Cells During Early Pregnancy in Mice Activation Competence in Uterine

The Journal of Immunology

GM-CSF Is an Essential Regulator of T Cell Activation Competence in Uterine Dendritic Cells during Early Pregnancy in Mice

Lachlan M. Moldenhauer,* Sarah N. Keenihan,* John D. Hayball,†,‡ and Sarah A. Robertson*

Uterine dendritic cells (DCs) are critical for activating the T cell response mediating maternal immune tolerance of the semialloge- neicfetus. GM-CSF (CSF2), a known regulator of DCs, is synthesized by uterine epithelial cells during induction of tolerance in early pregnancy. To investigate the role of GM-CSF in regulating uterine DCs and macrophages, Csf2-null mutant and wild-type mice were evaluated at estrus, and in the periconceptual and peri-implantation periods. Immunohistochemistry showed no effect of GM-CSF deficiency on numbers of uterine CD11c+ cells and F4/80+ macrophages at estrus or on days 0.5 and 3.5 postcoitum, but MHC class II+ and class A scavenger receptor+ cells were fewer. Flow cytometry revealed reduced CD80 and CD86 expression by uterine CD11c+ cells and reduced MHC class II in both CD11c+ and F4/80+ cells from GM-CSF–deficient mice. CD80 and CD86 were induced in Csf22/2 uterine CD11c+ cells by culture with GM-CSF. Substantially reduced ability to activate both CD4+ and CD8+ T cells in vivo was evident after delivery of OVA Ag by mating with Act-mOVA males or transcervical administration of OVA peptides. This study shows that GM-CSF regulates the efficiency with which uterine DCs and macrophages activate T cells, and it is essential for optimal MHC class II- and class I-mediated indirect presentation of reproductive Ags. Insufficient GM-CSF may impair generation of T cell-mediated immune tolerance at the outset of pregnancy and may contribute to the altered DC profile and dysregulated T cell tolerance evident in infertility, miscarriage, and preeclampsia. The Journal of Immunology, 2010, 185: 7085–7096.

endritic cells (DCs) have emerged as a critical cell lineage pression of cell surface markers suggests ontogenic relationships in the events underpinning successful embryo implan- between the two cell lineages, with expression of class II MHC, D tation and establishment of pregnancy, through their roles scavenger receptors including macrosialin and class A scavenger in decidual tissue remodeling and angiogenesis, and in the in- receptor (MSR1), sialoadhesin, and CD86 indicating that both duction of maternal–fetal tolerance (1, 2). This has led to a focus DCs and activated macrophages might act as APCs for uterine on identifying the factors that regulate DCs in the uterine endo- Ags (6). metrium, since deficiency in these cells is implicated in forms of In the uterus as in other mucosal tissues, the recruitment and infertility where the endometrium is not receptive to implanting function of DCs and macrophages appear to be regulated by a range embryos, or in preeclampsia where immune tolerance of the pla- of cytokines and chemokines, potentially including the 23-kDa centa is insufficient (3–5). DCs residing within the uterus express glycoprotein GM-CSF (also known as CSF2), which is a known CD11c and show a similar diversity in phenotype to analogous- regulator of DCs in other tissues. In the female reproductive cells in other mucosal tissues, but they vary in abundance and tract, epithelial cells lining the uterus are a potent source of relative composition at different times of the estrous cycle and GM-CSF in mice (8) and in women (9). Uterine epithelial cell during pregnancy, presumably to accommodate the changing Csf2 expression and GM-CSF protein synthesis fluctuate during functional demands of this tissue (6, 7). Most uterine DCs are the course of the ovarian cycle, under the positive regulatory in- CD8a2, but CD8a+ DCs are also present in early pregnancy (7). fluence of estrogen and inhibition by progesterone (10). If coitus Uterine DCs are accompanied by a large and fluctuating pop- occurs, expression of Csf2 is further upregulated when factors ulation of macrophages, and their heterogeneous pattern of ex- in the male seminal fluid, including TGFB1, bind receptors on luminal epithelial cells lining the cervix and uterus (11). This surge in GM-CSF production together with other cytokines and *Research Centre for Reproductive Health, School of Paediatrics and Reproductive chemokines induced by seminal fluid trigger a controlled in- Health, University of Adelaide; †Sansom Institute, School of Pharmacy and Medical Sciences, University of South Australia; and ‡Experimental Therapeutics Laboratory, flammation-like response within the uterine endometrium, asso- Hanson Institute, SA Pathology, Adelaide, Australia ciated with a further influx of DCs as well as neutrophils and Received for publication April 26, 2010. Accepted for publication September 22, macrophages, which persist in the stroma for several days before 2010. declining in number around the time of embryo implantation This work was supported in part by a Program grant and Fellowship from the (12–14). The DCs and macrophages recruited into the uterus after National Health and Medical Research Council, Australia. coitus express the a- and b-subunits of the GM-CSFR (15). A Address correspondence and reprint requests to Dr. Sarah A. Robertson, Research central role for GM-CSF in controlling their infiltration is in- Centre for Reproductive Health, School of Paediatrics and Reproductive Health, Uni- versity of Adelaide, Adelaide, SA 5005 Australia. E-mail address: sarah.robertson@ dicated by experiments showing that both DCs and macrophages adelaide.edu.au are attracted into the uterine stroma after in utero administration of Abbreviations used in this paper: DAB, diaminobenzidine tetrachloride; DC, exogenous GM-CSF to ovariectomized mice (15). Dysregulation dendritic cell; En, endometrium; LE, luminal epithelium; LN, lymph node; pc, post- of the uterine immune response may contribute to the mechanism coitum; PI, proliferation index; Treg, T regulatory; WT, wild-type. by which Csf2-null mutation leads to reproductive defects, with Copyright Ó 2010 by The American Association of Immunologists, Inc. 0022-1767/10/$16.00 a high rate of fetal loss particularly in male progeny (16, 17). www.jimmunol.org/cgi/doi/10.4049/jimmunol.1001374 7086 GM-CSF REGULATION OF UTERINE DENDRITIC CELLS

