FoxP3 with composition displayed as average percentage S.E.M. Statistical difference determined by (a) two-tailed unpaired Student’s t test or (b, c) non-parametric Kurskal–Wallis ANOVA with Dunn’s post test. American Journal of Reproductive Immunology 71 (2014) 391–400 ª 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
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suggesting that the effect of the ascending inflammatory stimulus reaches beyond the cervix. The reproductive behavior of the IL-6/ mouse is not entirely normal, notably in having increased fetal absorption and delayed parturition.25 Conversely, IL6 has been implicated in numerous pathologic pregnancies. Thus, our observations of IL-6 as a factor in determining local Treg composition may be part of a multifactorial response involving IL-6. Overall, our findings suggest that IL-6 plays a negative role in local Treg development. This finding would support the notion that local differentiation of na€ıve CD4+ T cells into Tregs is responsible for the differential composition in the cervix and distal uterus. We would further speculate that in the presence of IL-6, the reduced accumulation of Tregs is offset by an increase in local TH17 cells. In contrast to the reliable Treg identification through FoxP3 staining, TH17 identification has relied on quantification of IL-17 production or intranuclear ROR-cΤ staining, both of which require in vitro restimulation.26 We found that the small numbers of cells recovered from uterocervical tissues made such assays technically challenging. Furthermore, understanding potential alternate CD4+ subtypes’ fates will be important in future analyses. While the data here suggest that na€ıve CD4+ T cells differentiate in the local uteroplacental environment, our finding that there is a significant increase in the uterine draining lymph nodes’ (pelvic) Treg composition (Fig. 1c right) could reflect more centralized CD4+ T cell differentiation. As the IL-6/ mouse has delayed parturition and we find augmented levels of Tregs in the absence of IL-6, perhaps a natural diminishment in local maternal Tregs heralds the onset of labor by creating a local environment permissive for requisite inflammation. In fact, we find that in syngeneic matings, the peak of uterine Treg composition is at mid-gestation, with a diminishment by the conclusion of the pregnancy (Fig. 3b). In contrast, allogeneic pregnancy causes a persistence of local Treg enrichment. There doesn’t seem to be a difference in the timing of parturition between these two types of pregnancies, but perhaps the potency of the fetal allo-antigens is sufficient to promote inflammation-mediated initiation of the labor process, whereas in a syngeneic pregnancy, a diminishment of Tregs is required. Future exploration of this possibility in normal and pathologic pregnancies is needed. For example, an abnormal or premature decline in Treg composition at the UPI may be related to early increases in 398
inflammation (such as infection) and may contribute to pregnancy loss or preterm labor.27 While the fetus does not have a priori knowledge of its antigenicity, the relative degree of fetal antigenicity does play a role in determining the Treg composition at the UPI. This finding conveniently explains the hypothesis that a greater degree of immune tolerance must be invoked to protect highly antigenic fetuses from maternal immune attack. Thus, we find that the greatest amount of Treg accumulation occurs over allogeneic fetuses, followed by syngeneic males, and lastly syngeneic females, in part consistent with other reported observations.5 Variations in Treg composition of the UPI involving syngeneic females can be explained in part by the Treg composition of the neighboring UPI. The finding that increasing numbers of flanking males in syngeneic pregnancies increases the Treg composition of female UPIs is in line with other observations regarding the local influence of flanking males.28–30 Particularly, females flanked by males in utero have more ‘male’ characteristics than females not near males, with regards to size and subsequent behaviors. Such changes have been attributed to diffusion of local hormones characteristic of males.31 These observations provide additional insight into the complexity of local Treg accumulation. While driven by fetal antigenicity, other factors, such as neighboring antigenic environment, play a role in determining the cellular composition. Certainly, these specific data are unlikely to be directly applicable to human pregnancy as higher order multiple fetuses are uncommon, but other local conditions may influence the accumulation of maternal Tregs. The maintenance of immune tolerance at the UPI is essential for successful pregnancy outcomes. Understanding the factors that determine the local accumulation of Tregs at the UPI clarifies the natural mechanisms in place that provide immune tolerance. Immune-mediated poor pregnancy outcomes fall along a spectrum. That is, early disruption of tolerance likely leads to pregnancy loss (i.e., abortion),32 whereas later breakdown in tolerance leads to growth restriction and preterm birth.6,32 In line with this theoretical framework are reports of Treg transfers that prevent immune-mediated pregnancy loss,33,34 and that Tregs are essential to prepare the local environment to accept embryo implantation.35 Human pregnancy is more complex than rodent models, but many of the same principles of immune tolerance function in both scenarios. As reduced American Journal of Reproductive Immunology 71 (2014) 391–400 ª 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
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Treg composition at the UPI has been identified in pathologic human pregnancies (e.g., recurrent pregnancy loss36 and preeclampsia37,38), identification of specific disruptions known to influence local Treg accumulation may further our understanding. Lastly, pregnancy provides a physiologic situation in which immune tolerance is invoked and allows us to more fully appreciate the mechanisms that these cells employ to maintain immune homeostasis.
