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The Journal of Immunology, 2008, 180, 889 -897
Copyright © 2008 by The American Association of Immunologists, Inc.
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Pregnancy Allows the Transfer and Differentiation of Fetal Lymphoid Progenitors into Functional T and B Cells in Mothers1

Kiarash Khosrotehrani2,3,*,{dagger}, Michèle Leduc2,*, Véronique Bachy{ddagger}, Sau Nguyen Huu*, Michèle Oster*, Aicha Abbas§, Serge Uzan* and Sélim Aractingi*,{dagger}

* Université Pierre et Marie Curie, Paris VI, EA4053, Paris, France; {dagger} Assistance Publique-Hôpitaux de Paris, Service de Dermatologie, Hôpital Tenon, Paris, France; {ddagger} Institut National de la Santé et de la Recherche Médicale Unité 712, Hôpital Saint-Antoine, Paris, France; and § Assistance Publique-Hôpitaux de Paris, Service de Biochimie, Hôpital Tenon, Paris, France


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
T lymphocytes of fetal origin found in maternal circulation after gestation have been reported as a possible cause for autoimmune diseases. During gestation, mothers acquire CD34+CD38+ cells of fetal origin that persist decades. In this study, we asked whether fetal T and B cells could develop from these progenitors in the maternal thymus and bone marrow during and after gestation. RAG–/–-deficient female mice (Ly5.2) were mated to congenic wild-type Ly5.1 mice (RAG+/+). Fetal double-positive T cells (CD4+CD8+) with characteristic TCR and IL-7R expression patterns could be recovered in maternal thymus during the resulting pregnancies. We made similar observations in the thymus of immunocompetent mothers. Such phenomenon was observed overall in 12 of 68 tested mice compared with 0 of 51 controls (p = 0.001). T cells could also be found in maternal spleen and produced IFN-{gamma} in the presence of an allogenic or an Ag-specific stimulus. Similarly, CD19+IgM+ fetal B cells as well as plasma Igs could be found in maternal RAG–/– bone marrow and spleen after similar matings. Our results suggest that during gestation mothers acquire fetal lymphoid progenitors that develop into functional T cells. This fetal cell microchimerism may have a direct impact on maternal health.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Fetal cells enter the maternal circulation during pregnancy (1) and persist in some women for decades after delivery (2). This fetal cell microchimerism is a common physiological phenomenon because it has been reported during all human pregnancies and in 30–70% of women with prior pregnancy history (3, 4). In particular, the presence of lymphocytes of fetal origin in maternal circulation has been demonstrated in several studies (5). T cells have been isolated from the circulation or the skin of women affected or not with systemic sclerosis (4, 6). Fetal cells have indeed been detected in maternal circulating CD3+, CD4+, or CD8+ populations (T lymphocyte subsets) in, respectively, 70, 31, and 64% of tested women after pregnancy (5). In addition, T lymphocytes of fetal origin could be cloned from skin samples from scleroderma patients (7). However, studies analyzing fetal cell microchimerism in a variety of autoimmune diseases such as systemic sclerosis, primary biliary cirrhosis, or Sjögren syndrome failed to demonstrate a role for these cells (3, 8, 9, 10). Therefore, the immune consequences of the presence of fetal-derived semiallogenic T cells in maternal tissues are still debated. Besides T lymphocytes, B lymphocytes (CD19+), NK cells (CD56/16+), and monocytes (CD14+) of fetal origin have as well been detected in the circulation of a large proportion of healthy women that had children (5).

The detection of such a variety of lymphoid and monocytic fetal cells decades after delivery may reflect the activity of a common fetal progenitor acquired during pregnancy. Indeed, the presence of fetal hemopoietic progenitor cells in maternal blood during pregnancy has been suspected either by revealing their cell surface CD34+ expression or, functionally, as colony-forming progenitors following in vitro culture of maternal blood (11, 12, 13). The long-term persistence of these fetal-derived presumed progenitors has also been reported (14, 15). Fetal male cells could be detected in the flow cytometry-sorted CD34+CD38+ population, suggesting they were lymphoid progenitor cells (2). However, to date, no study has directly assessed the "stemness" of the fetal CD34+ cells persisting after pregnancy in terms of proliferation and differentiation capacity. In this study, we hypothesized that if mothers acquire lymphoid progenitors of fetal origin during pregnancy, fetal T and B cells should develop in the maternal thymus and bone marrow during and after gestation. We show in this study that in immunocompromised mice in which fetal wild-type lymphocytes have a survival advantage, as well as in immunocompetent mice, fetal-derived lymphocyte progenitors develop in the maternal thymus and bone marrow, in contact with maternal Ags. The resulting fetal cells are functional T and B lymphocytes in the periphery.