There are several lines of evidence implicating GM-CSF as mutation in the Csf2 gene (Csf22/2 mice) were generated using gene a regulator of DCs and macrophages in their ability to activa- targeting techniques in 129/OLA-derived E14 embryonic stem cells and were propagated from founder mice by mating with C57BL/6J mice as te T cell responses. GM-CSF influences DC and macrophage re- 2 2 previously described (26). Csf2 / breeding pairs were provided by cruitment into inflammatory sites, with transgenic overexpression A. Dunn (Ludwig Institute for Cancer Research, Melbourne, Australia). of Csf2 in tumor cells eliciting DC recruitment in vivo (18). The genotype of Csf22/2 and wild-type control mice were confirmed by GM-CSF is also important for effective Ag processing and pre- PCR of DNA extracted from blood or tail tissue of breeding pairs and senting function in APCs, with in vitro experiments showing that offspring. TCR transgenic C57BL/6 OT-I mice (37) (provided by W. Heath, Walter and Eliza Hall Institute for Medical Research, Melbourne, GM-CSF facilitates Ag uptake (19) and expression of MHC class Australia, and F. Carbone, Melbourne University, Melbourne, Australia) II and the costimulatory molecules CD80 and CD86 (20–22), and were crossed with B6.SJL-PtprcaPep3b/BoyJArc mice (Animal Resource this is linked with increased T cell activation (18, 20, 23, 24) and Centre, Perth, Western Australia) to generate OT-I mice with the CD45.1 + T cell-dependent Ab production (25). Experiments in Csf2-null marker (OT-I mice). The TCR of the CD8 OT-I T cells recognizes the mutant mice have demonstrated that although macrophage and chicken OVApeptide OVA257–264 (SIINFEKL) presented in the context of H- 2Kb MHC class I. Transgenic OT-II mice (also provided by W. Heath and F. DC populations are essentially intact when GM-CSF is absent (26, Carbone) express the TCR on CD4+ T cells that is reactive to the chicken 27), this cytokine influences mature macrophage and DC function OVA epitope OVA323–339 (ISQAVHAAHAEINEAGR), presented in the to affect the incidence and severity of some inflammatory and context of C57BL/6 I-Ab MHC class II. The transgenic C57BL/6 Act-mOVA autoimmune diseases, including arthritis, multiple sclerosis, glo- mice, which ubiquitously express a membrane-bound variant of OVA under control of the ActB promoter (Act-mOVA mice) (38), were provided by merulonephritis, and LPS-induced lung inflammation (28). The Dr. M. K. Jenkins (University of Minnesota, Minneapolis, MN). Act-mOVA impact of GM-CSF on the kinetics and outcome of inflammatory male mice heterozygous for OVAtransgene expression were bred from B6 3 and immune responses appears to depend on the tissue site and Act-mOVA intercrosses and identified by the presence of OVA protein eliciting stimulus. For example, in T cell-dependent peritonitis, detected by flow cytometry on peripheral blood cells using biotinylated rabbit but not in thioglycollate-elicited peritonitis, macrophage activa- anti-OVA Ab (Rockland Immunochemicals, Gilbertsville, PA). For experimental matings, adult (6–12 wk old) female wild-type or tion, phagocytosis, and Ag-presenting function are compromised Csf22/2 mice were placed one to three per cage with individual stud B6 or when GM-CSF is deficient (29). Act-mOVA males of proven fertility. The day of sighting of a vaginal plug The uterine response to pregnancy requires induction of a T cell (checked between 0800 and 1000 h) was designated day 0.5 postcoitum response, with paternal Ag-specific T cells within the maternal (pc). Mated females were removed from males and housed in groups of two to five females per cage. T cell repertoire showing evidence of Ag encounter from early in Some mated female mice were administered OVA257–264 or OVA323–339 pregnancy, even before embryo implantation occurs (30). This delivered into the uterus transcervically at 0900–1100 h on day 0.5 pc. The T cell response is activated at conception (31), when paternal Ags vaginal plug was removed under isofluorothane-induced anesthesia, and present in seminal fluid are presented indirectly by maternal bone the peptide was administered transcervically using a 3.5 Fr Tom Cat marrow-derived Ag-presenting cells to CD4+ and CD8+ T cells in catheter (Tyco Healthcare Group, Mansfield, MA) inserted through the cervix and into the uterus, positioned with the aid of a lubricated auroscope the para-aortic lymph nodes (LNs) draining the uterus (32). In (Heine Optotechnik, Herrsching, Germany). The University of Adelaide normal healthy pregnancies, activated paternal Ag-reactive T cells Animal Ethics Committee approved all experiments. do not develop antifetal immunity and instead become function- Abs ally anergic or hyporesponsive, leading to tolerance of the conceptus and also tissue grafts expressing paternal Ags (30, 33, 34). Rat mAbs 2F8 (reactive with scavenger receptor class A [MSR1, CD204]), Tolerance also requires expansion of the CD4+CD25+Foxp3+ FA11 (reactive with macrosialin), and 3D6 (reactive with sialoadhesin) were T regulatory (Treg) cell pool, and sufficient numbers of Treg cells provided as tissue culture supernatants by S. Gordon (University of Oxford, Oxford, U.K.). Rat mAbs as tissue culture supernatants generated from with paternal Ag specificity are essential for normal embryo im- hybridomasobtainedfrom the AmericanTypeCultureCollection(Rockville, plantation (35, 36). MD) were TIB120 (reactive with MHC class II) and F4/80. Biotin conju- The aim of this study was to use Csf2-null mutant mice to gation of TIB120 was performed in-house. mAb GL1 (reactive with CD86) determine the physiological role of GM-CSF in the recruitment wasobtainedfrom BD Pharmingen(San Jose, CA).Biotinlylatedanit-CD11c was obtained from eBioscience (San Diego, CA). HRP-conjugated goat anti- and/or activation and maturation of DCs and macrophages in the rat Ig, biotin-conjugated rabbit anti-rat Ig, and streptavidin-conjugated uterus during early pregnancy, and to define whether GM-CSF is HRP were obtained from Dako (Copenhagen, Denmark). For flow cytom- required for these uterine APC populations to present paternal etry, cells were labeled with PE-conjugated anti-F4/80 plus Alexa Fluor 488- seminal fluid Ag to activate the maternal T cell repertoire for conjugated anti-CD11c and then either allophycocyanin-conjugated anti- pregnancy tolerance. We show that although GM-CSF is not es- MHC class II (all eBioscience) and biotinylated anti-MSR1 (CD204; Serotec, Raleigh, NC) followed by PE-Cy5–conjugated streptavidin (BD sential for DC and macrophage recruitment into the uterus during Pharmingen) or, alternatively, allophycocyanin-conjugated anti-CD80 the estrous cycle or in the postcoital inflammatory response, these and biotinylated anti-CD86 (both from BD Pharmingen) followed by PE- cells express less MHC class II and MSR1 in the absence of Cy5–conjugated streptavidin. OVA-reactive T cells were labeled with GM-CSF. This phenotypic change is associated with reduced PE-conjugated anti-CD8a (BD Pharmingen), allophycocyanin-conjugated anti-CD45.1 (eBioscience), or PE-conjugated anti-CD4a (BD Pharmingen) capacity to present paternal Ags via MHC class II and cross- for 30 min at 4˚C in FACS buffer, followed by PE-Cy5–conjugated strep- + + presentation via MHC class I to Ag-specific CD4 and CD8 tavidin (BD Pharmingen). T cells. Impaired priming of paternal Ag-specific T cells may inhibit generation of maternal immune tolerance for pregnancy, Immunohistochemistry and it is likely to contribute to the reproductive defects seen in Immunohistochemistry was performed as previously described (6). Briefly, Csf2-null mutant mice and potentially in infertile and subfertile uteri freshly excised from estrous, day 0.5 pc, or day 3.5 pc mice were women. embedded vertically in OCT compound (Tissue Tex, Naperville, IL) and frozen immediately by immersion in liquid nitrogen-cooled isopentane. Air-dried tissue sections (7 mm thick) were fixed in ethanol and labeled Materials and Methods sequentially with mAbs, biotin-conjugated rabbit anti-rat Ig, and HRP- Mice conjugated streptavidin. Within an experiment, uterine sections from all mice were processed together and control sections were incubated in PBS All mice were bred and maintained in a pathogen-free facility at the with 10% normal mouse serum or isotype-matched mAbs. Bound HRP University of Adelaide Medical School Animal House on a 12-h light/dark was localized on sections with diaminobenzidine tetrachloride (DAB) cycle and were administered food and water ad libitum. Female and male (Sigma-Aldrich, St. Louis, MO). After counterstaining in Gill’s hema- wild-type C57BL/6 (H-2Kb) mice were bred in-house. Mice with a null toxylin (Sigma-Aldrich), sections were dehydrated in absolute ethanol, The Journal of Immunology 7087 cleared in Safsolvent (Ajax Chemicals, Auburn, Australia), and mounted Results in DePeX (BDH Laboratory Supplies, Poole, U.K.). Full-section high- The effect of GM-CSF deficiency on the abundance and resolution images were made using a NanoZoomer slide