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Acknowledgments The authors wish to acknowledge the support of Harvey L. Karp Discovery Award, a gift from the Scholars in Translational Medicine Program, the California Institute of Technology – University of California, Los Angeles Joint Center for Translational Medicine, and the Reproductive Scientist Development Program NIH/NICHD 2K12HD000849-26 (Moley).
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generation of pathogenic effector TH17 and regulatory T cells. Nature 2006; 441:235–238. Lin W, Haribhai D, Relland LM, Truong N, Carlson MR, Williams CB, Chatila TA: Regulatory T cell development in the absence of functional Foxp3. Nat Immunol 2007; 8:359–368. Nancy P, Tagliani E, Tay CS, Asp P, Levy DE, Erlebacher A: Chemokine gene silencing in decidual stromal cells limits T cell access to the maternal-fetal interface. Science 2012; 336:1317–1321. Erlebacher A: Mechanisms of T cell tolerance towards the allogeneic fetus. Nat Rev Immunol 2013; 13:23–33. Cardenas I, Means RE, Aldo P, Koga K, Lang SM, Booth CJ, Manzur A, Oyarzun E, Romero R, Mor G: Viral infection of the placenta leads to fetal inflammation and sensitization to bacterial products predisposing to preterm labor. J Immunol 2010; 185:1248– 1257. Woudwyk MA, Monteavaro CE, Jensen F, Soto P, Barbeito CG, Zenclussen AC: Study of the uterine local immune response in a murine model of embryonic death due to Tritrichomonas foetus. Am J Reprod Immunol 2012; 68:128–137. Alpdogan O, van den Brink MR: Immune tolerance and transplantation. Semin Oncol 2012; 39:629–642. Gasteiger G, Hemmers S, Firth MA, Le Floc’h A, Huse M, Sun JC, Rudensky AY: IL-2-dependent tuning of NK cell sensitivity for target cells is controlled by regulatory T cells. J Exp Med 2013; 210:1167–1178. Winger EE, Reed JL: The multiple faces of the decidual natural killer cell. Am J Reprod Immunol 2013; 70:1–9. Teles A, Zenclussen AC, Schumacher A: Regulatory T cells are baby’s best friends. Am J Reprod Immunol 2013; 69:331–339. Teles A, Thuere C, Wafula PO, El-Mousleh T, Zenclussen ML, Zenclussen AC: Origin of Foxp3(+) cells during pregnancy. Am J Clin Exp Immunol 2013; 2:222–233. Sharkey DJ, Macpherson AM, Tremellen KP, Mottershead DG, Gilchrist RB, Robertson SA: TGF-beta mediates proinflammatory seminal fluid signaling in human cervical epithelial cells. J Immunol 2012; 189:1024–1035. Robertson SA, Prins JR, Sharkey DJ, Moldenhauer LM: Seminal fluid and the generation of regulatory T cells for embryo implantation. Am J Reprod Immunol 2013; 69:315–330. Prins JR, Gomez-Lopez N, Robertson SA: Interleukin-6 in pregnancy and gestational disorders. J Reprod Immunol 2012; 95:1– 14. Ivanov II, McKenzie BS, Zhou L, Tadokoro CE, Lepelley A, Lafaille JJ, Cua DJ, Littman DR: The orphan nuclear receptor RORgammat directs the differentiation program of proinflammatory IL-17+ T helper cells. Cell 2006; 126:1121–1133. Racicot K, Cardenas I, Wunsche V, Aldo P, Guller S, Means RE, Romero R, Mor G: Viral infection of the pregnant cervix predisposes to ascending bacterial infection. J Immunol 2013; 191:934–941. Hurd PL, Bailey AA, Gongal PA, Yan RH, Greer JJ, Pagliardini S: Intrauterine position effects on anogenital distance and digit ratio in male and female mice. Arch Sex Behav 2008; 37:9–18. Nagao T, Wada K, Kuwagata M, Nakagomi M, Watanabe C, Yoshimura S, Saito Y, Usumi K, Kanno J: Intrauterine position and postnatal growth in Sprague–Dawley rats and ICR mice. Reprod Toxicol 2004; 18:109–120. Morley-Fletcher S, Palanza P, Parolaro D, Vigano D, Laviola G: Intrauterine position has long-term influence on brain mu-opioid receptor density and behaviour in mice. Psychoneuroendocrinology 2003; 28:386–400.
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31 Ryan BC, Vandenbergh JG: Intrauterine position effects. Neurosci Biobehav Rev 2002; 26:665–678. 32 Shima T, Sasaki Y, Itoh M, Nakashima A, Ishii N, Sugamura K, Saito S: Regulatory T cells are necessary for implantation and maintenance of early pregnancy but not late pregnancy in allogeneic mice. J Reprod Immunol 2010; 85:121–129. 33 Zenclussen AC, Gerlof K, Zenclussen ML, Sollwedel A, Bertoja AZ, Ritter T, Kotsch K, Leber J, Volk HD: Abnormal T-cell reactivity against paternal antigens in spontaneous abortion: adoptive transfer of pregnancy-induced CD4+ CD25+ T regulatory cells prevents fetal rejection in a murine abortion model. Am J Pathol 2005; 166:811– 822. 34 Yin Y, Han X, Shi Q, Zhao Y, He Y: Adoptive transfer of CD4+ CD25+ regulatory T cells for prevention and treatment of spontaneous abortion. Eur J Obstet Gynecol Reprod Biol 2012; 161:177–181.