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

Eight- to 12-wk-old wild-type C57BL/6 or immunodeficient RAG–/– (B6.129-RAG2tm1Fwa) female mice (Ly5.2 background) were mated to the following strains of males. Ly5.1 (B6.SJL-Ptprca Pep3b) males congenic for the CD45 locus. These males are homozygous for the CD45.1 allele (CD45.1/CD45.1) (CDTA). EGFP (C57BL/6-Tg(ACTbEGFP)) males transgenic for the enhanced GFP (EGFP)4transgene under the control of the chicken β-actin promoter, provided by Dr. M. Okabe (Osaka, Japan) (16). Marilyn (RAG2tm1FwaTg(TcraH-Y, TcrbH-Y)1Pas/Pas) male mice transgenic for the TCR {alpha}- and β-chain recognizing the I-Ab histocompatibility Ag loaded with a peptide from the H-Y male Ag (NAGFNSNRANSSRSS) (17). These TCR transgenic mice had a RAG–/– background (CDTA). OT2 (RAG2tm1Fwa-Tg(TcraTcrb)425Cbn) male mice transgenic for the TCR {alpha}- and β-chain that pairs with the CD4 coreceptor and is specific for chicken OVA 323–339 in the context of I-Ab. These TCR transgenic mice had a RAG–/– background (provided by O. Lantz, Institut Curie, Paris, France). UGFP (C57BL/6-Tg(UBC-GFP)30Scha) male mice transgenic for the GFP transgene under the direction of the human ubiqutin C promoter. RAG–/– males as controls. All animals were maintained in standard pathogen-free conditions and had access to food and water ad libidum. All animal protocols were performed according to current French regulations.

Tissue collection

Between day 14 and 17 of gestation or 4–6 wk after a third delivery, female RAG–/– mice mated with various male mice strains were sacrificed. Thymus, bone marrow, spleen, as well as plasma were collected in 1x PBS, 0.5% BSA, and 2 mM EDTA, or snap frozen in liquid nitrogen. Single-cell suspensions were obtained and further analyzed.

Flow cytometry (FACS)

One million cells from each lymphoid organ were used for the assay. Cells from thymus and spleen were stained with anti-CD45.1 coupled to FITC, anti-TCR β-chain, anti-CD3 or anti-CD127 (IL-7R) coupled to PE, anti-CD8 coupled to PerCP-Cy5, and anti-CD4 coupled to allophycocyanin Abs. When appropriate, we used anti-Va2 to detect the OT2 anti-OVA TCR. Similarly, bone marrow and spleen were labeled with anti-CD45.1 coupled to FITC, anti-IgM H chain coupled to PE, and anti-CD19 coupled to allophycocyanin. Before staining, all cells were incubated with unlabeled anti-CD32 (24G2) Ab (BD Pharmingen). Cells were analyzed on a FACSCalibur cytometer using the CellQuest Pro software (BD Biosciences) and analyzed using Flowjo. Extreme precaution was taken in the order of samples during acquisition. Samples from negative controls and test samples were always analyzed before wild-type animals to exclude carryover of acquisition from one sample to the other.

Magnetic cell sorting (MACS)

Cell suspensions (1–10 million cells) were incubated with anti-CD3 coupled with biotin, followed by anti-biotin Abs coupled to magnetic beads. Similarly, anti-CD4, CD8, or IgM Abs directly coupled to magnetic beads were used for positive selection (Miltenyi Biotec). Recovered cells were pelleted down and frozen at –80°C for further DNA extraction.

DNA extraction and real-time quantitative PCR

DNA extraction from cell pellets was performed, as recommended by manufacturer (Qiagen). Real-time quantitative PCR amplification of the egfp transgene was performed on sorted cells, as previously described. The number of amplified genome equivalent in the egfp assay was reported to the total number of genomes obtained after amplification of a shared genomic sequence of the apolipoprotein b gene to estimate the frequency of EGFP transgenic cells in the sample (18).