scanner (Hama- matsu Photonics, Hamamatsu City, Japan). DAB-labeled cells in the distribution of uterine DCs and macrophages at estrus and endometrial stroma of duplicate sections from each uterus were quantified during early pregnancy by video image analysis using VideoPro software (Faulding Imaging, Adelaide, Australia). The mean percentage positivity value [(mean area of To investigate the effect of GM-CSF deficiency on uterine DCs DAB stain/mean area of hematoxylin + DAB stain) 3 100] of $10 fields and macrophages, mAbs specific for the DC lineage marker CD11c, per section was calculated. Eosinophils were identified as endogenous the macrophage lineage marker F4/80, and the DC and macrophage + peroxidize cells present in sections stained with DAB in the absence of activation markers MHC class II, macrosialin, MSR1, sialoadhesion, primary Ab or detection reagents. and CD86 were used to compare the morphology, distribution, and Flow cytometric analysis of uterine DCs and macrophages abundance of mAb-reactive cells in uterine tissue from wild-type 2/2 ∼ Uteri were collected aseptically on day 0.5 pc and were trimmed of and Csf2 mice collected at estrus, on day 0.5 pc ( 12hafter mesentery and fat, slit longitudinally, and minced extensively using fine coitus), and on day 3.5 pc (just prior to blastocyst implantation) scissors. The tissue fragments of individual uteri were stirred gently at room (n = 5/group). temperature for 1 h in 2 ml/uterus collagenase/DNase (1 mg/ml type I In wild-type uterine tissue collected at estrus, CD11c+ cells were collagenase [Sigma-Aldrich] and 2.5 mg/ml DNase I [Sigma-Aldrich] in RPMI 1640 [Sigma-Aldrich] with 10% heat-inactivated FCS [JRH Bio- sparsely distributed within the endometrial stromal tissue (Fig. 1A, + sciences, Lenexa, KS]), 0.015 M HEPES (Sigma-Aldrich), and 0.05% 1B) whereas F4/80 macrophages were more abundant (Fig. 1E, sodium azide (Sigma-Aldrich), with disruption of tissue clumps by manual 1F). There was no effect of GM-CSF deficiency on the number 3 pipetting. Cells were then washed twice in ice-cold FACS buffer (1 PBS/ of either cell lineage (Fig. 2A,2B). In contrast, MHC class II+ 0.1% BSA/0.5% sodium azide), filtered through 70-mm mesh, and labeled 2/2 with flurophore-conjugated Abs to detect F4/80, CD11c, MSR1, MHC cells were reduced by 43% in Csf2 mice (p = 0.02) (Figs. 1I, class II, CD80 and CD86, or isotype-matched control mAbs for 30 min at 1J,2C). Cells expressing macrosialin, MSR1, or sialoadhesin 4˚C, followed by two washes in 2 ml of ice-cold FACS buffer after each were comparable in abundance and distribution to F4/80+ mac- labeling step. Labeled cells were analyzed by flow cytometry using the rophages, whereas CD86 was expressed by very few cells. There FACSCanto and FACSDiva software (BD Biosciences, San Jose, CA). was no effect of GM-CSF deficiency on the distribution or abun- Gates were established to exclude dead cells and debris. MHC class II, MSR1, CD80, and CD86 expression were assessed within the F4/80+ and dance of cells expressing these markers or on the population of CD11c+ populations. eosinophils, which were detected as endogenous peroxidize+ cells Adoptive transfer of OVA-reactive OT-I and OT-II TCR (Fig. 2H). Cells expressing these markers were also present in the transgenic T cells myometrial and mesometrial triangle regions of the uterus of both wild-type and Csf22/2 mice but were generally fewer in number Lymphocytes were prepared from OT-I and OT-II female mice by sterile than in the endometrium (data not shown). excision of LNs and mechanical dispersion between glass microscope + slides. OT-I and OT-II cells were suspended at 106 cells/ml in RPMI 1640 After coitus on day 0.5 pc, the numbers of CD11c cells were containing 10% FCS, 5 3 1025 M 2-ME, penicillin, and streptomycin increased 3.0-fold in the uterine endometrium of wild-type mice (RPMI-FCS) containing 0.1 ml of 5 mM CFSE (Molecular Probes, Eugene, and a similar 2.1-fold increase was seen in Csf22/2 mice (Figs. OR) in DMSO and incubated for 10 min at 37˚C. After washing in RPMI- 1C,1D,2A). F4/80+ macrophages were elevated 1.4-fold in wild- FCS and filtration through 70-mm mesh, 10 3 106 cells in 200 ml of in- complete RPMI 1640 (no FCS) were administered i.v. via the lateral tail type mice and accumulated predominantly in the stromal tissue vein into recipient wild-type and Csf22/2 mice at 1200 h on day 0.5 pc. immediately subjacent to the luminal and stromal epithelium, and 2/2 Flow cytometric analysis of OT-II and OT-I T cell responses a comparable 2.0-fold increase was seen in Csf2 mice (Figs. 1G,1H,2B). MHC class II+ cells were also elevated, regardless of To assess OT-II and OT-I T cell activation and proliferation, recipient mice GM-CSF presence or absence (2.1-fold and 4.0-fold in wild-type were killed 68 h after T cell transfer on day 3.5 pc and single-cell sus- 2/2 pensions were prepared from excised para-aortic (iliac) LNs that drain the and Csf2 mice, respectively) (Figs. 1K,1L,2C). Endometrial + + uterine tissue. Cells were labeled to detect CD4 (OT-II T cells) or CD8a and MSR1 and sialoadhesin cells were substantially elevated in CD45.1 (OT-I T cells), followed by PE-Cy5–conjugated strepavidin. All wild-type mice, with increases of 2.0-fold and 1.9-fold, respec- labeling was performed for 30 min at 4˚C in FACS buffer. Analysis was tively, at day 0.5 pc compared with estrus, but neither increase performed using a FACSCanto flow cytometer with FACSDiva software 2/2 (BD Biosciences). The proliferation of OT-I (CD8+CD45.1+CFSE+) and occurred in Csf2 mice (Figs. 1O,1P,2E,2F). MSR1 expres- OT-II (CD4+CFSE+) T cells populations was quantified by measuring sion was most notably affected by GM-CSF deficiency, with CFSE peaks, and proliferation was expressed as the proliferation index (PI) a 47% decrease in MSR1+ cells at day 0.5 pc in Csf22/2 mice (32, 39). compared with wild-type mice (p , 0.01) (Fig. 2E). Macrosialin In vitro culture of uterine cells with GM-CSF was unchanged after coitus in either wild-type or GM-CSF–de- To determine whether expression of APC activation markers could be ficient mice (Fig. 2D). The most strikingly change after coitus was + restored in vitro by exposure to GM-CSF, whole uterus was harvested from seen in CD86 cells, which increased regardless of Csf2 status 2 2 Csf2 / females at day 0.5 pc. The uterus was digested as described above (3.6-fold and 2.0-fold increase in wild-type and Csf22/2 mice, 3 6 and cells were resuspended at 1 10 cells/ml in 200 ml of complete respectively) (Figs. 1S,1T,2G). For each of these markers, RMPI 1640 (with 2 mM L-glutamine, 50 U/ml penicillin, 50 mg/ml streptomycin, 50 mM 2-ME [all from Sigma-Aldrich], and 10% FCS). labeled cells were seen to accumulate in the superficial endome- Recombinant mouse GM-CSF (R&D Systems, Minneapolis, MN) was trium immediately subjacent to luminal epithelial cells after coitus added to the culture at 20, 40, or 80 ng/ml. Following 24 h of incubation at (Fig. 1). Whereas eosinophils were not changed after mating in 2 2 37˚C at 5% CO2 cells were released from culture wells with 2 mM EDTA wild-type mice, their numbers increased .2-fold in Csf2 / mice (BDH Laboratory Supplies, Melbourne, Australia) and resuspended in + + FACS buffer. Cells were labeled with Abs against F4/80, CD11c, MHC (Fig. 2H). Numbers of F4/80 and MSR1 cells in the myome- class II, MSR1, CD80, and CD86, and expression of markers was analyzed trium and mesometrial triangle regions of the uterus also increased by flow cytometry as described above. on day 0.5 pc, albeit not as dramatically as in the endometrium, + + + Statistical analysis whereas numbers of class II MHC , CD11c , and CD86 cells in these tissue compartments remained similar to estrus (data not Data were analyzed by nonparametric Kruskal-Wallis H tests and Mann- Whitney U tests using SPSS for Windows version 17.0 (SPSS, Chicago, shown). IL). Differences between treatment groups were considered significant On day 3.5 pc, a further marked change in the number and when p , 0.05. appearance of uterine DCs and macrophages was evident. In wild- 7088 GM-CSF REGULATION OF UTERINE DENDRITIC CELLS type mice, the numbers of cells expressing CD11c remained similar (p , 0.05) (Fig. 2C), and the remaining MHC class II+ cells to day 0.5 pc, whereas F4/80+ macrophages declined to levels seen showed a reduced intensity of MHC class II staining (data not in estrus mice, and Csf22/2 mice showed similar staining patterns shown). Macrosialin+ cells, MSR1+ cells, and sialoadhesin+ cells (Fig. 2A,2B). In contrast, whereas the numbers of MHC class II+ in the endometrium all declined by between 50 and 80% com- cells remained similar from day 0.5 pc to day 3.5 pc in wild-type pared with day 0.5 pc, and this occurred regardless of Csf2 mice, in Csf22/2 mice MHC class II+ cells declined by day 3.5 to genotype status (Fig. 2D–F). In the case of both MSR1 and a level 57% lower than numbers in wild-type mice on day 3.5 pc macrosialin, the density of positive cells on day 3.5 pc was below