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35 Teles A, Schumacher A, Kuhnle MC, Linzke N, Thuere C, Reichardt P, Tadokoro CE, Hammerling GJ, Zenclussen AC: Control of uterine microenvironment by foxp3(+) cells facilitates embryo implantation. Front Immunol 2013; 4:158. 36 Sasaki Y, Sakai M, Miyazaki S, Higuma S, Shiozaki A, Saito S: Decidual and peripheral blood CD4+ CD25+ regulatory T cells in early pregnancy subjects and spontaneous abortion cases. Mol Hum Reprod 2004; 10:347–353. 37 Prins JR, Boelens HM, Heimweg J, Van der Heide S, Dubois AE, Van Oosterhout AJ, Erwich JJ: Preeclampsia is associated with lower percentages of regulatory T cells in maternal blood. Hypertens Pregnancy 2009; 28:300–311. 38 Santner-Nanan B, Peek MJ, Khanam R, Richarts L, Zhu E, Fazekas de St Groth B, Nanan R: Systemic increase in the ratio between Foxp3+ and IL-17-producing CD4+ T cells in healthy pregnancy but not in preeclampsia. J Immunol 2009; 183:7023–7030.
American Journal of Reproductive Immunology 71 (2014) 391–400 ª 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Maternal and Fetal Factors that Contribute to the Localization of T Regulatory Cells During Pregnancy Carrie M. Wambach1, Sonal N. Patel2, Daniel A. Kahn3 1
Division of Reproductive Endocrinology and Infertility, Department of OB/GYN, Cedars-Sinai Medical Center, Los Angeles, CA, USA; Division of Biology, California Institute of Technology, Pasadena, CA, USA; 3 Division of Maternal-Fetal Medicine, Department of OB/GYN, David Geffen School of Medicine, UCLA, Los Angeles, CA, USA 2
Keywords Allogeneic, cervix, interleukin-6, T regulatory, uteroplacental interface Correspondence Daniel A. Kahn, UCLA OB/GYN, Box 951740, 22-168 CHS, Los Angeles, CA 90095-1740, USA. E-mail: [email protected] Submission December 9, 2013; accepted February 5, 2014. Citation Wambach CM, Patel SN, Kahn DA. Maternal and fetal factors that contribute to the localization of T regulatory cells during pregnancy. Am J Reprod Immunol 2014; 71: 391–400 doi:10.1111/aji.12223
Problem To determine the interplay between fetal antigenicity and local maternal factors in determining reproductive tract T regulatory (Treg) cell accumulation during pregnancy. Method of study Examination of maternal Treg composition in the uterus, cervix, and uteroplacental interface (UPI) of murine syngeneic and allogeneic pregnancies and non-pregnant controls by flow cytometry. The impact of fetal antigenicity was defined by either fetal gender in syngeneic pregnancies or by allogeneic paternity. Impact of IL-6 on local Treg composition was determined using syngeneic pregnancies in IL-6/ females. Results An increased fraction of CD4+ T cells in the pregnant uterine lymphocytic infiltrate and draining pelvic lymph nodes are Tregs. Maternal IL-6 decreases Treg accumulation within the uterus and to a greater extent in the cervix in syngeneic pregnancy. Fetal antigenicity is matched by accumulation of Tregs to the UPI. Treg accumulation at the UPI of nonantigenic female fetuses is determined by the intrauterine position relative to male siblings. Conclusion Reproductive tract tissue Treg composition during pregnancy is influenced by maternal IL-6 and fetal antigenicity.