Functional assay

ELISPOT assays for IFN-{gamma} secretion were performed, as previously described (19). In each well, 500,000 responder total splenocytes were incubated 24 h in plates (Millipore) precoated with anti-IFN-{gamma} Ab (BD Biosciences), in the presence of 100,000 syngenic bone marrow-derived dendritic cells loaded with various amounts of the H-Y or the OVA peptide. Alternatively, responders were incubated with various quantities of allogeneic (H-2d) bone marrow-derived dendritic cells obtained from BALB/c mice after 1-wk culture in 200 U/ml GM-CSF. After 24-h incubation, plates were washed and revealed with a complementary biotin-coupled anti-IFN-{gamma} Ab (BD Biosciences). Streptavidin-conjugated HRP was added to the wells, and, after washing, revelation was performed by adding the substrate 5-bromo-4-chloro-3-indolyl phosphate plus NBT (R&D Systems).

Western blot

Plasma was obtained from circulating blood of sacrificed animals. Total plasma (4 µl) was loaded for each sample on a nondenaturating agarose gel and blotted to a nitrocellulose membrane. Serum Igs were revealed using an anti-mouse Ig Ab coupled to alkaline phosphatase. As a positive control, plasma from Ly5.1 mice was used after dilution (1/200).

Immunohistochemistry

RAG mice mated with UGFP male mice were sacrificed at day 14–17 of gestation. Thymus and spleen were collected. Mice were perfused with 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) via heart puncture. The tissues were quickly frozen.

The tissue blocks were sectioned (3–4 µm thickness) and then incubated for 20 min in PBS with 0.1% Triton X-100. Tissues were washed with 1x PBS, blocked with 20% normal goat serum, and incubated with a rabbit anti-GFP (1/40; Chemicon International) alone or combined with rat anti-CD45 (1/10; BD Pharmingen) for 3 h. Goat anti-rabbit Texas Red (1/100) was then used for the simple staining. Alternatively, goat anti-rabbit FITC (1/200) and goat anti-rat Texas Red (1/100) were used for double staining. Slides were then counterstained with 0.3 µg/ml 4',6'-diamidino-2-phenylindole and mounted. Each tissue section was then carefully searched for the presence of GFP+ fetal cells and observed under a fluorescence microscope (Leica Microsystems) with a QImaging digital camera (Media Cybernetics).

Statistical analysis

The frequency of fetal cells per million maternal cells was considered as a continuous variable. Groups of mice (RAG–/– vs wild-type) were compared using Student’s t test. Categorical variables (presence or absence of fetal double-positive (DP) cells in maternal thymus) were compared between different groups using Fisher’s exact test. A p value below 0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Lymphocytes of fetal origin can be found in maternal various lymphoid tissues during pregnancy

RAG–/– female mice were mated to EGFP+ males. As a result, all fetuses were heterozygous for the RAG locus and 50% were transgenic for the EGFP. During the third week of pregnancy, the thymus, spleen, and bone marrow from each individual RAG–/– mouse bearing transgenic fetuses were assessed for the presence of fetal cells. After magnetic sorting with anti-CD3 Ab, fetal-derived cells could be detected by egfp amplification in 7 of 10 thymuses and 5 of 10 spleens (Fig. 1). The frequency of the CD3+ fetal cells ranged from 29 to 4249 fetal per million maternal cells in the thymus and from 4 to 838 fetal per million maternal cells in the spleen. No amplification could be observed in genomic DNA obtained from virgin B6 or RAG–/– mice. We also amplified the egfp transgene in the sorted CD4, CD8, or IgM+ cells from maternal spleens (Fig. 1). Similarly, IgM+ fetal cells could be detected in the bone marrow in 4 of 10 mice. We also did these experiments in C57BL/6 wild-type female mice bearing transgenic fetuses at a similar gestational time point. In wild-type unsorted maternal tissues, we found fetal egfp+ cells in 5 of 13 thymuses, 2 of 13 spleens, and 4 of 13 bone marrow. After sorting for CD3, fetal cells could be detected in 4 of 13 thymuses and in 1 of 13 spleens (Fig. 1). Similarly, IgM+ fetal cells could be detected in the bone marrow in 1 of 13 mice. The number of fetal cells in sorted populations from wild-type mothers was significantly lower when compared with RAG–/– mothers in thymus (89 vs 786 CD3+ fetal per million CD3+ maternal cells, p = 0.03), spleen (5 vs 133 CD3+ fetal per million CD3+ maternal cells, p = 0.04), or bone marrow (4 vs 56 IgM+ fetal per million IgM+ maternal cells, p = 0.05) (Fig. 1).