FIGURE 1. Effect of GM-CSF deficiency on DC and macrophage localization in uterine endometrial tissue. Sections of uterine tissue collected from wild-type or Csf22/2 mice at estrus or on day 0.5 pc were labeled with mAb reactive with CD11c (A–D), F4/80 (E–H), MHC class II (I–L), scavenger receptor class A (MSR1) (M–P), or CD86 (Q–T). Images are representative of n = 5 mice/group. DAB and hematoxylin, original magnification 320. En, endometrium; LE, luminal epithelium. The Journal of Immunology 7089 that seen at estrus. Most notably, the density of CD86+ cells de- DCs and macrophages at this time. Individual uteri from wild-type creased by .75% between day 0.5 pc and day 3.5 pc (p = 0.01), mice and Csf22/2 mice were recovered on day 0.5 pc, enzymati- and this also occurred in Csf22/2 mice (Fig. 1F). Interestingly, cally digested to generate single-cell suspensions, and assessed by cells expressing MSR1 and CD86 in the day 3.5 pc uterus were flow cytometry. generally located in the deep endometrium, often adjacent to the CD11c+ cells and F4/80+ macrophages were assessed for MHC myometrium, and unlike earlier stages they frequently occurred in class II, MSR1, CD80, and CD86 expression (Figs. 3 and 4, re- clusters (data not shown). The density of F4/80+ and MSR1+ cells spectively), and activation marker expression was further classi- in the mesometrial triangle and the myometrium decreased from fied as low or high. The proportion of CD11c+ cells expressing day 0.5 pc to day 3.5 pc, whereas MHC class II+ and CD86+ cells MHC class II was decreased by 22% in Csf22/2 mice (Fig. 3C), were similar in number (data not shown). The distribution and and there was a decrease in the intensity of MHC class II+ ex- morphology of eosinophils and cells expressing other activation pression, with a 42% reduction in the percentage of CD11c+ MHC markers in the endometrium were comparable between genotypes, class IIhi cells in Csf22/2 mice (Fig. 3D). There was no effect of and no differences were evident in the mesometrial triangle or GM-CSF deficiency on the proportion of CD11c+ cells expressing myometrium on day 3.5 pc (data not shown). MSR1 (Fig. 3E,3F). Within the CD11c+ population there was an 8% reduction in CD80+ cells in Csf22/2 mice (Fig. 3G) and Effect of GM-CSF deficiency on expression of activation expression was less intense, with a 48% reduction in CD11c+ markers by uterine DCs and macrophages on day 0.5 pc CD80hi cells (Fig. 3H). CD86 also showed decreased intensity of Since the immunohistochemical study indicated that the effect of expression with 68% fewer CD11c+ CD86hi cells in Csf22/2 GM-CSF deficiency was most notable during the postcoital in- uterine tissue following coitus (Fig. 3J). flammatory response, we sought to further investigate the effect of The proportion of F4/80+ macrophages expressing MHC class GM-CSF deficiency on expression of activation markers by uterine IIhi was reduced by 50% in Csf22/2 mice (Fig. 4D). There was no