Introduction Failure of maternal immune tolerance of her semiallogeneic fetus results in poor pregnancy outcome. To protect her fetus from maternal immune attack, the activity of specialized CD4+ T cells that express the transcription factor FoxP3 (T regulatory cells) are normally invoked.1 T regulatory cells (Tregs) are essential for maintaining immune homeostasis, thus limiting autoimmunity.2 Pregnancy co-opts this fundamental mechanism to provide immune tolerance during pregnancy.3–6 Many groups have now shown American Journal of Reproductive Immunology 71 (2014) 391–400 ª 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
that interruption of Treg function during pregnancy leads to fetal antigen-specific poor pregnancy outcomes,3,6,7 thus highlighting the important role that these cells play in protecting the fetus from maternal immune attack. Our understanding of the conditions that govern the maternal Treg response during pregnancy is emerging. Experimental evidence points to Treg-mediated immune tolerance as a local phenomenon.8 Tregs have been shown to exert their suppressive effects by direct cell contact9 as well as through local soluble mediators such as IL-10.10 Thus, it is important 391
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to understand how Tregs accumulate at local sites where immune tolerance is needed, such as the maternal–fetal interface. Tregs are either thymically derived (natural Tregs) or are peripherally converted from na€ıve CD4+ T cells under special local conditions (peripheral Tregs).11 There are numerous differences between these two types of Tregs, although both exert suppressive effects.11 Specifically, it has been shown that na€ıve CD4+ T cells will become Tregs (express FoxP3) when exposed to T cell receptor stimulation in the context of local TGF-b.12 When inflammatory cytokines such as IL-6 or IL-1b are included in the local milieu, then na€ıve CD4+ T cells differentiate away from Tregs to become inflammatory TH17 cells.12 We, and others, have shown that pregnancy causes an expansion of peripheral Tregs in response to fetal antigens.3,5 As peripheral induction of na€ıve CD4+ T cells to become Tregs could occur within the target tissue, local tissue conditions [i.e., at the uteroplacental interface (UPI)] could play a role in determining local Treg accumulation. For example, an environment that is prone to inflammation could be less effective at fostering CD4+ T cell differentiation to Tregs.8,12 Thus, the development of maternal immune tolerance is hypothetically an interplay between local maternal conditions and the driving fetal antigens. Using a mouse-based model system, we sought to deconvolute the roles that local maternal factors and fetal antigenicity play in determining maternal Treg accumulation at the UPI. The localization of Tregs to the reproductive tract during pregnancy is complex, but using defined fetal antigens and genetically modified mice we were able to delineate the impact of local maternal inflammation and variable fetal antigenicity on local Treg accumulation. These observations provide a framework for understanding some of the driving factors behind the peripheral conversion of na€ıve CD4+ T cells to Tregs during pregnancy. Materials and methods Mice The colony was housed in a barrier facility used for transgenic mice generation. The female mice used were C57BL/6 (6–8 weeks old), C57BL/6-FoxP3GFP,13 and C57BL/6-IL-6/ (6–8 weeks old). The male mice used were C57BL/6 (8–12 weeks old) and Balb/c (8–12 weeks old). All mice were obtained from Jackson Laboratories (Bar Harbor, 392
ME, USA). Mice were subjected to syngeneic or allogeneic matings. Syngeneic matings: female C57BL/ 6 9 male C57BL/6, female C57BL/6-FoxP3eGFP 9 male C57BL/6-FoxP3-eGFP, or female C57BL/6IL-6/ 9 male C57BL/6. Allogeneic mating: female C57BL/6 or C57BL/6-FoxP3-eGFP 9 male Balb/c. Timed matings were defined from the day of vaginal plug observation and were denoted as days post-coitus (d.p.c.) = 0.5. Virginal (no mating) C57BL/6 female mice (6–8 weeks old) were used as controls. Mice were housed and handled in accordance with National Institutes of Health guidelines under Institutional Animal Care and Use Committeeapproved protocols (UCLA 2010-118-11). Tissue Lymphocyte Isolation Virgin or pregnant mice were euthanized at indicated gestational ages (d.p.c. = 12.5 or 18.5). Uteri were removed, and the number of fetuses was identified. Each fetal-placenta unit was isolated. Sharp dissection was used to isolate the maternal aspect of the decidua from each placenta. The cervix was also identified and carefully dissected from the uterine horns. To isolate lymphocytes, tissues from each placenta were individually processed by mechanical dissociation and enzymatic digestion. First, they were placed in a digestion solution [Hanks Balanced Salt Solution, 1.3 lM ethylenediaminetetraacetic acid (EDTA), 25 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) 100 U/mL collagenase, 0.1 mg/mL DNase I and 5 lM b-mercaptoethanol] and manually disrupted using a tissue homogenizer. Homogenized samples were incubated in the same buffer for 60 min at 37°C while shaking at 200 r.p.m. in an orbital incubator shaker (New Brunswick Scientific, Edison, NJ, USA). Samples were then strained to separate the lymphocytes from undigested tissue, and the strained lymphocytes were washed and re-suspended in a 44% Percoll solution. Percoll density gradient separation was then performed by layering cells over a 67% Percoll solution and centrifuging at 800 9 g at 20°C for 20 min. Purified lymphocytes were washed twice with phosphate-buffered saline (PBS), and cell pellets were re-suspended in 1% bovine serum albumin (BSA) in PBS in preparation for cell staining and flow cytometric analysis. The same protocol was applied to cervical and uterine tissues in non-pregnant controls. Inguinal and pelvic/periaortic lymph nodes were collected and single-cell suspensions were prepared by passing the tissue through a 70-lm polypropylene American Journal of Reproductive Immunology 71 (2014) 391–400 ª 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
