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research-article |
Cancer Research UK Institute for Cancer Studies,3 University of Birmingham, Division of Reproductive and Child Health,4 Birmingham Women's Hospital, Birmingham, B15 2TT United Kingdom
ABSTRACT
Recent studies have demonstrated that fetal cells can be detected in the maternal circulation during virtually all human pregnancies. These fetal cells can engraft and may be isolated for many decades after pregnancy, leading to a state that may be maintained by the passage of pregnancy-associated progenitor cells. The clinical consequences of fetal cell microchimerism are unclear but may be potentially detrimental or valuable to the mother. One possibility is the generation of an alloreactive immune response by the mother to antigens expressed by the fetus; for example, the HY protein encoded by the Y chromosome. To test this we have screened a cohort of women with a range of parity histories within 8 yr of their last pregnancy for the presence of an HY-specific CD8+ T-cell response. Fluorescent HLA-peptide (HY) tetramers were used to stain short-term T-cell cultures from these women for analysis by flow cytometry. Responses were detected in 37% of women with a history of pregnancies that produced males, and this value rose to 50% in women with two or more pregnancies that produced males. HY-specific CD8+ T cells also could be detected directly in the peripheral blood of women with a history of at least two pregnancies that produced males. These HY-specific CD8+ T cells produced interferon gamma (IFNG) following peptide stimulation, demonstrating their functional capacity. In conclusion, our data indicate that alloreactive CD8+ T cells are generated frequently following normal pregnancy and retain functional capability for years following pregnancy.
cytokines, immunology, parturition, pregnancy
Studies over the last few years have indicated that fetal cells circulate in the bloodstream of pregnant women and can persist for many years postpartum [1]. Indeed, this fetal cell microchimerism is likely to occur in all pregnancies [2]. Fetal cells pass into the maternal circulation during pregnancy, and it has been estimated that the number of fetal cells in the second trimester is around 1–6 cells/ml [3]. Increased fetomaternal transfer occurs following termination of pregnancy, and women with a history of fetal loss have been reported to exhibit higher levels of fetal cell microchimerism [3]. Further transfer occurs at parturition, but thereafter the number of fetal cells declines, although some studies indicate that microchimerism can be detected in up to 90% of healthy women following pregnancy [4].
Fetal cell microchimerism has been defined as the persistence of fetal cells in maternal organs without any apparent graft-versus-host reaction or graft rejection [5]. However, several lines of evidence suggest that an immune response against paternally-derived antigens present on fetal tissue may be stimulated by normal pregnancy. Cellular and humoral immune responses against paternal antigens have been described following both murine and human pregnancy [6–12], and pregnancy history is a risk factor for the induction of graft-versus-host disease following stem cell transplantation from a female donor [13, 14].
Potential fetal alloantigens include minor histocompatibility antigens (mHAg) encoded by proteins derived from the HY chromosome or polymorphic genes encoded on autosomes [15]. Maternal T cells specific for mHAg peptides derived from HY protein or the HA1 and HA2 antigens encoded by autosomal genes have been identified in women following T-cell culture in vitro [16, 17]. However, there currently is no information on how the priming of such responses is influenced by pregnancy history or the frequency of such cells in peripheral blood.
In this study we have used a large cohort of women with differing parity status to determine the frequency of priming of CD8 T-cell responses to mHAg HY peptides (FIDSYICQV and SPSVDKARAEL) in peripheral blood mononuclear cells (PBMCs) taken within a few years of pregnancy. These two immunodominant peptides were chosen since they are presented by the common Caucasian HLA-alleles HLA-A2 and HLA-B7, respectively.
Thirty-five female donors were selected with differing parity status (either nulliparous, primiparous, or multiparous) and were between ages 18 and 44 yr (median age, 33.5 yr). All donors expressed the HLA-A*0201 and/or HLA-B*0702 genotype and were recruited to the study following informed consent (approved by the South Birmingham Research Ethics Committee). A record of pregnancy and miscarriage history was obtained from each donor. Between 20 and 120 ml of heparinized blood was collected on one or more occasions during the postnatal period. The interval from last pregnancy to the time of study varied between 6 wk and 8 yr.
Generation of T-Cell Lines Against Minor Histocompatibility Antigen Peptides
T-cell cultures were set up according to the adaptation of a previous method [18]. Briefly, fresh PBMCs were isolated by Ficoll-Isopaque density centrifugation and incubated with 30 µg/ml HY peptide (FIDSYICQV or SPSVDKARAEL; Invitrogen, UK) in serum-free RPMI 1640 media for 1 h. Cells were then resuspended in RPMI complete medium (containing penicillin/streptomycin (50 µg/ml), L-glutamine (2 mM; Invitrogen, UK), 10% human serum (HD Supplies, UK) with the addition of interleukin 7 (IL7; 25 ng/ml), and IL15 (2 ng/ml) (Peprotech, UK) . These cells were cultured for up to 2 wk. From Days 3–5 onward cultures were supplemented with IL2 (100 U/ml; Chiron, UK), and media was changed every 3–4 days with IL2-containing complete media.