FIGURE 2. Effect of GM-CSF deficiency on uterine endometrial DC and macrophage number and activation phenotype. The percentage positivity for mAb- reactive cells in the uterine endometrium of wild-type (open bars) or Csf22/2 mice (hatched bars) at estrus, on day 0.5 or day 3.5 pc, was quantified by video image analysis. Data are the means 6 SEM percentage positivity for CD11c (A), F4/80 (B), MHC class II (C), macrosialin (D), scavenger receptor class A (MSR1) (E), sialoadhesin (F), CD86 (G), or endogenous peroxidize (eosinophils) (H)(n = 5 mice/group). The effects of Csf2 genotype and stage of early pregnancy were determined by Kruskal-Wallis one-way ANOVA and Mann-Whitney U test. *p , 0.05, significant effect of Csf2 genotype at a given time point; a,bp , 0.05, different superscripts indicate differences between time points in wild-type mice. x,yp , 0.05, different superscripts indicate differences between time points in Csf22/2 mice. 7090 GM-CSF REGULATION OF UTERINE DENDRITIC CELLS

FIGURE 3. Effect of GM-CSF deficiency on MHC class II, scavenger receptor class A (MSR1), CD80, and CD86 expression by uterine CD11c+ cells. Uterine cells prepared from WT mice or Csf22/2 mice on day 0.5 pc were labeled with mAb reactive with CD11c, MHC class II, MSR1, CD80, and CD86 and analyzed by flow cytometry. Representative histograms of CD11c+ cells (A) or isotype-matched negative controls (B) are shown, with gates set to discriminate between MHC class IIlo and MHC class IIhi, MSR1lo and MSR1hi, CD80lo and CD80hi, or CD86lo and CD86hi cells. The numbers at the top of histograms are the percentages of cells within each gate. C–J, Percentages of CD11c+ cells expressing MHC class II+ (C), MHC class IIhi (D), MSR1+ (E), MSR1hi (F), CD80+ (G), CD80hi (H), CD86+ (I), and CD86hi (J). Data are means 6 SEM from wild-type mice (open bars, n = 7–21) and Csf22/2 mice (hatched bars, n = 10–22). The effect of Csf2 genotype was determined by Mann-Whitney U test. *p , 0.05, significant effect of Csf2 genotype. WT, wild-type. effect of GM-CSF deficiency on expression of MSR1, CD80, or metry. There was no change in MHC class II or MSR1 expression CD86 in F4/80+ macrophages (Fig. 4E–J). in CD11c+ or F4/80+ cells (data not shown). CD11c+ cells cultured To further investigate whether expression of activation markers in the presence of 80 ng/ml GM-CSF showed a 31% increase in uterine CD11c+ and F4/80+ cells is regulated by GM-CSF, uter- in CD80hi expression (Fig. 5A) and a 36% increase in CD86hi ine cells were recovered by enzymatic digestion from day 0.5 pc expression (Fig. 5B). CD86 expression was also increased in the Csf22/2 females and cultured with or without recombinant GM- F4/80+ population, with a 40% increase in F4/80+ CD86hi cells in CSF for 24 h. MHC class II, MSR1, CD80, and CD86 expression the presence of 40 ng/ml GM-CSF compared with control cells by cultured CD11c+ and F4/80+ cells were assessed by flow cyto- cultured without GM-CSF. The Journal of Immunology 7091

FIGURE 4. Effect of GM-CSF deficiency on MHC class II, scavenger receptor class A (MSR1), CD80, and CD86 expression by uterine F4/80+ macrophages. Uterine cells prepared from WT mice or Csf22/2 mice on day 0.5 pc were labeled with mAb reactive with F4/80, MHC class II, MSR1, CD80, and CD86 and analyzed by flow cytometry. Representative histograms of F4/80+ macrophages (A) or isotype-matched negative controls (B) are shown, with gates set to discriminate between MHC class IIlo and MHC class IIhi, MSR1lo and MSR1hi, CD80lo and CD80hi, or CD86lo and CD86hi cells. The numbers at the top of histograms are the percentages of cells within each gate. C–J, Percentages of F4/80+ macrophages expressing MHC class II+ (C), MHC class IIhi (D), MSR1+ (E), MSR1hi (F), CD80+ (G), CD80hi (H), CD86+ (I), and CD86hi (J). Data are means 6 SEM from wild-type mice (open bars, n = 7–21) and Csf22/2 mice (hatched bars, n = 10–22). The effect of Csf2 genotype was determined by Mann-Whitney U test. *p , 0.05, significant effect of Csf2 genotype. WT, wild-type.