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mesh cell strainer (BD) in PBS on ice. Spleens were harvested, and single-cell suspensions were prepared by passing the tissue through a metal screen (300 lm). Red blood cell (RBCs) were lysed by room temperature incubation in a hypotonic solution [0.83% NH4Cl, 0.02 M Tris (pH 7.6)]. Flow Cytometric Analysis Gating strategy was based on lymphoid tissues processed in parallel. To identify Tregs in C57BL/6 and C57BL/6-IL-6/ mice, fluorescently labeled monoclonal antibodies to CD4 [fluorescein isothiocyanate (FITC)] and FoxP3 (APC) (Biolegend, San Diego, CA, USA) and a FoxP3 staining kit (eBioscience, San Diego, CA, USA) were used. In C57BL/6-FoxP3-eGFP mice, Tregs were identified as a percentage of the CD4-APC-positive population. Extracellular and intracellular staining was done according to the manufacturer’s instructions. Analysis was performed on a BD Accuri C6 with post-acquisition analysis performed with Flowjo (Treestar, Palo Alto, CA, USA). Fetal Gender Identification Fetal tissue was placed in 75 lL Base Solution (0.025 M NaOH, 0.2 mM EDTA, pH 12) and incubated at 95°C for 30 min. To this was added 75 lL of Neutralization Solution (0.04 M Tris–HCl, pH 5.0). The supernatant was used as a DNA template in a polymerase chain reaction (PCR) reaction (Platinum Amplitaq; Invitrogen, Grand Island, NY, USA) with primers for murine SRY 5 0 -TTGTCTAGAGAGCAT GGAGGGCCATGTCAA-3 0 and 30 -AGCCTCCCGAT TTCACAGTGTCTCCTCACC-50 . In the same reaction, control primers for Fabpi 50 -CCTCCGGAGAGCAGCGATTAAAAGTGT-30 and 30 -TAGAGCTTTGCCACATC ACAGGTCATTCA-50 were included. The amplicons of SRY, 268 bp, and Fabpi, 455 bp were evaluated by agarose gel electrophoresis. Statistical Analysis Normality was determined using D’Agostino and Pearson omnibus test. Differences in normally distributed populations were statistically analyzed using unpaired Student’s t test or analysis of variance (ANOVA) with Bonferroni/Dunn post-test. Nonparametric analysis was done with a Mann–Whitney test. Analysis was accomplished with PRISM (Graphpad, La Jolla, CA, USA). American Journal of Reproductive Immunology 71 (2014) 391–400 ª 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Results Analysis of lymphocyte populations isolated from the reproductive tract required mechanical and enzymatic disruption. Identification of Tregs in uterine tissue as CD4+ FoxP3+ cells was done as shown in Fig. 1a. We found T lymphocytes to be an overall low frequency occurrence at the UPI in accordance with other published observations.14 Thus, we applied strict gating criteria based on isotype controls to the samples. In addition, the same gating strategy also identified CD4+ FoxP3+ cells more readily in lymphoid tissues (Fig. 1b). We did not find an effect of allogeneic pregnancy on the Treg composition of the CD4+ population in inguinal lymph nodes or spleen (Fig. 1c left and middle) in accordance with our previous observations in syngeneic pregnancies.6 In contrast to syngeneic pregnancies, we found a significant increase in the Treg composition in the uterine draining pelvic lymph nodes (Fig. 1c right) in allogeneic pregnancies. Local Treg Composition of the Uterocervical Lymphocyte Population Changes with Pregnancy Compared with non-pregnant controls, we found that the reproductive tract lymphocyte non-T cell population increases with pregnancy (data not shown) consistent with previously published observations.14,15 When examining the T-cell composition of the uterocervical lymphocyte populations, we found no significant change in the percentage of CD4+ T cells (Fig. 2a,b) as a consequence of pregnancy. When examined more closely, we found that the composition of the CD4+ population shifted within the uterine environment to significantly increase the percentage of cells that expressed FoxP3 (Fig. 2c), consistent with the notion of local immune suppression during pregnancy. Conversely, we found that within the lymphocyte population of the pregnant cervix, we found a significant decrease in the Treg composition (Fig. 2d). Local Maternal Inflammatory Response Drives Decreased Cervical Treg Composition The finding that the local intracervical Treg population decreases with pregnancy led us to consider local factors that could disrupt tissue differentiation of na€ıve CD4+ T cells into Tregs. Many lines of investigation have found that the cervix and uterus 393
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are subjected to ascending inflammatory stimuli from the vagina.16,17 It has been shown that na€ıve CD4+ T cells can differentiate into FoxP3-expressing Tregs under the local influence of TGF-b.12 Conversely, when the same na€ıve CD4+ T cells are exposed to TGF-b and IL-6, the result is differentiation away from Tregs towards the highly inflammatory TH17 cell.12 To address the hypothesis that normally occurring local maternal cervical inflammation (presumably in response to vaginal microflora) could include IL-6 production that would inhibit Treg accumulation, we evaluated uterocervical lymphocyte populations from wild type (IL-6+/+) and deficient (IL-6/) mice during syngeneic pregnancy. As we had seen previously, in wild-type mice there was a decrease in cervical Treg composition with wild-type pregnancy (Fig. 2e right). Consistent with our hypothesis, we found that the overall uterocervical Treg population increases in the 394
Fig. 1 Flow cytometric gating scheme for the identification of T regulatory cells in murine uterine tissues. (a) Uterine tissues were isolated by dissection from the underlying placental tissue from mice (C57BL/ 6) that had undergone timed allogeneic matings. Lymphocytes were isolated by collagenase digest followed by Percoll density centrifugation. Flow cytometric gating strategy involved identification of lymphocyte population by size/granularity, single cell discrimination; CD4 (fluorescein isothiocyanate)- and FoxP3 (APC)-positive lymphocyte population was identified by isotype controls. (b, c) Application of gating strategy to lymphoid organs from virgin (n = 8) C57BL/6 females or pregnant (n = 10) C57BL/6 females (d.p.c. = 18.5) that had undergone an allogeneic mating with a Balb/c male. Statistical difference determined by two tailed Mann–Whitney (N.S. non – significant) test.
