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The Journal of Immunology, 2006, 177: 7567-7578.
Copyright © 2006 by The American Association of Immunologists, Inc.

Paternal Antigen-Bearing Cells Transferred during Insemination Do Not Stimulate Anti-Paternal CD8+ T Cells: Role of Estradiol in Locally Inhibiting CD8+ T Cell Responses1

Matthew M. Seavey and Tim R. Mosmann2

David H. Smith Center for Vaccine Biology and Immunology, and Department of Microbiology and Immunology, University of Rochester Medical Center, NY 14642


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Maternal immunological tolerance of the semiallogeneic fetus involves several overlapping mechanisms to balance maternal immunity and fetal development. Anti-paternal CD8+ T cells are suppressed during pregnancy in some but not all mouse models. Since semen has been shown to mediate immune modulation, we tested whether exposure to paternal Ag during insemination activated or tolerized anti-paternal CD8+ T cells. The uterine lumen of mated female mice contained male MHC I+ cells that stimulated effector, but not naive, CD8+ T cells ex vivo. Maternal MHC class I+ myeloid cells fluxed into the uterine lumen in response to mating and cross-presented male H-Y Ag to effector, but not naive, CD8+ T cells ex vivo. However, neither unprimed nor previously primed TCR-transgenic CD8+ T cells specific for either paternal MHC I or H-Y Ag proliferated in vivo after mating. These T cells subsequently responded normally to i.p. challenge, implicating ignorance rather than anergy as the main reason for the lack of response. CD8+ T cells responded to either peptide Ag or male cells delivered intravaginally in ovariectomized mice, but this response was inhibited by systemic estradiol (inducing an estrus-like state). Subcutaneous Ag induced responses in both cases. Allogeneic dendritic cells did not induce responses intravaginally even in ovariectomized mice in the absence of estradiol. These results suggest that inhibition of antiallogeneic responses is restricted both locally to the reproductive tract and temporally to the estrous phase of the menstrual cycle, potentially decreasing the risk of maternal immunization against paternal Ags during insemination.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Maternal antifetal responses are regulated at several points before and during gestation (1, 2, 3, 4). Allogeneic paternal gene products, especially MHC class I (MHC I),3 are expressed on the placental trophoblast (5, 6, 7). However, the issue of whether anti-paternal CD8+ T cells respond to placental Ags during gestation remains controversial. In normal mice, anti-paternal CD8+ T cells were neither activated nor suppressed during pregnancy (Ref. 8 and also K. Trejo-Oliver, M. M. Seavey, L. J. Guilbert, and T. R. Mosmann, unpublished observations). Results from TCR-transgenic models have been contradictory. In two studies, the numbers or functions of anti-paternal CD8+ T cells were reduced during pregnancy (9, 10). These results may have been influenced by the unusual presence of extremely large numbers of anti-paternal CD8+ T cells in the TCR-transgenic mice. In a third model, using either TCR-transgenic mice directly, or adoptive transfer of smaller numbers of TCR-transgenic cells to mimic a normal response, anti-paternal CD8+ T cells were neither primed nor tolerized during pregnancy (Ref. 11 and also M. M. Seavey and T. R. Mosmann, unpublished observations). Finally, anti-paternal CD8+ T cells were activated during pregnancy in a fourth TCR-transgenic model (12).

Several mechanisms are thought to prevent antipaternal immunological attack at the maternal-fetal interface. These include sequestering fetal MHC I Ag (i.e., placental barrier), expressing the tryptophan-metabolizing enzyme IDO (2, 13, 14), T regulatory cells (15), immunosuppressing APC subsets possibly producing IL-10 (16), expressing FasL on trophoblast (17, 18), expressing various negative regulators of costimulation such as PD-L1/2 (19) and B7-H1 (20), and additional mechanisms (21, 22, 23, 24, 25, 26). This complexity of protective mechanisms and Ag exposure to paternal Ags during pregnancy may contribute to the variable results obtained for CD8+ T cell antipaternal reactivity in different experimental systems. If different mechanisms induce tolerance and ignorance to paternal Ags, then differences in the relative contribution of each mechanism in the various models could account for the discrepancies seen. Another aspect of this complexity is that mothers are potentially exposed to paternal Ag at two distinct stages of pregnancy. In addition to the expression of MHC I Ags on fetal cells starting at day 9.5 of pregnancy (7), these Ags are also expressed on paternal cells that are introduced in the ejaculate into the mother during insemination (1).

Because maternal immune responses against semen Ags, such as sperm acrosomal-head Ags (27) or MHC Ags (28, 29), could be deleterious for the ensuing pregnancy or future fertility, it is not surprising that several mechanisms appear to inhibit damaging responses against semen Ag, or induce immune responses that may actually enhance fertility with allogeneic males (21). Soluble immunomodulatory factors such as TGFbeta or PGE2 in the seminal fluid may prevent inappropriate antipaternal responses and promote successful pregnancy (1, 30, 31).

The hormonal environment may also regulate antipaternal T cell responses in the female reproductive tract (FRT). During the estrogen-dominated stage of the menstrual cycle (estrus), when the female is most receptive to mating, rat and mouse CD4+ T cell responses in the FRT are suppressed by a mechanism possibly acting through an APC, and this effect can be reproduced by estradiol treatment of ovariectomized (OVX) mice (32, 33, 34, 35). Intravaginal (IVAG) immunization during estrus (but not diestrus) also reduced subsequent delayed-type hypersensitivity responses (36). Human CTL activity in the uterus is present during the proliferative but not the secretory phase (postovulatory period) of the menstrual cycle and is greatest in uterine tissues from postmenopausal women (37). Down-regulation of T cell priming or activity during the secretory phase corresponds to the time when fertilization and implantation are most likely to occur.

Given the importance of balancing between the induction of beneficial CD8+ T cell responses against sexually transmitted pathogens and inhibition of harmful antipaternal responses during allopregnancy, we focused on the exposure of CD8+ T cells to paternal Ag as a consequence of mating. We took advantage of two TCR-transgenic mouse models to track the presence of paternal APC and Ag and also to measure the in vivo response of T cells specific for paternal Ag. Although low frequencies of paternal cells capable of activating alloantigen-specific CD8+ T cells were transferred into the mother during insemination, and maternal APC acquired and cross-presented male Ags, neither naive nor memory paternal-specific CD8+ T cells divided in vivo. Antipaternal cells were not tolerized. Consistent with previous CD4+ T cell findings, estradiol strongly suppressed in vivo CD8+ T cell responses, and this suppression was restricted to IVAG but not s.c. immunization. Thus, our results are most consistent with a model in which estrogen helps to maintain CD8+ T cell ignorance to paternal Ags in semen when insemination occurs during the periovulatory phase of the menstrual cycle.