Effect of GM-CSF deficiency on MHC class II-restricted and GM-CSF deficiency on the capacity of uterine DCs and macro- MHC class I-restricted Ag presentation phages to present Ag and activate T cells, we used a model pa- Previously we showed that maternal bone marrow-derived APCs ternal Ag system employing male mice that produce transgenic are critical for both the MHC class I-restricted cross-presentation OVA ubiquitously under an Actb promoter, which results in OVA and direct MHC class II-restricted presentation of paternal seminal secretion into the seminal fluid (32). After mating females with fluid Ags at the time of conception (32). To evaluate the effect of Act-mOVA males to expose the uterus to seminal fluid OVA, 7092 GM-CSF REGULATION OF UTERINE DENDRITIC CELLS

FIGURE 6. Effect of GM-CSF deﬁciency on MHC class II-restricted presentation of Ags in the uterus. Wild-type mice or Csf22/2 mice were mated with Act-mOVA males, or alternatively were mated with B6 males

and then administered OVA323–339 peptide transcervically on day 0.5 pc. Both groups received CFSE-labeled OT-II T cells on day 0.5 pc, and OT-II T cell proliferation in para-aortic LNs harvested 68 h later was quantiﬁed by ﬂow cytometry and the PI was calculated. Representative histograms from a wild-type (A) and a Csf22/2 mice (B) mated with Act-mOVA males are shown, with PI values given in the top left corner. C and D, Mean 6 SEM PI values (from 7–14 mice/group) from WT (open bars) and Csf22/2 mice (hatched bars) mated with Act-mOVA males (C) or mated with B6

males and then administered OVA323–339 peptide transcervically (D). The effect of Csf2 genotype was determined by Mann-Whitney U test. *p , 0.05, signiﬁcant effect of Csf2 genotype. WT, wild-type.

FIGURE 5. Effect of recombinant GM-CSF on activation marker ex- followed by CFSE-labeled OT-II T cells. When OT-II T cells were + + pression by uterine CD11c cells and F4/80 cells in vitro. Uterine cells recovered from the para-aortic LNs 68 h later, proliferation was 2/2 prepared from Csf2 mice on day 0.5 pc were cultured with 0, 20, 40, or significantly reduced in Csf22/2 mice compared with wild-type 80 ng/ml recombinant GM-CSF for 24 h, then harvested and labeled with mice, corresponding to a 67% reduction in the average number mAb reactive with CD11c, F4/80, CD80, and CD86 and analyzed by flow of cell divisions (Fig. 6D). Similarly, to determine whether GM- cytometry. Data are the means 6 SEM percentage of CD11c+ cells classified as CD80hi (A) or CD86hi (B) and the percentage of F4/80+ cells CSF deficiency alters MHC class I-mediated presentation of OVA classified as CD86hi (C)(n = 3–6 wells/treatment group, from n = 6 mice). peptide to OT-I T cells, OVA257–264 peptide was administered to 2/2 The effect of GM-CSF concentration was analyzed by Mann-Whitney the uterine cavity of mated wild-type or Csf2 mice, and CFSE- U test. a,bp , 0.05, significant differences between treatment groups. labeled OT-I T cells were transferred. When OT-I T cells were recovered from the para-aortic LNs 68 h later, proliferation was 2/2 + + significantly reduced in Csf2 mice compared with wild-type OVA-specific CD4 OT-II T cells or CD8 OT-I T cells were mice, corresponding to a 78% reduction in the average number CFSE-labeled and transferred into mated females. The extent of of cell divisions (Fig. 7D). Ag presentation was determined by quantifying the number of cycles of proliferation, as indicated by CFSE dye dilution, in OT-II T cells and OT-I T cells recovered 68 h later from the para- Discussion aortic LNs that drain the uterus (32). Uterine DCs and macrophages are important participants in the The average PI of transgenic OT-II T cells recovered from the events of embryo implantation and establishing pregnancy. Their 2 2 para-aortic LNs of wild-type mice was reduced in Csf2 / mice roles include processing and presentation of Ags from seminal fluid compared with wild-type mice (Fig. 6A,6B), corresponding to and the conceptus to activate the T cell-mediated immune tolerance a 68% reduction in the average number of cell divisions for each vital for progression of gestation (32, 33). Variation in the size and parent OT-II cell (Fig. 6C). A similar reduction in PI of transgenic phenotype of these populations is linked with infertility, mis- 2 2 OT-I T cells was seen in Csf2 / mice (Fig. 7A,7B), with a 65% carriage, and disorders of pregnancy such as preeclampsia (3–5). reduction in the average number of cell divisions (Fig. 7C). In this study we have identified GM-CSF as a key cytokine con- This decrease in OT-II T cell and OT-I T cell proliferation in trolling macrophage and DC populations in the uterine tissue, in response to OVA Ag in seminal fluid indicates that maternal particular their expression of activation markers MHC class II and GM-CSF deficiency results in reduced presentation of OVA peptide the scavenger receptor, MSR1, CD80, and CD86. We show that to reactive T cells. To assess whether GM-CSF deficiency causes the changes in APC populations due to GM-CSF deficiency se- reduced MHC class II-mediated Ag presentation when OVA verely compromise the functional capacity of the uterus to acti- peptide is delivered to the female reproductive tract, thereby vate a T cell-mediated immune response to reproductive Ags. averting the requirement for Ag uptake and processing, the OVA Regulation by GM-CSF appears to be most critical during the peptide OVA323–339 was administered by the transcervical route controlled inflammatory response elicited by male seminal fluid 2 2 to the intraluminal cavity in mated wild-type or Csf2 / mice, at coitus (12, 13), when GM-CSF expression by epithelial cells The Journal of Immunology 7093