Generation of HLA Class I/HY Peptide Tetrameric Complexes
Phycoerythrin (PE)-complexed HLA-peptide tetramers containing the HY mHAg peptides FIDSYICQV and SPSVDKARAEL were refolded with HLA-A*0201 and HLA-B*0702 protein, respectively, according to established protocols [19].
Detection of HY-Specific CD8+ T Cells by Staining with HLA-Peptide Tetramer
Fresh PBMCs or cultured T-cell lines were stained with HY-specific tetramers at 37°C for 15 min and washed twice in MACS buffer (PBS/0.5% BSA/2 mM EDTA; Sigma, UK). In order to enrich the population of cells that stained with tetramer, cells were further incubated with anti-PE magnetic beads for 20 min at 4°C, washed twice, and passed over two MS MACS columns (Miltenyi Biotech, Bisley, UK). In some instances, the AUTOMACS (Miltenyi Biotec) was used on the "posseld" program. In all cases, some of the sample prior to column selection was retained for analysis. Both pre-enriched and enriched samples were stained with CD8 FITC (clone SFC12 Thy2D3) or CD8 PC5 (clone B9.11; both from Beckman Coulter, UK), washed, and resuspended in MACS buffer for analysis by flow cytometry (EPICS-XL; Beckman Coulter). Prior to fluorescence-activated cell sorting (FACS) analysis cells were stained with propidium iodide (1 µg/ml; Sigma) to exclude dead cells.
This assay was performed using the Miltenyi Biotec interferon gamma (IFNG) cytokine secretion and enrichment protocol [20]. Briefly, PBMCs were rested overnight in culture plates at a concentration of 1 x 107/ml. The next day cells were incubated with 10 µg/ml HY-peptide (FIDSYICQV or SPSVDKARAEL) for 3 h at 37°C/5%CO2. Cells were harvested and labeled with catch reagent (a bispecific antibody that binds at one end to CD45 expressed on leucocytes, leaving a free binding site at the other end for IFNG) for 5 min and then diluted to a concentration of 1 x 106 cells/ml in warm RPMI complete medium incubated at 37°C for 45 min on a rotator. After incubation cells were washed and subsequently labeled with a PE-conjugated anti-IFNG antibody for 10 min on ice. They were subsequently washed and enriched using anti-PE beads and magnetic selection prior to CD8 counterstaining and FACS analysis, as described above.
Determination of the T-Cell Frequency Following PE-Bead Enrichment
In order to determine the original precursor frequency of antigen-specific T cells from the enriched population obtained after magnetic selection of tetramer or IFNG-positive cells, the following calculation was used [20]. The percentage of antigen-specific CD8+ T cells in the total CD8+ population = (total number of antigen-specific CD8+ T cells after enrichment / total number of CD8+ T cells before enrichment) x 100.
CD8+ T cells from HY-specific T-cell lines were cloned by limiting dilution over irradiated (40 Gy) allogeneic PBMCs, Epstein-Barr virus lymphoblastoid cell line (LCL), and phytohemagglutinin (PHA; 5µg/ml; Sigma) in complete media containing IL2 (100 U/ml; Chiron), IL4 (5 ng/ml), and IL7 (5 ng/ml; Peprotech). Tetramer-staining clones were tested for lysis of HY peptide and HA1 (irrelevant peptide)-loaded female LCLs and male LCLs (HY positive) as targets in standard 4-h chromium release assays [21].
Statistical analysis was performed using Prism (Graphpad). A P value of <0.05 was considered statistically significant.
HY-Specific CD8+ T-Cell Lines Can Be Generated from Women with a History of a Pregnancy That Produced a Male
PBMCs were obtained from 35 women with a range of pregnancy histories. A total of 19 subjects had a history of a single pregnancy that produced a male child, whereas 8 donors had had two or more pregnancies that produced male children. Several women in both groups had also had pregnancies that produced a female child. Four women were studied who had no pregnancy history, and another four had a history of pregnancies that produced only female children. Donors were studied at intervals between 2 mo and 8 yr following their last delivery.
PBMCs were stimulated with HY peptide for 14 days and then stained with HY-specific HLA-peptide tetramers. Tetramer-binding cells were enriched by staining with anti-PE beads, followed by magnetic selection. HY-specific CD8+ T cells were generated from 10 (37%) of the 27 donors with a history of at least one pregnancy that produced a male. There was a trend for more women having an HY-specific CD8 response if they had had two or more boys (50% [4 of 8]), versus one boy (32% [6 of 19]); however, this was not statistically significant (P = 0.32, Fisher exact test). No HY-specific CD8+ T cells were found in women without a pregnancy that produced a male.