This striking demonstration of the ability of estradiol to inhibit CD8+ T cell priming is consistent with the principle that the suppression of antipaternal responses, necessary for successful pregnancy, should be limited to the shortest time and most restricted location possible to minimize the opportunities for pathogens to take advantage of immunosuppression and establish infection. These data may have implications for the design of vaccine strategies for IVAG vaccine administration.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice

All mouse strains C57BL/6 (B6; H2b), B10.D2 (H2d), B10.BR (H2k), B10 (H2b), and B6.mOVA-transgenic mice were purchased from The Jackson Laboratory. All female mice were 6–8 wk of age and male studs were aged for several months before use. OVX female C57BL/6 mice were purchased from Taconic Farms. The anti-Ld TCR-transgenic (2C) mice backcrossed onto a C57BL/6 background were a gift from J. Schneck’s (Johns Hopkins University, Baltimore, MD) laboratory. The B6 2C-transgenic mice were backcrossed onto a B6.SJL background to obtain CD45.1+ 2C-transgenic T cells. The anti-H-Y Ag TCR-transgenic mice on a C57BL/6 RAG–/– background were obtained from Taconic Farms. OT-I TCR-transgenic mice were a gift from N. Crispe (University of Rochester, New York) and were maintained on a B6.SJL CD45.1+ background. All animals were maintained under pathogen-free conditions and in compliance with national and institutional guidelines. All animal experiments were approved by the University of Rochester University Committee on Animal Resources.

Media, cell lines, and peptides

All ex vivo work was performed using RPMI 1640 (Mediatech) containing penicillin-streptomycin (Mediatech), L-glutamine (Mediatech), 10% FBS (Perbio; HyClone), and 2-ME (Sigma-Aldrich). All peptides were obtained from Invitrogen Life Technologies, dissolved in DMSO or 100% ethanol, and stored at –80°C. The peptide pSYGL (SIYRYYGL) binds to H2Kb and is an alternate specificity of H2Ld-restricted 2C TCR-transgenic T cells (38). The peptide pSMCY (KCSRNRQYL) (39, 40) binds to H2Db and stimulates H-Y TCR-transgenic CD8+ T cells. The peptide pSIINFEKL (SIINFEKL) binds to H2Kb and stimulates OT-I TCR-transgenic CD8+ T cells.

Matings and hormone replacement

Female mice used for the retrieval of ejaculate (Figs. 1 and 2) were treated with fertility hormones to induce a synchronized estrus state. Briefly, female mice were injected with a 5-IU dose of pregnant mare serum gonadotropin (Sigma-Aldrich) i.p. in PBS 48 h before being mated, then given 5 IU of human chorionic gonadotropin hormone (Sigma-Aldrich) i.p. in PBS on the day of mating or immunization. For matings (Figs. 3 and 4), one pair of female mice were placed randomly into cages with stud males and mated for 3 h. Vaginal plug detection was used as an indicator of successful mating and was considered 0.5 day of pregnancy. Each of the mated mice was separated into an individual cage for the remainder of the experiment. OVX mice used for all IVAG immunization experiments (Figs. 5–10) were injected with either PBS or water-soluble cyclodextrin-encapsulated 17beta-estradiol (Sigma-Aldrich) at 1 µg/mouse per day i.p. in 200 µl of PBS. Mice were treated with hormones starting 3 days before immunization and continuing for the duration of the experiment. Estrous state was confirmed by visual observance of swelling of the vaginal labia.


Figure 1
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  		FIGURE 1. Paternal APC in the reproductive tract after mating stimulated effector but not naive anti-paternal CD8+ T cells ex vivo. A (upper panels), B6 female mice were mated with B10.D2 (H2d) males for 3 h or not mated (NM). Male ejaculate cells flushed from the uteri of vaginal plug-positive females at 3 hpc or 12–16 hpc were analyzed by flow cytometry. Flushed cells were analyzed by first gating on forward and side scatter parameters (left panels), then gating on 7-AAD low/intermediate H2Dd-positive cells (center panels) or isotype control (Iso)-positive cells (right panels). A representative mouse from each group is shown in the flow cytometry plots. Lower panels, Bar graph shows the relative percentage of paternal H2Dd-positive cells from total cells flushed from uteri of mated females in groups of at least three mice (mean ± SEM) per time point; *, p < 0.05. B, B6 female mice were mated with B10.D2 (H2d) males for 3 h. Male ejaculate cells were then flushed from the uteri of mated females and analyzed by flow cytometry. Cells were gated on forward scatter, side scatter and 7-AAD (left and center), and then analyzed for paternal and maternal MHC I. C, B6 female mice were either not mated (NM) or mated for 3 h with target allogeneic B10.D2 (H2d) or third-party B10.BR (H2k) males. Male ejaculate cells were flushed from the uteri of mated B6 female mice, then titrated into reverse ELISPOT assays containing either sorted naive 2C cells (IL-2; upper panel) or effector 2C cells (IFN-{gamma}; lower panel). Graph shows mean ± SEM of groups of at least five mice; *, p < 0.05. Five hundred bone-marrow-derived H2d or H2k DC per well were used as positive and negative controls, respectively. D, Testicles from a B10.D2 male were homogenized, stained, and analyzed by flow cytometry. Live cells were gated as 7-AAD low events; dead cells gated as positive for 7-AAD (histogram). Upper panel graph, The number of IFN-{gamma} spots (i.e., number of APC) per million testicular cells in a reverse ELISPOT assay for presorted testicular cells from each male strain; lower panel graphs, sorted live (left) or dead (right) cells in reverse ELISPOT assays. Two mice per group shown.

 

Figure 2
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  		FIGURE 2. Maternal MHC I+ cells entered the uterine lumen after mating and cross-presented male H-Y Ag to CD8+ T cells ex vivo. A (upper panels), B6 female mice were mated with B10.D2 (H2d) males for 3 h, and cells flushed from the uteri of vaginal plug-positive females were analyzed by flow cytometry. Flushed cells were analyzed by first gating on forward and side scatter (left panel), then were analyzed by further gating on 7-AAD and H2Db (center panel) or isotype control (Iso, right panels). A representative mouse from each group is shown. Lower panels, Relative percentage of maternal H2Db-positive cells in groups of at least three mice per time point shown (mean ± SEM); *, p < 0.05. B, B6 female mice were either not mated (NM) or mated with B10.D2 (H2d) males. B10.D2 (H2d) females were mated with either B6 (H2b) or B10.D2 (H2d) males. Cells flushed from the uteri of 12–16 hpc-mated female mice were titrated into reverse ELISPOT assays containing either sorted naive anti-H-Y Ag CD8+ TCR-transgenic T cells (IL-2; upper panel) or effector anti-H-Y Ag CD8+ TCR-transgenic cells (IFN-{gamma}; lower panel). Mean ± SEM of groups of at least three mice are shown; *, p < 0.05. Five hundred bone marrow- derived male H2b or female H2b DC per well were used as positive and negative controls, respectively.