neutrophils. Alternatively, these cells could be precursors to conventional DCs, analogous to the CD11c+ MHC class II2 cells reported in bone marrow and lymphoid tissues (42–44). Conversely, the reduced MSR1 expression evident by immunohistochemistry was not supported by flow cytometry data. The reduction in MSR1 staining might be due to diminished cyto- plasmic MSR1 within DCs and macrophages in the postcoital uterus, rather than less surface MSR1 expression, or possibly fewer cells expressing MSR1 but not F4/80 or CD11c. Previously we reported that the accumulation of DCs in the implantation site in part stems from immature CD11b+ monocytic precursors recruited at estrus and during the postcoital inflammatory response, which differentiate into CD11b2 MHC class II+ cells (6). The present study indicates that GM-CSF is a key cytokine in regulating this phenotypic maturation, with absence of the postcoital GM-CSF surge causing a persisting deficit in MHC class II+ APCs through the preimplantation period. CD11b+CD8a2 DC precursors have been shown to be differentially responsive to FIGURE 7. Effect of GM-CSF deficiency on MHC class I-restricted GM-CSF–regulated expansion and maturation into MHC class IIhi 2/2 cross-presentation of Ags in the uterus. Wild-type mice or Csf2 mice DCs (45). Their altered phenotype in the absence of GM-CSF could were mated with Act-mOVA males or alternatively were mated with B6 be expected to compromise DC function during the postimplan- males and then administered OVA peptide transcervically on day 257–264 tation period when DCs begin to process Ags released by invading 0.5 pc. Both groups received CFSE-labeled OT-I T cells on day 0.5 pc, and OT-I T cell proliferation in para-aortic LNs harvested 68 h later was placental trophoblasts (32, 33) and have an essential role in regu- quantified by flow cytometry and the PI was calculated. Representative lating the vascular changes that ensure an adequate decidual re- histograms from wild-type (A) and a Csf22/2 mice (B) mated with Act- sponse and placental cell invasion (2, 46). mOVA males are shown, with PI values given in the top left corner. C and The presence of otherwise normal numbers of DCs and mac- D, Mean 6 SEM PI values (from 8–14 mice/group) from WT (open bars) rophages in the GM-CSF–deficient uterus during early pregnancy 2 2 and Csf2 / mice (hatched bars) mated with Act-mOVA males (C)or is explained by the array of other chemokines and cytokines ex- mated with B6 males and then administered OVA257–264 peptide trans- pressed in the uterus, including CSF1, RANTES, MIP1a, MIP1b, cervically (D). The effect of Csf2 genotype was determined by Mann- and MCP1 (47–49). These data are consistent with studies in other U p , Csf2 2 2 Whitney test. * 0.05, significant effect of genotype. WT, tissues of Csf2 / mice showing that in steady-state conditions, wild-type. the numbers of myeloid and DC cell populations in LNs and peripheral organs are essentially normal (27, 50). lining the female reproductive tract is strongly induced (40). The Using OVA as a surrogate paternal Ag, we showed in in vivo periconceptual period is a crucial time in the cascade of events experiments that induction of T cell responses in the uterus is driving maternal adaptation to pregnancy, since the first exposure severely compromised in GM-CSF–deficient mice. OVA Ag pres- to paternal Ags occurs in the context of seminal fluid. Activation ent in seminal fluid or delivered transcervically during the post- of T cells reactive with paternal Ags occurs within the 72 h after coital inflammatory response showed a very limited ability to coitus (31, 32) and is associated with expansion of the Treg cell drive CD4+ T cell and CD8+ T cell proliferation. This indicates pool that confers immune tolerance when the embryo implants that maternal GM-CSF is required for optimal presentation of some 4 d later (36). Disruption of the immune adaptation initiated seminal fluid Ag via both the MHC class II- and MHC class during this period causes allogeneic pregnancy to fail (35) and I-restricted pathways. Because there was no major change in the is the mechanism underpinning predisposition to fetal loss in absolute numbers of DCs and macrophages, we conclude that abortion-prone CBA 3 DBA/2 mice (41). impaired Ag capture, processing, and/or presentation pathways In the absence of GM-CSF, there were no substantial changes in within individual APCs likely explain their deficient ability to the kinetics of inflammatory cell recruitment and activation after activate T cells. coitus. The greatest change was seen in cells expressing the scav- Our finding of substantially reduced T cell activation in the enger receptor MSR1, which were reduced in number by 50% in uterus contrasts with other reports showing no effect or only a the absence of GM-CSF. The postcoital inflammatory response modest reduction in APC competence to generate T cell responses abates in wild-type mice by the time of embryo implantation on in Csf22/2 mice. Ag-specific T cell responses to methylated BSA day 3.5 pc. A comparable decline in uterine leukocytes was seen administered to the peritoneal cavity developed normally in in Csf22/2 mice at this time, but MHC class II+ cells were abnor- Csf22/2 mice; however, macrophages from peritoneal exudates of mally diminished and were 2-fold lower in GM-CSF–deficient immunized mice elicited a lower proliferative response in allo- mice by the time of implantation on day 3.5 pc. geneic splenic T cells (29). In contrast, APCs from Csf22/2 mice In contrast to the immunohistochemical analysis, flow cytometry activated comparable responses to wild-type APCs in wild-type revealed that the proportion of both DCs and macrophages ex- CD4+ T cells, and an effect of Csf2-null mutation on CD4+ T cell pressing MHC class II, as well as their intensity of MHC class II proliferation in response to keyhole limpet hemocyanin immuni- expression, was reduced in the absence of GM-CSF on day 0.5 pc. zation was attributed to a defect in Csf22/2 T cells as opposed to This is consistent with the interpretation that GM-CSF defi- APCs (51). Similarly, s.c. immunization with OVA in Csf22/2 ciency causes a population-wide reduction in the intensity of sur- mice did not cause any detectable change in CD4+ OT-II pro- face MHC class II expression on a per cell basis in both DCs and liferation or the production of OVA-specific Ig, unless mice were macrophages. More than 40% of CD11c+ cells within the uterine also Flt3-ligand deficient (52). Taken together, these observations tissue at day 0.5 pc are MHC class II2, and it is conceivable that suggest that the extent of APC functional deficiency in Csf22/2 a proportion of the CD11c+ cells may be macrophages or mice depends on the site of Ag delivery and the nature of the local 7094 GM-CSF REGULATION OF UTERINE DENDRITIC CELLS inflammatory environment, and they indicate that APCs in the cytokine expression profile of spleen CD11c+ DCs, with reduced uterus may be unusually dependent on this cytokine. Since uterine production of IL-6 and IL-23 required to drive Th17 T cell differ- GM-CSF synthesis is controlled by ovarian steroid hormones as entiation (62), indicating that exploration of GM-CSF–regulated well as seminal fluid factors (10, 11), this provides a mechanism cytokine synthesis in uterine DCs is warranted. through which local APCs can respond to the changing