absence of maternal IL-6 and that the difference between uterine and cervical Treg composition was abolished (Fig. 2e left). Fetal Antigenicity and Length of Gestation Influence Local Treg Accumulation at the Uteroplacental Interface The ‘strength’ of a particular tissue antigen has been ascribed to the time to which significant graft rejection occurs in a transplant.18 The maternal immune system during syngeneic pregnancies only has to cope with the presence of male fetuses due to the expression of a minor transplant antigen; H-Y.6 We have previously shown that during syngeneic pregnancies the mother develops a protective Treg response to H-Y.6 Similarly, others have shown that allogeneic pregnancies lead to protective Treg-mediated responses to those fetal antigens.3,5 As alloantigens American Journal of Reproductive Immunology 71 (2014) 391–400 ª 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
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Fig. 2 Helper T cell composition within the uterus and cervix changes with pregnancy. Uterine (n = 8) or cervical (n = 4) tissue was isolated from euthanized virgin C57BL/6 female mice (6–8 weeks old), from uterine tissue not in direct placental contact (n = 8), or from cervical tissue (n = 4) from pregnant C57BL/6 that had undergone a timed syngeneic mating (day 18.5 post observed vaginal plug). Lymphocytes were isolated by mechanical disruption of the tissue, followed by collagen digest and Percoll density centrifugation, and lastly stained for CD4 and FoxP3. Flow cytometric analysis was carried out for CD4 (a, b) or CD4/FoxP3 (c, d) with composition displayed as average percentage S.E.M. Statistical difference was determined by two tailed Mann–Whitney (N.S. non-significant) test. Maternal IL-6 production influences local distribution of Tregs within uterocervical tissues during pregnancy. Uterine or cervical tissue was isolated from euthanized pregnant (e) wildtype C57BL/6-IL-6+/+ female mice (6–8 weeks old; n = 8) and C57BL/6 – IL-6/ female mice (6–8 weeks old; n = 8) that had undergone a timed syngeneic mating (day 18.5 postobserved vaginal plug). Lymphocytes were isolated by mechanical disruption of the tissue, followed by collagenase digest and Percoll density centrifugation, and lastly stained for CD4 and FoxP3. Flow cytometric analysis was carried out for CD4/FoxP3 with composition displayed as average percentage S.E.M. Statistical difference determined one-way ANOVA with Bonferroni– Dunn post-test (*,†P < 0.01, N.S. nonsignificant).
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are more provocative in terms of tissue rejection than H-Y antigen, we hypothesized that more local Tregs would be found in allogeneic pregnancies than in synAmerican Journal of Reproductive Immunology 71 (2014) 391–400 ª 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
geneic pregnancies in the maternal uterine tissues in contact with the fetus. When we examined the lymphocytes isolated from the UPI, we found a significant 395
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increase in the percentage of Tregs in allogeneic pregnancies as compared with syngeneic pregnancies (Fig. 3). We were surprised to find that over the course of a syngeneic pregnancy there was a peak in uteroplacental Treg composition at mid-gestation (Fig. 3b). In contrast, highly antigenic (allogeneic) pregnancies had a sustained accumulation of Tregs evident from d.p.c. 12.5 onwards (Fig. 3b).