 

Figure 3
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  		FIGURE 3. Lack of anti-paternal CD8+ T cell responses after mating. A, Memory CFSE-labeled 2C cells were adoptively transferred into females that were mated with third-party B10.BR (H2k) or target-allogeneic B10.D2 (H2d) males. Proliferation of 2C cells was measured by flow cytometry at 3.5 days postcoitus. DC-immunized nonmated females were used as positive controls. B, Memory CFSE-labeled anti-H-Y Ag CD8+ T cells were adoptively transferred into females that were unmated or mated with syngeneic males. Proliferation of anti-H-Y Ag T cells was measured by flow cytometry at 3.5 days postcoitus. Peptide-immunized nonmated females were used as positive controls. A and B (upper panels), Dot plots show transferred transgenic CD8+ cells from H2d-mated (A) or H2b-mated (B) females before immunization. One representative mouse is shown from groups of at least three mice (except the positive control in A, two mice per group). Lower panels, Mean ± SEM of the gated CFSElow populations from the upper panels are shown. SP, Spleen; PA, para-aortic lymph nodes; MES, mesenteric lymph nodes; PLN, nondraining popliteal lymph nodes.

 

Figure 4
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  		FIGURE 4. Anti-paternal CD8+ T cells were not tolerized by mating. A, Memory CFSE-labeled 2C cells were adoptively transferred into B6 females that were mated with third-party B10.BR (H2k, center panels) or target-allogeneic B10.D2 (H2d, right panels) males immunized with target allogeneic B10.D2 DC at 3.5 days postcoitus, and spleens were analyzed at 6.5 days postcoitus. Results from one representative mouse per group are shown. B, Memory CFSE-labeled anti-H-Y Ag CD8+ T cells were adoptively transferred into B6 females that were unmated or mated with syngeneic males, immunized with pSMCY peptide at 3.5 days postcoitus, and spleens were analyzed at 6.5 days postcoitus. Results from one representative mouse from a group of least three mice are shown. A and B, CFSE histogram plots of spleen cells gated on transferred T cells (left panels).

 

Figure 5
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  		FIGURE 5. Allogeneic DC-induced CD8+ T cell responses by i.p., but not IVAG immunization. Antipaternal 2C cells were transferred into female hosts and immunized with allogeneic DC either i.p. or IVAG. Draining lymphoid tissues were analyzed for 2C proliferation 3 days postimmunization. SP, Spleen; PA, para-aortic lymph nodes. Upper panels, Histogram of gated 2C cells from one representative mouse from groups of three mice. Lower panel, Mean ± SEM of 2C proliferation; *, p < 0.05.

 

Figure 6
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  		FIGURE 6. Estradiol inhibited CD8+ T cell responses to IVAG peptide immunization. CFSE-labeled 2C cells from naive mice were transferred into OVX female hosts that were treated with either saline (A; PBS) or E2 or progesterone (B) or E2 for 3 days before being immunized with pSYGL peptide in HBSS either i.p. or IVAG. Draining lymph tissues were analyzed for 2C proliferation 3 days postimmunization. A (upper panels), Histogram of gated 2C cells from one representative mouse from groups of three mice; lower panels, mean ± SEM of 2C proliferation; *, p < 0.05. Draining nodes for positive control s.c. immunization were the cervical, brachial, and axial nodes, and for IVAG immunization were the para-aortic lymph nodes. B, Proliferation of 2C cells in response to peptide immunization IVAG in para-aortic lymph nodes of progesterone or E2-treated mice (mean ± SEM); *, p < 0.05; n = 3/group.

 

Figure 7
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  		FIGURE 7. Estradiol did not directly inhibit the proliferation of Ag-specific CD8+ T cells in vitro. CFSE-labeled 2C cells were incubated with the whole spleen pulsed with pSIYRYYGL peptide in different concentrations of estradiol for 6 days. The graph shows mean ± SEM.

 

Figure 8
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  		FIGURE 8. Estradiol-induced suppression of anti-paternal CD8+ T cell responses was localized to the reproductive tract and did not lead to tolerance. CFSE-labeled 2C cells were transferred into OVX hosts that were treated with either saline (PBS) or E2 for 3 days before being immunized either IVAG or s.c. with pSYGL peptide in HBSS. Upper panels, Proliferation of 2C cells 3 days after peptide immunization. Draining nodes for s.c. immunizations were the brachial, cervical, and axial nodes; for IVAG immunizations, the para-aortic lymph nodes. CFSE histogram plots were gated on transferred cells. Plots show a representative mouse from a group of three mice; lower panels, mean ± SEM of 2C proliferation; *, p < 0.05; #, p < 0.05.

 

Figure 9
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  		FIGURE 9. Estradiol selectively inhibits anti-paternal CD8+ T cell responses in the FRT over a wide range of Ag concentrations. CFSE-labeled OT-I cells were transferred into OVX hosts that were treated with either saline or E2 for 3 days before being immunized either IVAG or s.c. with different doses of pSIINFEKL peptide in HBSS. Upper panels, Proliferation of OT-I cells 3 days after peptide immunization. Draining nodes for s.c. immunization were the brachial, cervical, and axial nodes; for IVAG immunizations, the para-aortic lymph nodes were used. Plots were gated on transferred cells. Lower panels, OT-I proliferation in response to different peptide amounts.

 

Figure 10
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  		FIGURE 10. Male reproductive tissue homogenates induce a CD8+ T cell response after IVAG immunization, but only in the absence of estradiol. CFSE-labeled OT-I cells were transferred into OVX hosts that were treated with either saline (PBS) or E2 for 3 days before being immunized IVAG with either B6 or transgenic B6.mOVA male testicular homogenates (one-fiftieth of combined two testicles) in 10 µl of HBSS. Upper panels, Flow cytometry data from a representative mouse treated with PBS and immunized IVAG with B6 male testicular homogenates. Dot plots (left) show the gate used to identify OT-I cells for the CFSE histograms (right). Lower panels, Proliferation of OT-I cells measured as the percentage of OT-I cells that divided at least once; *, p < 0.05; mean ± SEM; n = 3/group.