The frequency of HY-specific CD8+ T cells within the T-cell lines represented between <0.01% and 3% of the total CD8+ population (Fig. 1A and Table 1), and a magnetic enrichment step was used to confirm the specificity of the response in all cases (Fig. 1B). In this example, 0.07% of CD8+ T cells stained for tetramer (Fig. 1A), and this population was enriched to 76% (1000-fold increase; Fig. 1B) using anti-PE beads and magnetic column selection.
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Direct Detection of HY-Specific CD8+ T Cells in the Peripheral Blood of Donors
As HY-specific CD8+ T cells could be isolated after in vitro culture, it was considered important to see whether they could be detected directly ex vivo. Both HLA-peptide tetramers and the IFNG secretion assay in conjunction with the magnetic enrichment step were used for quantification of HY-specific responses.
Only 7 of the 10 original donors in whom HY-specific CD8+ T cells had been detectable following T-cell culture were available for study, and so 3 additional female donors (patients 11–13) with a history of pregnancies that produced males were recruited (Table 2). The detection limit of antigen-specific cells with and without magnetic selection was taken as 0.0001% and 0.01%, respectively [20]. HY-specific CD8+ T cells were detected in six of the donors, all of whom had a history of at least two pregnancies that produced males. The frequency of HY-reactive T cells ranged between 0.0001% and 0.03% of the CD8+ T-cell pool. No HY-specific T cells were seen in women with a history of just one or no pregnancies that produced males.
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Comparable results were obtained when the analysis was performed with either HLA-peptide tetramers or the IFNG secretion assay; five of the six women in whom HY-specific T cells were detected by tetramers also demonstrated a positive IFNG response. No IFNG response was detected in the six women in whom no HLA-peptide tetramer staining was observed. An example of HLA-peptide tetramer staining and IFNG secretion analysis on peripheral blood is shown in Figure 2.
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A panel of HY-specific T-cell clones was isolated from donor 3 by limiting dilution analysis. HY tetramer-positive clones were assessed for their ability to lyse target cells expressing the HY peptide. T-cell clones were able to kill target cells loaded with HY peptide (10 µg/ml). Moreover, they also lysed unpulsed male LCL targets, indicating that they are able to recognize the HY peptide following endogenous antigen processing. (Fig. 3A). The functional avidity of T-cell clones was assessed by peptide titration, which revealed marked differences in the cytotoxic capacity of individual clones in response to peptide dilution (Fig. 3B). Clone C8 demonstrated lysis of target cells at a 1000-fold lower peptide concentration than clone G12. This difference in avidity of target cell recognition between clones was reflected in increased lysis of male target cells by clone C8 compared with clone G12 (Fig. 3A).
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Transplacental passage of fetal cells into the maternal circulation is now accepted as a physiologic feature of human pregnancy, and fetal cells have been found in the tissues of the majority of pregnant women who have been examined [1, 22, 23]. Fetal cells can persist in the mother for periods of up to 27 yr following delivery [24], and this had led to the suggestion that fetal stem cells may enter the maternal circulation and tissues, possibly reflecting the enrichment of primitive haemopoietic progenitors in fetal blood [25]. The immunologic consequences of such transfer have not been studied extensively, but observations in murine and human studies have suggested that maternal immune responses may be triggered by fetal cells carrying paternal antigens. Many different immunologic processes operate to limit the immunogenicity of fetal cells to the mother, including expression of nonclassical MHC class I molecules in the fetal trophoblast, which can bind to inhibitory NK receptors and protect the fetus from immune attack [26].
A wide range of paternal antigens are expressed on fetal cells, including proteins of the major histocompatibility antigen complex, which will show some degree of mismatch in virtually all pregnancies. Indeed, a humoral immune response to HLA protein is often detected following pregnancy [27], although T-cell immunity is difficult to demonstrate due to a paucity of knowledge concerning HLA-derived peptides that may be presented through maternal HLA alleles.
Minor histocompatibility antigens are peptides that are presented by HLA alleles and act to stimulate allogeneic immune responses in donors who are matched for major histocompatibility antigens. These peptides trigger the allogeneic immune responses that elicit graft-versus-host disease following stem cell transplantation and have been subject to considerable scientific investigation [15]. As mHAg peptides are presented by specific HLA alleles, they represent a useful target with which to study the presence of a cellular allogeneic immune response following pregnancy. HY-specific T cells have been reported previously in parous women. James et al. [16] identified HY-specific T cells in a single donor with a history of three pregnancies that produced males after multiple rounds of in vitro stimulation with peptide. Verdijk et al. [17] identified mHAg-specific T cells in four of seven multiparous women following dual FACS sorting, although precursor frequencies within peripheral blood were not determined. We chose to study mHAg peptides derived from HY proteins, as their presence is indicated clearly by the gender of the fetus and requires no further genetic testing. Several mHAg peptides have been characterized that arise from HY proteins, but the two immunodominant peptides, FIDSYICQV and SPSVDKARAEL, are derived from the SMCY protein and are presented by the HLA-A2 and HLA-B7 alleles, respectively. These were chosen because of the high frequency of these HLA alleles within the population. In addition, these peptides show ubiquitous tissue expression and thus may act as effective immunogens [28].