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Neighboring Antigenic Fetuses can Influence Local Treg Populations at the Uteroplacental Interface We found that allogeneic pregnancies have a greater Treg composition at the UPI than syngeneic pregnancies (Fig. 3a). On closer examination, we found that a small, but significant, increase in uteroplacental Tregs was found in syngeneic pregnancies involving the male fetuses, compared to female fetuses (Fig. 4a). This finding was consistent with previous observations regarding maternal Treg immune response to H-Y antigen during syngeneic pregnancies.6 Occasionally, we noticed increased Treg accumulation in the UPI of female fetuses in syngeneic matings. This finding led us to the hypothesis that Treg populations at male fetuses were spreading to neighboring female fetuses possibly through diffusion. In fact, when we considered the gender of flanking fetuses, we found that female uteroplacental lymphocyte populations with increasing numbers of neighboring males had increasing Treg percentages (Fig. 4b). In contrast, the degree to which male fetuses were isolated from neighboring males did not impact the local Treg population (Fig. 4c). Discussion In this study, we provide additional insight into the complexity of Treg accumulation at the UPI. We have found that the lymphocytic infiltrate characteristic of pregnancy changes in terms of the CD4+ T cell composition to include a greater percentage of Tregs. We find that maternal IL-6 plays a role in determining local Treg composition. Lastly, we find that the degree of fetal antigenicity plays a role in accumulation of Tregs to the UPI, and that local fetal antigenicity can influence bystander accumulation. Histologic evaluation of the UPI has found that a characteristic lymphocytic infiltrate, which includes a large number of uterine natural killer (NK) cells, occurs during pregnancy.15 CD4+ T cell subsets play many diverse roles in guiding the ‘theme’ of a local 396
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Fig. 3 Treg accumulation at the uteroplacental interface is influenced by fetal antigenicity. (a) Lymphocytes were isolated from the uterus directly opposite the placenta by careful dissection; tissues were from pregnant C57BL/6-FoxP3-eGFP females that had undergone a timed syngeneic (n = 31 uterine samples) mating with male C57BL/6 mice, or allogeneic (n = 10 uterine samples) mating with male Balb/c mice. Pregnant females were euthanized 18.5 days post-coitus (d.p.c.). (b) Treg composition of the uterine lymphocytes was determined from pregnant C57BL/6-FoxP3-eGFP mice that had undergone a timed syngeneic or allogeneic (Balb/c male) mating, and evaluated either as virgin (n = 8), d.p.c. 12.5 (n = 12), or d.p.c. 18.5 (n = 12). Lymphocytes were recovered from the maternal tissue by mechanical disruption, followed by collagenase digestion and Percoll density centrifugation, and lastly stained for CD4. Flow cytometric analysis was carried out for CD4/FoxP3 with composition displayed as average percentage S.E.M. Statistical difference determined by two-tailed unpaired Student’s t test.
immune response.8 Thus, we are not surprised to find that the overall CD4+ T cell percentage of the lymphocytic infiltrate does not change with pregnancy, but rather that the composition of the CD4+ population shifts to include a greater percentage of Tregs to meet the challenge of requisite immune tolerance. While not addressed in this study, the large increase in uterine NK cells during American Journal of Reproductive Immunology 71 (2014) 391–400 ª 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
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pregnancy may be a target of Treg-mediated local immune control. Recent evidence has suggested that Tregs can directly control NK cell activity.19 As uterine NK cells are phenotypically distinct from those found in the periphery, it is not clear if those findings are applicable.20 It is clear that uterine NK cells are necessary to support pregnancy, but retain the potential to cause pathology.20 Perhaps local Tregs modulate NK cell activity to favor a permissive environment for pregnancy. Experimental evidence supports the possibility that pregnancy invokes peripherally induced Tregs.3,21,22 We find that within the reproductive tract a variation in Treg composition exists, with fewer Tregs in the cervix as compared to the more distal uterus during pregnancy. As the vagina is heavily colonized under normal conditions, and the intrauterine environment is sterile, the microbial interface is at the
cervix. Furthermore, the cervix is an important site for priming of the maternal immune response to paternal (e.g., seminal fluid) antigens.23,24 Contained within this polymicrobial flora are inflammatory stimuli that play a role in modulating the normal maternal local immune response.16,17 One of the consequences of the ascending inflammatory stimuli could be the production of IL-6. We hypothesized that the observed decrease in Treg accumulation within the cervix compared with the uterus could be a result of local maternal IL-6 preventing local na€ıve CD4+ T cells from becoming Tregs. Our observation that a deficiency of maternal IL-6 abolishes the difference in cervical versus distal uterine Treg composition suggests that local maternal IL-6 is a determinant of Treg composition. Furthermore, we found that there was a significant increase in the uterine Treg composition in the IL-6/ mouse,
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Fig. 4 Local fetal antigen influences regional accumulation of Tregs at the uteroplacental interface (UPI). Treg composition of the UPI lymphocytes was determined from pregnant C57BL/6-FoxP3-eGFP mice that had undergone a timed syngeneic mating and evaluated at d.p.c. 18.5. Fetal gender was determined by PCR. Lymphocytes were recovered from the maternal tissue by mechanical disruption, followed by collagenase digestion and Percoll density centrifugation, and lastly stained for CD4. (a) Comparison of Treg accumulation at female UPI (n = 20) vs male UPI (n = 25). (b) Determination of influence of local males on Treg accumulation at non-antigenic (female) UPI, with zero males flanking (n = 2), one flanking male (n = 7), or two flanking males (n = 3). (c) Determination of influence of local males on Treg accumulation at antigenic (male) UPI, with zero males flanking (n = 2), one flanking male (n = 4), or two flanking males (n = 3). Flow cytometric analysis was carried out for CD4/FoxP3 with composition displayed as average percentage S.E.M. Statistical difference determined by (a) two-tailed unpaired Student’s t test or (b, c) non-parametric Kurskal–Wallis ANOVA with Dunn’s post test. American Journal of Reproductive Immunology 71 (2014) 391–400 ª 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