Figure 1
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  		FIGURE 1. Fetal-derived lymphocytes in various maternal lymphoid tissues during pregnancy. Cell suspensions from various maternal lymphoid tissues from 10 RAG–/– female (RAG) mice bearing EGFP+ fetuses were compared with 13 C57BL/6 (wild-type (WT)) mice bearing EGFP+ fetuses sacrificed at gestational age 14–17. Cells were sorted using various T or B lymphocyte markers. Positively selected cells were then assessed for the presence of fetal cells using egfp transgene amplification by real-time quantitative PCR. Points represent the log of the number of fetal cells per million maternal cells in each individual mouse.

 
At a later time point during gestation (17–21 days), the same experiments in wild-type female mice bearing transgenic fetuses could detect fetal cells in 8 of 25 unsorted thymuses and 2 of 25 unsorted spleens.

T cells of fetal origin develop in the maternal thymus during and after pregnancy

To further characterize CD3+ fetal cells in maternal thymus, we used flow cytometry to detect and phenotype the fetal cells. We mated RAG–/– mice to Ly5.1 males. All fetuses from such mating are heterozygous for the rag locus and have a normal phenotype. Besides, they all have the paternal CD45.1 allele on their leukocytes. In 3 of 33 RAG–/– maternal thymuses examined during pregnancy, we found the presence of DP (CD4+CD8+) thymocytes that displayed the fetal CD45.1 Ag (Fig. 2). Fetal cells also expressed the CD3 (or the TCR β-chain; data not shown). In each experiment, a RAG–/– mouse mated to a RAG–/– male at a similar gestational stage (n = 7) as well as male RAG–/– mice (n = 13) or RAG–/– retired breeders (n = 15) were used as controls and never displayed any CD45.1+ DP cells, excluding contamination from sample to sample.


Figure 2
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  		FIGURE 2. RAG–/– maternal thymus allows the development of fetal DP T lymphocyte precursors acquired during gestation. RAG–/– mice were mated to wild-type Ly5.1 males (middle column). At day 14–17 of gestation, maternal thymus was recovered and analyzed for the presence of fetal CD45.1+ cells and compared with the thymus of RAG–/– females mated to RAG–/– males (left column) or to Ly5.1 males (right column). Gated CD4+CD8+ DP or CD4+CD3+ T cells expressed the fetal marker CD45.1 in RAG–/– mice mated to Ly5.1 males.

 
Similarly, wild-type mice were mated to UGFP males and assessed for the presence of fetal GFP+ cells at day 14–17 of gestation. We found DP (CD4+CD8+) fetal thymocytes in 2 of 14 wild-type thymuses. Wild-type mice (n = 6) were used as controls and did not display GFP+ staining (Fig. 3).


Figure 3
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  		FIGURE 3. C57BL/6 maternal thymus allows the development of fetal DP T lymphocyte precursors acquired during gestation. C57BL/6 mice were mated to UBC-GFP males. At day 14–17 of gestation, maternal thymus was recovered and analyzed for the presence of fetal GFP+ cells and compared with the thymus of C57BL/6 females or with UBC-GFP males. Gated GFP+ fetal cells were distributed in CD4+CD8+ DP, CD4+CD8–, or CD4–CD8+ SP T cells.

 
In addition, the presence of fetal cells in maternal thymus was confirmed by immunostaining. We collected the thymus and the spleen from a RAG–/– female mouse after mating to a UGFP male at day 14–17 of gestation. We identified GFP+ fetal cells in the thymus, but not in the spleen. The fetal cells formed groups of four to eight cells, suggesting division of a common progenitor (Fig. 4A). Double staining revealed mostly CD45+ fetal cells (Fig. 4B). However, a few CD45– cells of fetal origin could be observed.