 
Flow cytometry

All FITC-, R-PE-, PE-Cy5.5-, PE-Cy7-, allophycocyanin-, allophycocyanin-Cy7-, and biotin-conjugated Abs or streptavidin were purchased from either BD Pharmingen or eBioscience. Alexa Fluor 405-conjugagted anti-mouse CD8{alpha} (5H10) was obtained from Caltag Laboratories. Ab clones were used as follows: anti-mouse: IAd (AMS-32.1), IAb (AF6-120.1), IAk (11-5.2), Dd (34-2-12), Kb (AF6-88.5), Kk (36-7-5), Kd (SF1-1.1) isotype control rat IgG2a (R35-95), F4/80 (BM8), CD46b (DX5), IgG1 (A85-1), TCRbeta (H57-597), CD3 (17A2), CD11b (M1/70), CD11c (N418), CD16/32 (2.4G2), CD44 (IM7), CD45.1 (A20), CD45.2 (104), CD45R/B220 (RA3-6B2), CD62L (MEL-14), CD90.2 (53-2.1), CD127 (A7R34), V{alpha}2 (B20.1), and Vbeta5 (MR9-4). The 7-aminoactinomycin D (7-AAD) was obtained from Calbiochem. The anti-mouse H-Y- transgenic TCR clonotype Ab clone T3.70 was obtained from eBioscience. The anti-mouse 2C-transgenic TCR clonotypic Ab-producing hybridoma clone 1B2 was a gift from J. Schneck (Johns Hopkins University). All samples were analyzed on a BD LSR II flow cytometer.

Collection of postcoitus cells from mated females

We designed the mating experiments to generate a narrow window of insemination times. Mice were left alone to mate for 3 h and then checked for vaginal plugs. Because mating could have occurred at any time during the 3-h window, mice harvested immediately after plug detection were designated as 0–3 h postcoitus (hpc). Alternatively, mice were harvested 12–16 h after vaginal plug detection to obtain maternal cells of the decidual reaction. The uteri were removed and flushed with 20 U/ml heparin (Abraxis Pharmaceutical Partners) in PBS. Cells were washed using HBSS (Mediatech) with 1% FBS (HyClone).

Reverse ELISPOT assay

Numbers of APC were measured by titrating different concentrations of APC populations with constant, saturating numbers of TCR-transgenic T cells in ELISPOT assays for IL-2 or IFN-{gamma} detection. T cells only secrete cytokines on APC stimulation, and therefore the number of spots in this assay represented the number of APC, not the number of T cells. Sorted naive CD8+ T cells (secreting IL-2 but not IFN-{gamma} upon activation) were used to detect APC that could activate naive T cells, and 5 days in vitro-activated effector CD8+ T cells (secreting IFN-{gamma}) were used to detect APC that could restimulate effector T cells. ELISPOT wells were coated with 2 µg/ml purified anti-mouse IFN-{gamma} (AN18) in 1x PBS or 2 µg/ml purified anti-mouse IL-2 (JES6-1A12) in 1x PBS. Constant numbers (30,000/well) of 2C or OT-I TCR-transgenic CD8+ T cells were added to each well and APC were titrated across the plate in RPMI 1640 plus 10% FCS and 2 ng/ml human IL-2 (eBioscience). After overnight incubation, cytokines were detected by the addition of 50 µl of biotinylated anti-mouse IFN-{gamma} (XMG1.2) at 2 µg/ml in PBS/0.1% polyoxyethylene (20) sorbitan monolaurate/1% BSA (PBSTB; Sigma-Aldrich), or anti-mouse IL-2 (JES6-5H4) at 2 µg/ml in PBSTB to each well. Both coating and detection Ab pairs were purchased from eBioscience. After overnight (16 h) incubation at 37°C in 5% CO2, plates were washed in 1x PBS/0.1% polyoxyethylene (20) sorbitan monolaurate, and 1 µg/ml alkaline phosphatase-conjugated streptavidin (The Jackson Laboratory) in PBSTB was incubated on the plate for 30 min at 25°C, then washed off using 1x PBS/0.1% polyoxyethylene (20) sorbitan monolaurate. Plates were developed using the alkaline phosphatase substrate kit III (Vector Laboratories). Spots were counted using the Immunospot CTL scanner and counting software (CTL Laboratory).

Dendritic cells (DC)

Bone marrow-derived DC were generated by methods described elsewhere (41). Briefly, bone marrow was obtained from the tibia of mice, washed in complete medium, and cultured in complete medium plus 100 U/ml IL-4 and 100 U/ml rGM-CSF (eBioscience). Bone marrow-derived DC were activated overnight using 1 µg/ml LPS (Sigma-Aldrich).

Adoptive transfers and reisolation of transgenic cells

All transgenic cells used for adoptive transfers were isolated from the spleen and lymph nodes. Tissues were homogenized using a rubber plunger over a metal mesh strainer in sterile HBSS and filtered several times over sterile cell strainers. RBC were lysed using ammonium chloride-RBC lysis buffer. Cells were labeled with CFSE (Sigma-Aldrich). Five million transgenic TCR-positive T cells in whole spleen were transferred into female hosts i.p. in sterile HBSS. This procedure was used for both the 2C and anti-H-Y Ag-transgenic T cell systems. To isolate the transferred cells, we removed organs of interest, loosely ground the organs using a wire mesh screen and rubber plunger over HBSS to avoid cellular damage, and then filtered the suspension over a cell strainer to remove debris.

Immunizations

For all IVAG immunizations, female mice were treated with fertility hormones as described above. Immunization was performed using a micropette; 10 ìl of HBSS solution was instilled into the upper cervicovaginal region. Mice were anesthetized for the entire procedure using 2,2,2-tribromoethanol at 240 mg/kg of total body weight. Two days postimmunization, spleen, nondraining lymph nodes (popliteal), and draining lymph nodes (para-aortic) were harvested and the transgenic cells were collected for analysis. For s.c. immunizations, mice were injected in the back of the neck with Ag in 100 µl of PBS; cervical, brachial, and axial lymph nodes were used as draining lymph nodes. For i.p. immunizations, mice were injected with Ag in 200 µl of PBS.