functional GM-CSF is an essential cytokine for normal reproduction, with requirements of this dynamic tissue, according to stage of the defects in Csf22/2 mice ranging from altered ovarian function reproductive cycle and pregnancy. GM-CSF is likely to act in (63, 64), compromised preimplantation embryo development (65), concert with other APC-regulating cytokines, modulated by the dysregulated placental morphogenesis (16, 66), and increased indirect actions of ovarian steroid hormones on macrophages and cidence of fetal growth restriction and death in the late gestation and DCs (53, 54). perinatal period (16, 17). The present study suggests that one po- This effect on Ag-presenting function, together with the ob- tential cause for these reproductive anomalies might be failure to servation of reduced MSR1 expression in Csf22/2 mice, suggests establish an optimal T cell response to pregnancy, due to impaired that GM-CSF may influence Ag capture mediated by scavenger presentation of reproductive Ags to the T cell repertoire. receptors. MSR1 is part of the larger scavenger receptor super- Our data using the surrogate paternal Ag OVA indicate that family, comprised of pattern recognition receptors that mediate indirect presentation via both the MHC class II pathway to CD4+ internalization of an array of proteins and other materials (55). cells, as well as MHC class I-mediated cross-presentation to CD8+ Msr1 is upregulated in mature and differentiated CD11c+ DCs T cells, is substantially impaired in the absence of GM-CSF. This by GM-CSF (56); however, we are unaware of previous reports would compromise adaptation in both the CD4+ T cell compart- of scavenger receptor dysregulation in Csf22/2 mice. Blocking ment required to expand the CD4+ Treg cell pool (67) and in the scavenger receptor superfamily members prevents the activation CD8+ T cell compartment where generation of regulatory cells of OVA-reactive CD4+ T cells, indicating that MHC class II-re- with suppressive function (68) as well as anergic responses (34) stricted Ag presentation relies upon Ag capture by these mole- occur. Deficient T cell tolerance fits with the observation that in cules (57). It has been suggested that scavenger receptor family pregnant Csf22/2 mothers, male fetuses are more likely to die or members are also important in Ag cross-presentation by DCs (58); become growth restricted in utero compared with female progeny however, a specific requirement for MSR1 is contentious since (16), since male fetuses comprise a greater antigenic challenge DCs from MSR1-deficient mice can cross-present as effectively as than do females. The interpretation that GM-CSF facilitates an DCs from wild-type mice (59, 60). appropriate T cell response to pregnancy is consistent with ex- To evaluate whether a defect in Ag uptake and/or processing periments showing that administration of exogenous GM-CSF might explain the reduced T cell response to OVA Ag in seminal in the preimplantation period can normalize fetal loss rates in fluid, we used OVA peptides that do not require intracellular abortion-prone CBA 3 DBA/2 mice and is associated with ex- processing for presentation to OT-II and OT-I TCRs. However, pansion of CD8+ T cells with suppressive function (68). Admin- administration of OVA peptides to the uterus failed to overcome istration of bone marrow-derived DCs from normal fertile mice to the compromised Ag presentation seen with whole OVA protein abortion-prone CBA 3 DBA/2 mice similarly reduces the mis- in GM-CSF–deficient mice, indicating that Ag capture and/or carriage rate, implying that GM-CSF–mediated rescue of fetal loss intracellular processing do not fully explain the defect. is operating through a DC-mediated mechanism (69). A key marker of APC activation and maturity is MHC class II, As well as generating T cell tolerance, DCs and macrophages are and failure to upregulate MHC class II could reasonably also important for the extensive tissue remodeling and angiogenic contribute to defective Ag presentation to CD4+ T cells through changes necessary for the uterine decidual response and for pla- insufficient availability of MHC molecules to form MHC–OVA cental invasion and development (2, 46). Secretion of vascular complexes and engage in TCR ligation. Within a range of in vitro endothelial growth factor by decidual DCs is essential for the model systems, exogenous GM-CSF has been shown to induce development of an adequate blood supply to the developing pla- surface MHC class II expression, through both transcriptional and centa (2). Although this study has focused on the Ag-presenting posttranscriptional mechanisms (22, 61), particularly in CD11b+ function of uterine DCs and macrophages, the additional possi- CD11c+ DCs where GM-CSF induces translocation of cytoplas- bility that GM-CSF influences DC or macrophage capacity to mic MHC class II to the cell surface (45). However, earlier studies support vascular development or otherwise remodel reproductive on MHC class II expression in Csf22/2 mice did not show tissues cannot be excluded. any difference in MHC class II expression, in Ag-elicited perito- Collectively, the data presented in this study indicate that neal cells (29), in skin-draining LNs (52), or in spleen APCs (62). GM-CSF is critically important for uterine DCs and macrophages to We have previously reported that GM-CSF–deficient mice have reach maturity and attain competence to present Ag and activate less MHC class II expression on macrophages and DCs within T cells during the periconceptual phase of early pregnancy, when the ovary (63), suggesting that MHC class II expression on fe- maternal immune tolerance of reproductive Ags is induced. APCs in male reproductive tract DCs may be particularly responsive to this the uterus and elsewhere in the female tract appear to be distin- cytokine. guished by the extent of their dependence on this cytokine, perhaps GM-CSF is known to upregulate DC expression of costimulatory because of their ontogenic relationship with monocyte precursors molecules CD80 and CD86 (21, 22), which are critical for stabi- recruited during the controlled inflammatory response to seminal lizing the peptide–MHC–TCR interaction and generating strong fluid. Their functional deficiency in the absence of GM-CSF is likely Ag-specific T cell responses. By immunohistochemistry we did to be the consequence of reduced expression of surface MHC class II not detect an effect of GM-CSF deficiency on CD86 in the en- and scavenger receptor MSR1. These results raise the question of dometrium, consistent with data on costimulatory molecules CD40, the significance of Csf2 expression in regulating DCs in the human CD80, or CD86 in DCs from skin-draining LNs (52) or spleen (62). uterus. Our findings underscore the importance of evaluating However, by flow cytometry, expression of CD80 and CD86 was whether GM-CSF contributes to the altered numbers and pheno- clearly decreased in the uterine CD11c+ population from Csf22/2 types of DCs seen in women experiencing reproductive patholo- mice, and this phenotype could be rescued by ex vivo culture with gies (3–5). Because in women, as in mice, GM-CSF synthesis by GM-CSF. Interestingly, Csf2-null mutation is reported to perturb the reproductive tract epithelial cells is responsive to male seminal The Journal of Immunology 7095

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