Figure 4
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  		FIGURE 4. Fetal CD45+ cells form clusters in maternal thymus during gestation. RAG–/– female mice were mated to UBC-GFP male. During gestation, animals were sacrificed and thymus was assessed for the presence of fetal GFP+ cells. A, Horizontal panels on the right, Photomicrograph showing the presence of fetal cells using anti-GFP immunofluorescence (red) on a RAG–/– maternal thymus (x1000 original magnification). Two vertical left panels, Display a positive (UBC-GFP transgenic mouse) and a negative control (RAG–/–) with the anti-GFP immunofluorescence staining (x400 original magnification). B, Combined staining of GFP (green) and CD45 (red). Presence of GFP+CD45– (yellow arrow) or GFP+CD45+ (white arrows) fetal cells GFP in maternal thymus (x1000 original magnification). Two vertical left panels, Display a positive (UBC-GFP transgenic mouse) and a negative control (RAG–/–) with the anti-GFP and anti-CD45 immunofluorescence staining (x400 original magnification).

 
To further confirm that fetal CD4+CD8+ DP cells were immature and developed in maternal thymus, we examined different markers of differentiation such as IL-7R (CD127) and the level of TCR expression. In RAG–/– as well as in wild-type maternal thymus, we observed the presence of fetal GFP+IL-7R– and GFP+IL-7R+ cells (Fig. 5A). The GFP+IL-7R– population was CD4+CD8+ DP. In contrast, the GFP+IL-7R+ population consisted in majority in single-positive (SP) CD4+ or CD8+ fetal thymocytes. In accordance, the fetal CD4+CD8+ DP population had a low level of TCR expression, whereas the CD4+ or CD8+ SP population expressed the TCR at high levels (Fig. 5B).


Figure 5
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  		FIGURE 5. Fetal thymocytes express various stage differentiation markers in maternal thymus. A, Flow cytometry of C57BL/6 maternal thymus bearing UBC-GFP transgenic fetuses at day 14–17 of gestation. Gated GFP+IL-7R+ or GFP+IL-7R– fetal cells were analyzed for CD4 or CD8 expression. The IL-7R+ fetal cells (R1) were in majority SP (CD4+ or CD8+), whereas the IL-7R– fetal cells (R2) were mainly DP (CD4+CD8+) cells. B, Flow cytometry of RAG–/– maternal thymus bearing Ly5.1+/– fetuses at day 14–17 of gestation. SP or DP cells based on their CD4 or CD8 expression were gated and assessed for their level of TCR expression. The DP (CD4+CD8+) fetal cells (R1) expressed the TCR at low level, and the SP (CD4+ or CD8+) fetal cells (R2 and R3) expressed the TCR at high level.

 
The development of fetal thymocytes in maternal thymus during pregnancy prompted us to ask whether this phenomenon could persist long-term after delivery. We therefore assessed RAG–/– mice that gave birth three times to RAG+/– CD45.1+/– pups. Animals were sacrificed 4–6 wk after the third delivery. Two of eight RAG–/– mice displayed CD4+TCR-β+ and CD8+TCR-β+ fetal cells in the thymus (Fig. 6). No TCR+ or CD45.1+ cells could be recovered from thymuses of RAG–/– mice mated three times with RAG–/– males. We also examined RAG–/– retired breeders (n = 15) and never found any TCR+ or CD45.1+ cell (data not shown). Similarly, in another mouse model, RAG–/– female mice were mated to OT2 males. Four to 6 wk after a third delivery, two of nine thymus displayed DP (CD4+CD8+) thymocytes that expressed the fetal V{alpha}2 marker (Fig. 7). CD4+ V{alpha}2+ SP fetal cells could also be retrieved in the same thymuses displaying higher levels of TCR expression. CD8+ V{alpha}2+ cells were absent, suggesting a positive selection of the fetal T lymphocytes.


Figure 6
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  		FIGURE 6. Long-term persistence of T cells of fetal origin in maternal thymus. Four to 6 wk after a third delivery of Ly5.1+/– pups, RAG–/– maternal thymus was recovered and assessed for the presence of CD45.1-expressing fetal cells. We compared RAG–/– female mice mated three times to Ly5.1 males (middle column) with RAG–/– mice mated to RAG–/– males (left column) and to Ly5.1 control mice (right column). Gated CD4+CD8– (R1) and CD4–CD8+ (R2) thymic cells were further assessed for the expression of CD45.1 and the β-chain of the TCR.

 

Figure 7
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  		FIGURE 7. TCR transgenic fetal cells develop in the maternal thymus after three gestations. RAG–/– were mated to OT2 transgenic males. After a third delivery, comparisons were made with RAG–/– and with male OT2 mice. Gated CD4+CD8+ (R1), CD4+CD8– (R3), and CD4–CD8+ (R2) thymic cells were further assessed for the expression of CD4 and the V{alpha}2 chain of the TCR.