Isolation and sort of male testicular cells

Paired testicles from B10.D2 (H2d), B10.BR (H2k), B10 (H2b), or transgenic B6.mOVA males were removed and glass homogenized to release cells. Cells and debris were pelleted by centrifugation at 10,000 x g using a Sorvall RC-26 Plus centrifuge (Sorvall). To separate live vs dead testicular cells, cells were stained with 7-AAD and FACS sorted in HBSS containing 1% FCS, 1 mM EDTA, and 10 U/ml DNase (Roche) to prevent clumping. Gates were set on 7-AAD high/intermediate cells for dead and 7-AAD low cells for live. Cells were sorted into cell culture medium.

Software and statistical analysis

Significance was determined using the Mann-Whitney, nonparametric test; significance was considered for a p < 0.05. Statistical software used was the GraphPad Prism biostatistics software (version 4.0a, 2003). All flow cytometry data were analyzed using the analytical software FlowJo (version 6.3.1, 2005).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
We used two TCR-transgenic CD8+ T cell models (2C and anti-H-Y Ag) to analyze direct, cross-presented, and peptide responses. The 2C TCR-transgenic T cells (42) were used to analyze direct antipaternal responses to H2Ld presenting a ubiquitous selfpeptide or antipeptide response against the synthetic peptide SIYRYYGL (pSYGL) presented by H2Kb (38). Anti-H-Y male Ag-specific TCR-transgenic T cells (43) were used to analyze direct antipaternal responses to an H-Y peptide presented by H2Db on paternal cells, the same peptide cross-presented on H2Db after uptake and processing by maternal cells or antipeptide responses against the synthetic peptide KCSRNRQYL (pSMCY).

Paternal APC in the uteri of mated females

We first tested whether paternal cells crossed into the FRT upon ejaculation. Several reports suggested that somatic cells from the father entered the semen, and that even spermatozoa expressed low levels of MHC I (44, 45, 46). If these cells could penetrate into the mother, they could potentially stimulate antipaternal T cells directly (47). We measured the abundance of these cells and their ability to stimulate maternal host CD8+ T cells.

B6 female mice were treated with fertility hormones and placed with allogeneic B10.D2 (H2d) males for mating. After 3 h, the female mice were checked for vaginal plugs indicative of insemination. Cells flushed from the uteri of mated B6 females contained very low numbers of cells that stained positive for paternal MHC I (Fig. 1A; *, p < 0.05). The numbers of paternal MHC I+ cells increased by 12–16 hpc (Fig. 1A; *, p < 0.05), suggesting either further activation or migration out of the vaginal plug into the uterine lumen. As might be expected from the diversity of cell types in semen, the MHC levels were heterogeneous. The cells flushed from the uterus after mating are expected to represent a subset of the cells from the male reproductive tract, partly because of selective transfer into semen and partly because of selective survival.

To determine whether maternal cells had acquired paternal MHC and appeared as paternal cells in this analysis, cells flushed from a 0–3 hpc uterus from a H2b female mated to a H2d male were stained for both maternal and paternal MHC I (Fig. 1B). The majority (73%) of cells expressing paternal MHC did not express maternal MHC I (H2Db).

To determine whether these paternal MHC I+ cells were functional APC, cells flushed from the uterus 0–3 hpc were tested for the ability to stimulate CD8+ T cells directly ex vivo (Fig. 1C; n = 5/group) in a novel reverse ELISPOT assay, in which APCs were titrated into a saturating number of in vitro-activated 2C TCR-transgenic CD8+ T cells. Because antipaternal alloreactive cells can be found in both naive and effector populations, we tested whether the paternal cells from the uterus could stimulate sorted naive or in vitro-generated effector anti-paternal CD8+ T cells. Cells flushed 0–3 hpc from the uteri of B6 females mated with B10.D2 (H2d) males stimulated effector T cell IFN-{gamma} secretion, whereas cells from third-party B10.BR (H2k)-mated or nonmated females did not (Fig. 1C, bottom panel; *, p < 0.05). However, these flushed cells did not stimulate sorted naive 2C cells to produce IL-2 (Fig. 1C, top panel), although the naive 2C cells could be stimulated efficiently by bone marrow-derived DC from H2d, but not H2k male mice. Thus, paternal cells present in the uterine lumen after mating could stimulate maternal previously activated but not naive 2C CD8+ T cells in vitro.

To test whether viable paternal APC or dead cells and debris were the immunostimulatory fraction of the flushed cells, we used male testicular cells as a model for the semen cells and separated live cells from dead cells and debris for reverse ELISPOT assays. Live male cells stimulated effector 2C-transgenic T cells ex vivo whereas dead cells (high/intermediate 7-AAD) showed no Ag-specific stimulation by H2d vs H2b or H2k testicular cells (Fig. 1D; n = 2/group). As shown in Fig. 1B, effector but not naive 2C cells were stimulated by the male APC (data not shown). These results suggest that live, but not dead, paternal APC from the semen could potentially stimulate maternal CD8+ T cells after insemination. However, the low frequency of paternal cells found in the uterus after mating suggests that either the paternal cells were rapidly cleared or migrated into the reproductive tissue.

Maternal leukocytes flux into the uterine lumen and cross-present paternal Ag

In addition to the direct presentation of paternal Ags by paternal APC in the uterine lumen, the large quantity of cellular debris observed during FACS analysis (e.g., Fig. 1A) and in the vaginal plug suggests that large amounts of male Ags are available in the uterus. Neutrophils, DC, and macrophages enter the uterine lumen as part of the decidual reaction (48, 49, 50). Neutrophils help DC in processing Ag for cross-presentation (51), and DC and macrophages may take up, process, and present male Ag to maternal T cells. Thus, we tested whether maternal cells could acquire and process male Ags in vivo and consequently cross-present male H-Y Ag to specific CD8+ T cells ex vivo. The minor H Ag, derived from the Hya/HYA gene(s) on the Y chromosome, can elicit potent cytotoxic CD8+ T cell responses in vivo (40). Anti-H-Y TCR-transgenic CD8+ T cells recognize an H-Y Ag-derived peptide exclusively in the context of H2Db (39), and therefore cross-presentation of male Ags by infiltrating maternal myeloid cells (48) was tested by mating H2Db females with H2Dd males. In this mating combination, the male Ag could only be presented to H-Y TCR-transgenic T cells by maternal APC.