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suggesting that the effect of the ascending inflammatory stimulus reaches beyond the cervix. The reproductive behavior of the IL-6/ mouse is not entirely normal, notably in having increased fetal absorption and delayed parturition.25 Conversely, IL6 has been implicated in numerous pathologic pregnancies. Thus, our observations of IL-6 as a factor in determining local Treg composition may be part of a multifactorial response involving IL-6. Overall, our findings suggest that IL-6 plays a negative role in local Treg development. This finding would support the notion that local differentiation of na€ıve CD4+ T cells into Tregs is responsible for the differential composition in the cervix and distal uterus. We would further speculate that in the presence of IL-6, the reduced accumulation of Tregs is offset by an increase in local TH17 cells. In contrast to the reliable Treg identification through FoxP3 staining, TH17 identification has relied on quantification of IL-17 production or intranuclear ROR-cΤ staining, both of which require in vitro restimulation.26 We found that the small numbers of cells recovered from uterocervical tissues made such assays technically challenging. Furthermore, understanding potential alternate CD4+ subtypes’ fates will be important in future analyses. While the data here suggest that na€ıve CD4+ T cells differentiate in the local uteroplacental environment, our finding that there is a significant increase in the uterine draining lymph nodes’ (pelvic) Treg composition (Fig. 1c right) could reflect more centralized CD4+ T cell differentiation. As the IL-6/ mouse has delayed parturition and we find augmented levels of Tregs in the absence of IL-6, perhaps a natural diminishment in local maternal Tregs heralds the onset of labor by creating a local environment permissive for requisite inflammation. In fact, we find that in syngeneic matings, the peak of uterine Treg composition is at mid-gestation, with a diminishment by the conclusion of the pregnancy (Fig. 3b). In contrast, allogeneic pregnancy causes a persistence of local Treg enrichment. There doesn’t seem to be a difference in the timing of parturition between these two types of pregnancies, but perhaps the potency of the fetal allo-antigens is sufficient to promote inflammation-mediated initiation of the labor process, whereas in a syngeneic pregnancy, a diminishment of Tregs is required. Future exploration of this possibility in normal and pathologic pregnancies is needed. For example, an abnormal or premature decline in Treg composition at the UPI may be related to early increases in 398
inflammation (such as infection) and may contribute to pregnancy loss or preterm labor.27 While the fetus does not have a priori knowledge of its antigenicity, the relative degree of fetal antigenicity does play a role in determining the Treg composition at the UPI. This finding conveniently explains the hypothesis that a greater degree of immune tolerance must be invoked to protect highly antigenic fetuses from maternal immune attack. Thus, we find that the greatest amount of Treg accumulation occurs over allogeneic fetuses, followed by syngeneic males, and lastly syngeneic females, in part consistent with other reported observations.5 Variations in Treg composition of the UPI involving syngeneic females can be explained in part by the Treg composition of the neighboring UPI. The finding that increasing numbers of flanking males in syngeneic pregnancies increases the Treg composition of female UPIs is in line with other observations regarding the local influence of flanking males.28–30 Particularly, females flanked by males in utero have more ‘male’ characteristics than females not near males, with regards to size and subsequent behaviors. Such changes have been attributed to diffusion of local hormones characteristic of males.31 These observations provide additional insight into the complexity of local Treg accumulation. While driven by fetal antigenicity, other factors, such as neighboring antigenic environment, play a role in determining the cellular composition. Certainly, these specific data are unlikely to be directly applicable to human pregnancy as higher order multiple fetuses are uncommon, but other local conditions may influence the accumulation of maternal Tregs. The maintenance of immune tolerance at the UPI is essential for successful pregnancy outcomes. Understanding the factors that determine the local accumulation of Tregs at the UPI clarifies the natural mechanisms in place that provide immune tolerance. Immune-mediated poor pregnancy outcomes fall along a spectrum. That is, early disruption of tolerance likely leads to pregnancy loss (i.e., abortion),32 whereas later breakdown in tolerance leads to growth restriction and preterm birth.6,32 In line with this theoretical framework are reports of Treg transfers that prevent immune-mediated pregnancy loss,33,34 and that Tregs are essential to prepare the local environment to accept embryo implantation.35 Human pregnancy is more complex than rodent models, but many of the same principles of immune tolerance function in both scenarios. As reduced American Journal of Reproductive Immunology 71 (2014) 391–400 ª 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
LOCALIZATION OF TREGS TO THE UPI
Treg composition at the UPI has been identified in pathologic human pregnancies (e.g., recurrent pregnancy loss36 and preeclampsia37,38), identification of specific disruptions known to influence local Treg accumulation may further our understanding. Lastly, pregnancy provides a physiologic situation in which immune tolerance is invoked and allows us to more fully appreciate the mechanisms that these cells employ to maintain immune homeostasis.
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Acknowledgments The authors wish to acknowledge the support of Harvey L. Karp Discovery Award, a gift from the Scholars in Translational Medicine Program, the California Institute of Technology – University of California, Los Angeles Joint Center for Translational Medicine, and the Reproductive Scientist Development Program NIH/NICHD 2K12HD000849-26 (Moley).
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