 
One possible explanation for the presence of CD45.1+ T cells in RAG maternal thymus might have been the persistence and migration of paternal lymphocytes from the sperm. To rule out this hypothesis, we performed similar experiments using Marilyn males. This mouse line had a RAG–/– background and was transgenic for the {alpha}- and β-chains of a TCR specific for the H-Y (male) Ag. As a consequence, Marilyn male mice do not have any T lymphocytes in the periphery due to thymic deletion of their autoreactive (anti-H-Y) T cells. RAG–/– female mice were mated to Marilyn males. After three gestations, animals were sacrificed and evaluated for the presence of T lymphocytes in maternal thymus (data not shown). In CD4+CD8+ thymic cells, we found TCR β-chain-expressing lymphocytes in three of four tested mice.

Overall, these experiments allowed us to observe the differentiation of fetal thymocytes in maternal thymus in 12 of 68 tested mice during or after gestation compared with 0 of 51 control mice (p = 0.001).

T cells of fetal origin in maternal spleen display allogenic or Ag-specific activity during and after pregnancy

Given the presence of fetal cells in maternal thymus, we analyzed spleen as a secondary lymphoid organ to assess the presence and the functionality of fetal T cells. Because different hemopoietic populations (lymphocytes, monocytes, and granulocytes) do not have the same size, we evaluated the side scatter intensity of CD45.1+ cells in the spleen of RAG–/– mice mated with male Ly5.1. The CD45.1+ cell population was mostly (57%) lymphoid during gestation (data not shown). After three gestations, fetal cells were again mostly lymphocytes (75%). However, in one mouse, 61% of the CD45.1+ cells were monocytes (data not shown).

We next aimed to better characterize the lymphocytic population of fetal origin. In 2 of 33 maternal spleen fetal CD4+ or CD8+ T cells were detected during gestation (Fig. 8). Similarly, in the spleen of mice that had three gestations, fetal-derived T cells could be detected in 3 of 14 mice (data not shown). No TCR+ or CD45.1-positive cells could be recovered from spleen or thymuses of RAG–/– mice mated three times with RAG–/– males. We also examined RAG–/– retired breeders (n = 15) and never found any TCR+ or CD45.1+ cell (data not shown). Interestingly, in RAG–/– mice mated three times to OT2 males, we detected the presence of V{alpha}2+ cells in 2 of 9 spleens, but they did not express CD4 or CD8 costimulatory molecules (Fig. 9).


Figure 8
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  		FIGURE 8. T cells of fetal origin can be found in the maternal spleen during gestation. During gestation (day 14–17), maternal spleen was recovered and CD3+ cells were sorted using MACS and assessed for the presence of fetal CD45.1+ cells. We compared RAG–/– female mice mated to Ly5.1 males (middle columns; two examples provided; only one has detectable fetal cells; circle) with RAG–/– mice mated to RAG–/– males (left column) and to unsorted splenocytes from Ly5.1 males (right column).

 

Figure 9
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  		FIGURE 9. TCR transgenic fetal cells develop in the maternal spleen after three gestations. RAG–/– were mated to OT2 transgenic males. Splenocytes were recovered and assessed for the presence of TCR V{alpha}2-expressing cells in the CD4+ population. Comparisons were made with RAG–/– mice and with male OT2 mice.

 
To assess whether T lymphocytes of fetal origin were functional, we tested their capacity to drive an allogeneic reaction. Total splenocytes of RAG–/– mice mated three times to Ly5.1 males (H-2b) were incubated with bone marrow-derived dendritic cells from BALB/c mice (H-2d). The level of activity of responders was compared with a wild-type Ly5.1 mouse and with RAG–/– mice that gave birth three times to RAG–/– fetuses. Two of eight mice displayed significant levels of IFN-{gamma}-producing splenocytes as compared with the negative controls, showing the influence of prior pregnancies on the allogeneic response (Fig. 10). However, fetal-derived T lymphocytes could not be isolated from background in these animals due probably to their low frequency (data not shown).