Twelve to 16 h after vaginal plug detection, mice were sacrificed and the uteri were flushed to collect cells that had entered during the semen-induced decidual reaction. The number of maternal cells expressing high levels of MHC I (Fig. 2A, top panel) was significantly increased at 12–16 hpc compared with 0–3 hpc (Fig. 2A; *, p < 0.05). To test whether the maternal cells that entered the lumen had acquired male H-Y Ag that could be cross-presented to CD8+ T cells, we analyzed the flushed 12–16 hpc uterine cells in our reverse ELISPOT assay using anti-H-Y TCR-transgenic CD8+ T cells as responders (Fig. 2B; n = 5/group). Uterine cells from females that carried the appropriate MHC (H2Db) efficiently cross-presented male Ag to effector anti-H-Y CD8+ T cells, inducing IFN-{gamma} production (Fig. 2B, bottom panel; *, p < 0.05), but did not stimulate sorted naive anti-H-Y CD8+ T cells to produce IL-2 (Fig. 2B, top panel). To control for nonspecific responses, we used various mismatched MHC haplotype matings. Since the anti-H-Y TCR-transgenic CD8+ T cells recognized processed H-Y Ag in the context of H2Db only, we used MHC congenic strains of mice that expressed either the correct (H2Db) or incorrect (H2Dd) MHC for presentation to the anti-H-Y Ag CD8+ T cells. Thus, the presentation of processed H-Y Ag could be restricted to either male or female cells. Only when the female cells expressed the correct MHC and the male cells supplied the H-Y Ag were T cells restimulated (Fig. 2B). Since the reverse ELISPOT assay detects Ag presentation by live but not dead cells (Fig. 1D), it is likely that this APC activity represents live maternal APC that have processed male Ag from either live or dead paternal cells. Thus, male H-Y Ag was transferred during insemination and could be cross-presented by maternal cells to stimulate effector, but not naive anti-H-Y Ag-specific CD8+ T cells.

Lack of anti-paternal CD8+ T cell responses after mating

Because both paternal and maternal APC were detectable in the uterine lumen after insemination and could restimulate effector T cells ex vivo (Figs. 1 and 2), we tested whether maternal anti-paternal memory CD8+ T cells were reactivated in vivo after allogeneic insemination.

Transgenic 2C or H-Y CD8+ T cells were adoptively transferred into female recipients that were subsequently immunized i.p. with activated bone marrow-derived DC from H2Ld+ or syngeneic male mice, respectively. The populations of transferred cells clonally expanded and then contracted to form a population of resting memory cells by day 21. On day 22, lymphoid tissues were collected and isolated memory cells were small, resting, and uniformly CD44high (data not shown). These memory cells were CFSE labeled and retransferred into new female recipients that were subsequently mated. Tissues were harvested at day 3.5 of pregnancy. Both the 2C and anti-H-Y Ag-transgenic CD8+ T cells remained undivided in mice mated with males that carried the cognate Ag or with third-party allogeneic male mice (Fig. 3). A similar lack of response was observed with naive 2C and anti-H-Y Ag CD8+ T cells (data not shown). However, the CD8+ T cells used for the transfer experiments proliferated in vivo upon rechallenge with peptide Ag or Ag-bearing DC injected i.p. in nonmated hosts (Fig. 3; positive control). This suggests that the CD8+ memory T cells were functional, but were not restimulated in vivo as a consequence of mating.

To test whether the Ag-specific T cells had been tolerized during the mating and would not respond when rechallenged with exogenous paternal Ag, we adoptively transferred memory CD8+ transgenic T cells into female mice mated with Ag-expressing or control male mice. Three days after mating, the female mice were immunized i.p. with DC expressing H2Ld (2C) or the H-Y Ag peptide pSMCY (anti-H-Y). At 6.5-day postcoitus, spleens were harvested and processed for flow cytometry. The transferred 2C memory cells in the mated females proliferated to a similar extent in response to the DC challenge, after previous exposure to semen APC expressing either the cognate Ag or a third-party control Ag (Fig. 4A). Similarly, mating with Ag-expressing male mice did not alter the subsequent proliferative response of anti-H-Y-transgenic T cells to DC immunization (Fig. 4B). Anti-H-Y Ag T cells proliferated less than 2C cells in these positive controls, possibly due to affinity differences between the two transgenic TCRs or to the Ag amount or APC involved in the two immunizations. In either case, implantation of the allogeneic blastocyst did not provide an alternative source of paternal Ags as MHC I is not expressed until day 9.5, and the mated but nonimmunized groups did not show any T cell proliferation or tolerance upon DC challenge.

Although we cannot exclude the possibility that a subpopulation of CD8+ T cells was tolerized, these results suggest that a substantial portion of the memory cells were not rendered refractory to in vivo restimulation with exogenous Ag even after mating with males that carried the cognate Ag.

Allogeneic DC induced Ag-specific CD8+ T cell responses by i.p., but not IVAG immunization

The lack of activation of CD8+ T cells by allogeneic mating (Fig. 3), even in the presence of paternal and maternal APC, suggests that either these APC have diminished APC function in vivo compared with the in vitro assay or that fully active APC do not reach lymphoid tissue. We used DC immunization to determine whether stronger paternal APC administered vaginally could stimulate an immune response (i.e., was the lack of response due to weak APC or a weakly immunogenic route). CFSE-labeled, 2C-transgenic T cells were transferred into female mice, which were then immunized either IVAG or i.p. with H2Ld+ male (B10.D2) DC. Strong proliferation of 2C cells was induced by an i.p. immunization but not IVAG immunization with the same numbers of DC (Fig. 5; *, p < 0.05), suggesting that intact paternal APC may not readily penetrate to lymphoid organs or that the Ag-presenting function of the DC was suppressed in the FRT.

Peptide Ags could induce CD8+ T cell responses by IVAG immunization, but only in the absence of estradiol

The results in Figs. 2 and 3 also suggest that indirect Ag presentation is ineffective by the IVAG route. To test this more precisely, we first used a peptide model system to determine whether immunizing with an immunogenic peptide IVAG could stimulate the proliferation of 2C TCR-transgenic T cells in vivo. Initial results using fertility hormone-primed mice showed variable responses (data not shown). Because the estrous cycle affects Ag presentation to CD4+ T cells in the FRT (33), we tested the effect of hormone treatment on CD8+ T cell responses to IVAG peptide immunization.

CFSE-labeled, 2C-transgenic T cells were transferred into OVX female mice treated with either 17beta-estradiol (E2) to mimic the hormone environment of estrus or with PBS as a control. The mice were then immunized either IVAG or i.p. with pSYGL peptide. Intraperitoneal immunization induced strong 2C proliferation in both groups of mice, whereas IVAG immunization induced strong responses in control mice, but much lower responses in E2-treated mice (Fig. 6A; *, p < 0.05). The 2C cells also proliferated in response to Ag in progesterone-treated mice (mimicking the hormone environment of diestrus, Fig. 6B; *, p < 0.05).