Our data reveal that HY antigens can prime a CD8+ T-cell response in at least 37% of women who have previously had one or more pregnancies that produced a male. This is likely to be an underestimate, given that only two peptides were chosen for study. The probability of detecting HY-specific CD8+ T cells was increased, although not statistically, when the mother had more than one pregnancy that produced a male, rising to 50% compared with 32% of women with a single pregnancy that produced a male. This is likely to reflect the greater immunologic challenge in multiparous donors, and humoral immunization to paternal antigen is also more commonly detected in women with multiple pregnancies [27]. Unfortunately, we could not address whether T-cell priming to HY occurred during pregnancy or at delivery, since samples were only available postnatally; this, however, would be of interest in future studies.
The prevalence of HY-specific CD8+ T-cell priming was determined by in vitro expansion of T cells in the presence of antigen. This approach was adopted due to the anticipated low frequency of antigen-specific T cells in peripheral blood and has been used widely to detect T-cell responses to viruses or tumor antigens [18, 29, 30]. The relatively high frequency of donors from whom HY-specific immune responses could be elicited led us to determine the frequency of HY-specific T cells directly in the peripheral blood. This was determined by the use of HLA-peptide tetramers and the IFNG secretion assay with magnetic bead enrichment, which increased the sensitivity of detection from 0.01% to 0.0001%. The absolute frequency of such HY-specific T cells ranged between 1:3000 and 1:1 000 000 of the CD8+ T-cell pool, which is comparable to frequencies reported for T-cell responses to nonpersistent viruses and tumor antigens.
The finding of significant numbers of HY-specific T cells in the peripheral blood of parous female donors raises interesting questions as to the functional capability of the cells. This may at least partly depend on the persistence of HY antigen in individual donors. As indicated above, evidence now indicates that fetal cells can persist in maternal tissues and thus may act to restimulate T-cell immunity. The IFNG secretion assay does not allow the detection of naive or anergic T-cell responses, and our data thus demonstrate for the first time that HY-specific T cells circulate within the peripheral blood in a functional state. As previously reported, HY-specific T-cell clones could also be expanded in vitro and have demonstrated the ability to lyse male but not female target cells [17].
The coexistence of an alloreactive immune response together with the presence of antigen indicates a potential risk of immunopathology. Indeed, the persistence of fetal cells has been suggested as a contributory cause to the generation of a range of diseases, such as systemic sclerosis and systemic lupus erythematosus [31–33]. However, it is also possible that immune mechanisms operate in vivo to suppress such alloreactive immune responses. The role of CD4+CD25+ regulatory T cells may be of particular importance, and such cells have been shown to be increased during pregnancy [34]. Their potential role also is suggested by experimental data from murine models that show that syngeneic male skin grafts are tolerated in multiparous mice but not in naive mice [35]. This immunologic tolerance is not due to clonal deletion, as memory HY-specific T cells can be identified in the periphery of multiparous but not naive mice [16]. Furthermore, multiparous mice are impaired in their ability to generate cytolytic cells to HY antigen in vitro following in vivo immunization [36], and adoptive transfer of HY-specific T cells to naive mice can transfer immunologic tolerance [37]. The antigenic specificity of regulatory T cells in this system remains unclear, but it is possible that mHAg-specific cells themselves may contain a regulatory subpopulation [38].
There has been considerable interest in the possibility that male antigen-specific immune responses may contribute to the development of secondary recurrent miscarriage due to a negative impact on the survival of a subsequent pregnancy. Such a proposal is strengthened by the observation that women with a history of recurrent miscarriage and a male firstborn child have a significantly lower chance of a second successful pregnancy than those whose first child was a girl [39].
Our study reveals that functional HY-specific CD8+ T cells are found very commonly in women with a history of a pregnancy that produced a male and can be detected from as early as 6 wk postdelivery to at least 8 yr postpartum. HY-specific T cells are present at a frequency comparable to the cellular immune response to many other antigens. The clinical significance of this alloreactive immune response deserves further attention with regard to its potential detrimental or beneficial effects on the host.
FOOTNOTES
1Supported by the Leukemia Research Fund. 
Correspondence: 2 FAX: 44 121 414 4486; e-mail: k.p.piper{at}bham.ac.uk
Received: 13 July 2006.
First decision: 10 August 2006.
Accepted: 14 September 2006.
REFERENCES
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