To determine whether the lack of a proliferative response to IVAG peptide was due to a direct effect of estradiol on CD8+ T cell activation or proliferation, we tested the effect of estradiol on the proliferative response of CD8+ T cells to Ag-peptide stimulation in vitro. Proliferation of either 2C (Fig. 7) or OT-I (data not shown) naive T cells was not reduced even by local E2 concentrations as high as 1 ng/ml, suggesting that the E2-induced block in IVAG immunization of our antipaternal T cells was not due to a direct affect on the T cells or spleen APC but rather some mechanism in vivo involving access to Ag, effects on a particular subset of APC, or indirect effects on T cell or APC function.

Estradiol treatment prevents vaginal but not s.c. antipaternal responses, without inducing tolerance

Because rat CD4+ T cell responses are inhibited during estrus by selective reduction of the Ag-presenting ability of cells in the FRT but not at other sites (33, 52, 53), and estradiol treatment neither inhibited nor augmented the 2C-transgenic T cell response to allogeneic DC administered IVAG or i.p. (data not shown), we therefore tested whether estradiol inhibited mouse CD8+ T cell responses only in the FRT. CFSE-labeled 2C cells were transferred into OVX estradiol-treated or control mice that were then immunized with peptide Ag either IVAG or i.p. The 2C cells in the draining lymph nodes proliferated in control hosts when Ag was administered either in the FRT (IVAG) or at a distant s.c. site, but in mice treated with E2, proliferation was only observed after s.c., not IVAG immunization (Fig. 8, left and center panels). Estradiol also inhibited CD8+ T cell responses in a second TCR-transgenic model using OT-I cells (data not shown). This experiment confirmed the suppression of 2C responses by estradiol and showed that this suppression was effective in the FRT but not at a distant site.

To test whether this lack of Ag-specific CD8+ T cell proliferation was due to tolerance or ignorance, CFSE-labeled 2C cells were transferred into OVX estradiol-treated or control mice that were then immunized IVAG with Ag peptide. After 3 days, the mice were restimulated i.p. with the same peptide. After another 3 days, the 2C cells in each group had proliferated similarly in response to the i.p. stimulation, suggesting that most of the 2C cells had not been tolerized by the initial IVAG peptide priming in the presence of estradiol (Fig. 8, right panels).

Estradiol-induced inhibition of anti-paternal CD8+ T cell responses in the FRT was not due to differences in tissue Ag saturation

Although these results suggested that estradiol selectively suppressed IVAG antipeptide responses, we also considered the possibility that IVAG immunization might be a more sensitive measure of generalized inhibition if this site had a higher peptide dose requirement. To test this, CFSE-labeled OT-I cells were transferred into OVX estradiol-treated or control mice that were then immunized IVAG or s.c. with different amounts of peptide Ag. The Ag dose-response curves at the two sites were similar, with responses detected down to 80 ng peptide, measured either in the spleen (data not shown) or draining lymph nodes (Fig. 9). Estradiol inhibited antipaternal responses over the whole range of peptide concentrations tested, up to 50 µg of peptide per mouse. Thus, IVAG administration is an efficient route for peptide immunization, and estradiol-mediated suppression of CD8+ T cell responses is tightly restricted to the vaginal site.

Anti-paternal CD8+ T cell responses can be primed IVAG by male cells in the absence of estradiol

Because mice normally do not mate out of estrus, when estradiol levels are high, it is difficult to test the antigenicity of semen Ags via mating in the absence of estradiol. As a surrogate for actual mating, we introduced testicular homogenates from mOVA-transgenic males by artificial insemination in the presence and absence of estradiol in OVX mice that had received adoptively transferred CFSE-labeled OT-I cells. When B6.mOVA male cells and debris were transferred IVAG, OT-I cells in PBS-treated mice responded by proliferating (Fig. 10; *, p < 0.05). However, when the mice were treated with estradiol this response was suppressed. This confirms results with peptide Ags and shows that delivery of physiological amounts of paternal Ags IVAG can prime CD8+ T cells in the mother and that estradiol prevents this response.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Our results support the model that anti-paternal CD8+ T cells fail to be stimulated during insemination, despite the presence of paternal Ag and paternal APC in the FRT after mating. This may be due to multiple mechanisms. First, paternal APC or activated DC do not activate CD8+ T cells when immunogenic numbers of APC (measured by i.p. immunization) are administered by the IVAG route, suggesting either that whole cells are blocked from entering the maternal tissues or that APC are functionally inhibited as they enter maternal tissues. Second, although peptide Ags can activate CD8+ T cells at equal efficiency via IVAG and s.c. routes, only the IVAG immunization is strongly inhibited by estradiol at levels that are present during estrus, when mating normally occurs. Estradiol inhibited CD8+ T cell proliferation in response to DC and peptide immunization. Although we cannot exclude the possibility that the CD8+ T cells were partially activated by APC in the presence of estradiol, such activation would have to occur at an early state, since estradiol also prevented changes in light scattering and up-regulation of activation markers such as CD44 and the early activation marker CD69 (data not shown). Because estradiol inhibited anti-paternal CD8+ T cell activation in three different TCR-transgenic models, this appears to be a robust and general effect.

The effect of estradiol on CD8+ T cell priming may be indirect, since estradiol did not inhibit the activation of CD8+ T cells in vitro. Estrogen has pleiotropic effects, especially on cells of the immune system, and it can inhibit B cell development (54, 55, 56), induce thymic atrophy (57), reduce developing T cell populations (58), induce monocyte apoptosis (59), and inhibit DC differentiation (60). Estrogens increase the production of biologically active TGFbeta from uterine epithelial and vaginal cells, and anti-TGFbeta Abs ablate the estradiol-induced suppression of Ag presentation by rat vaginal cells to CD4+ T cells (34, 61). Estradiol treatment and pregnancy increase the populations of T regulatory cells (62, 63), which can control homeostatic proliferation (64). Estradiol can also up-regulate PD-1 on several APC types (19). Ligation of PD-1 via either PD-L1 or PD-L2 can down-regulate effector T cell function and proliferation (65). This may be related to the ability of estradiol to promote APC that can suppress delayed-type hypersensitivity responses in vivo (66). Another potential mechanism affecting both T cells and APC is the homing of T cells to lymph nodes and APC from the FRT to the draining lymph nodes. Estradiol reduces expression of chemokine receptors CCR2 and CXCR3, as well as reducing migratory activity in response to MIP-1{alpha} and MCP-1 (59).

Other mechanisms may also contribute to the inhibition of maternal anti-paternal CD8+ T cell responses after insemination. Seminal fluid contains several immunomodulatory factors that dampen the maternal immune response to semen Ags; in particular, TGFbeta inhibits the proliferation and effector function of T cells (67, 68, 69). In addition, alternative-activated APC populations that have been shown to play a role in tolerance during pregnancy and can be found at mucosal sites involved in oral tolerance may also play a role in the reproductive tract to control antipaternal responses and may contribute to the results seen in our system (19, 70).

The cell types in semen and the maternal uterine infiltrate that presented MHC and H-Y Ags are not known; however, the H-Y Ag must be processed to be presented in the context of H2Db when the male is of H2Dd origin. Although DC cross-present Ag more effectively than macrophages (71), the uterine APC stimulated effector but not naive CD8+ T cells, whereas activated DC stimulated both types of T cell. Thus, the cross-presenting uterine APC may be relatively immature DC with weak costimulatory molecule expression, or macrophages that have acquired processed Ag during degradation of the high levels of dead male cells present in the uterus after mating. Also, it is possible that maternal APC may be prone to death or dysfunctional in migration since the primary cells involved in this decidual reaction are phagocytes that are thought to ensure the clearance of microorganisms and seminal debris remaining in the tract after insemination (72). Direct presentation of male MHC Ags may also have been mediated by seminal or testicular macrophages or immature DC that entered the semen upon ejaculation (73, 74, 75), since the paternal APC in uterine flushes only activated effector CD8+ T cells. However, neither direct nor cross-presenting APC stimulated responses of either naive or effector/memory CD8+ T cells in vivo. This suggests that paternal APC and maternal APC that enter the uterine lumen 12–16 h after mating may not traffic back into the tissue or may be suppressed by some unknown mechanism.

The APC that primes CD8+ T cell responses to IVAG-administered peptide in vivo may be a conventional DC, because even CD8+ T cells from unprimed mice are effectively induced to proliferate. Although CD4+ T cell help is required for several aspects of CD8+ T cell responses (76, 77), the high frequency of Ag-specific CD8+ T cells in our adoptive transfer model may override a requirement for help (78), at least for initial proliferation of the cells.

Adoptive transfer of large numbers of CD8+ TCR-transgenic T cells may not accurately represent the natural response of low frequencies of Ag-specific T cells in a normal host population during a viral infection (79). However, the normal frequency of alloreactive T cells can be as high as 10% among naive, effector, and memory T cell populations. Thus, the adoptive transfer of substantial numbers of TCR-transgenic T cells may be appropriate to monitor alloresponses. The frequencies of TCR-transgenic CD8+ T cells in our experiments ranged from 0.5 to 2% of host CD8+ T cells after transfer (M. M. Seavey and T. R. Mosmann, unpublished observations), which is below the expected frequency of endogenous CD8+ T cells specific for allogeneic MHC I.

At least three possible mechanisms may explain why systemic administration of estradiol selectively reduces T cell responses in the FRT but not peripherally. First, estradiol may physically alter the FRT, for example, by increasing the layers of tissue during estrus vs diestrus and, thus, reducing penetration of Ag (80, 81). Second, estradiol may inhibit the Ag-presenting capacity of the vaginal APC but not APC at the s.c. immunization site (52). Third, estradiol may induce local suppressor/regulatory cells in the vaginal environment (63) that temporarily block CD8+ T cell activation without inducing long-term tolerance. These potential mechanisms are all consistent with our finding that CD8+ T cells are neither stimulated nor tolerized by peptide immunization in the presence of estradiol.

The focusing of estradiol-mediated inhibition of CD8+ T cell activation in both location and time may help to optimize the maternal compromise between fetal protection and pathogen elimination. The selectivity of the estradiol-induced block of CD8+ T cell activation for vaginal rather than systemic Ag exposure may inhibit responses that might otherwise harm the developing fetus, while helping to reduce the risk of inhibiting antipathogen immune responses beneficial to the survival of the mother. Limitation of this inhibition to the short estradiol-dominated period of the estrous cycle may also help to minimize the risk. However, during infection by sexually transmitted pathogens this mechanism may need to be overridden, for example, by strong inflammatory signals. The estradiol-induced CD8+ T cell inhibition pathway must be precisely balanced: deficiencies might lead to compromised fertility if antipaternal responses are not blocked sufficiently, whereas inhibition for an increased duration or over a wider area might compromise resistance to sexually transmitted pathogens.

It seems unlikely that this estradiol-induced block of anti-paternal CD8+ T cell immune responses reduces immune surveillance during ovarian or endometrial cancer, since these antipaternal cells were not tolerized after mating. Thus, during diestrus (low estradiol) antitumor activities could continue unabated.

Thus, cells presenting paternal Ag are present in the reproductive tract after mating, but multiple regulatory mechanisms appear to mediate the absolutely critical function of preventing antipaternal reactions during pregnancy while maintaining responses against infections. Separate mechanisms may prevent responses to cellular Ags and soluble Ag fragments after insemination. These mechanisms are reinforced by several additional mechanisms dealing with the possible exposure to paternal Ag during embryo implantation, placental expression of gene products, or exposure during parturition to provide comprehensive and multilayered protection of the fetus from maternal immune recognition and damage.


   Acknowledgments
 
We thank Drs. Nick Crispe, Alexandra Livingstone, James Kobie, and Beena John for reviewing this manuscript; Nathan Laniewski and Dr. James Kobie and the Human Immunology Center at the University of Rochester for cell sorting help with flow cytometry; and Drs. Nick Crispe and Johnathan Schneck for the OT-I and 2C TCR-transgenic mice.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by the National Institutes of Health Grant AI051869 and the Multiple Sclerosis Society Grant NS51869. Back

2 Address correspondence and reprint requests to Dr. Tim R. Mosmann, David H. Smith Center for Vaccine Biology and Immunology, University of Rochester Medical Center, 601 Elmwood Avenue, Box 609 Rochester, NY 14642. E-mail address: Tim_Mosmann{at}urmc.rochester.edu Back

3 Abbreviations used in this paper: MHC I, MHC class I; FRT, female reproductive tract; OVX, ovariectomized; IVAG, intravaginal; 7-AAD, 7-aminoactinomycin D; hpc, hours postcoitus; E2, 17beta-estradiol; DC, dendritic cell. Back

Received for publication March 7, 2006. Accepted for publication September 12, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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L. M. Moldenhauer, K. R. Diener, D. M. Thring, M. P. Brown, J. D. Hayball, and S. A. Robertson
Cross-Presentation of Male Seminal Fluid Antigens Elicits T Cell Activation to Initiate the Female Immune Response to Pregnancy
J. Immunol., June 15, 2009; 182(12): 8080 - 8093.
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