Figure 10
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  		FIGURE 10. RAG–/– maternal splenocytes display IFN-{gamma}-producing cells in response to an allogenic stimulus. Splenocytes from RAG–/– mice (H-2b) were recovered 4–6 wk after a third gestation (after coupling with Ly5.1 males) and incubated with various numbers of bone marrow-derived dendritic cells (H-2d) (n = 6). The frequency of IFN-{gamma}-secreting cells was compared with that obtained in splenocytes from wild-type Ly5.1 males or RAG–/– mice mated three times with RAG–/– males. Two of the eight tested mice (RAG18 and 19) displayed IFN-{gamma}-expressing cells. Six other RAG–/– mice coupled three times to Ly5.1 males had background level of IFN-{gamma}-producing cells comparable to the RAG–/– females that were mated to RAG–/– males.

 
Similarly, we assessed the capacity of fetal cells to develop Ag-specific T cell responses. Total splenocytes of RAG–/– female mice mated three times to Marilyn males were incubated with bone marrow-derived dendritic cells from congenic mice (C57BL/6) loaded with various quantities of the H-Y Ag peptide. A high frequency of IFN-{gamma}-secreting cells was found in two of four of these animals when compared with RAG–/– mice mated only with RAG–/– males (Fig. 11A). In addition, a clear dose response could be established by varying the peptide concentration for at least one of the animals. However, identification of TCR+ cells in the spleen of these animals could not be performed due to low frequency and high background (data not shown).


Figure 11
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  		FIGURE 11. Fetal lymphocytes develop Ag-specific T cell responses in maternal spleen after delivery. RAG–/– female mice were mated three times to Marilyn (A), OT2 (B), or RAG–/– males. Four to 6 wk after a third delivery, 500,000 splenocytes were recovered and cultured in presence of 100,000 syngenic bone marrow-derived dendritic cells loaded with various quantities of the H-Y peptide (A) or syngenic splenocytes loaded with the OVA peptide (B). A, Male and female Marilyn splenocytes were also used as negative and positive control, respectively. Two of four previously mated RAG–/– mice (RM32 and RM26) had IFN-{gamma}-secreting T cells in response to H-Y stimulation. B, OT2 splenocytes were used as positive control. Two of five previously mated RAG–/– mice (ROT2*1 and ROT2*4) had IFN-{gamma}-secreting T cells in response to OVA Ag stimulation.

 
Similarly, splenocytes from RAG–/– female mice mated three times to OT2 males demonstrated the presence of a specific reaction against OVA 323–339 peptide presented by syngenic splenocytes from RAG–/– mice: two of nine mice had a response level higher than the negative control (RAG–/– splenocytes) (Fig. 11B).

Ab-producing B lymphocytes of fetal origin can be detected in maternal spleen during and after pregnancy

IgM+CD19+ cells of fetal origin were tracked in animals during and after pregnancy. In the spleen of RAG–/– mice mated to Ly5.1 males, after MACS enrichment of splenocytes for cell surface IgM, CD45.1+CD19+ cells could be detected during pregnancy between day 14 and 17 (2 of 33 tested mice) (Fig. 12A). Similar findings could be made more frequently (7 of 13 tested mice) in the spleen of RAG–/– mice bred to Ly5.1 males, 4 wk after a third delivery. Importantly, in 1 of the 13 tested mice, CD19+IgM+ cells of fetal origin could be as well detected in the bone marrow. We also assessed the presence of fetal c-kit+ cells in the bone marrow, but failed to detect any using flow cytometry. We never recovered any IgM+CD19+ cell in RAG–/– mothers mated to RAG–/– males during or after pregnancy (Fig. 12B). We finally tested RAG–/– mice that gave birth to wild-type pups for the presence of serum Igs. A Western blot analysis could find low levels of Abs in two of seven tested mice. In these mice, Igs were restricted in heterogeneity, suggesting a mono- or oligoclonal expansion of B cells. No Igs could be detected in the sera from RAG–/– mice mated to RAG–/– males (Fig. 12C).


Figure 12
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  		FIGURE 12. Fetal-derived B cells are detectable in the bone marrow and spleen in RAG–/– mice mated with Ly5.1 males during and after gestation. RAG–/– female mice were mated to Ly5.1 or to RAG–/– males (middle and left columns, respectively). During gestation (A) or after three matings (B), animals were sacrificed and spleen and bone marrow were assessed for the presence of fetal-derived IgM-expressing B lymphocytes. Results were compared with wild-type Ly5.1 males. The presence of CD19+IgM-expressing cells of fetal origin (CD45.1+) could be detected. C, In addition, a Western blot analysis was performed on the plasma of RAG–/– mice that were mated three times either to wild-type Ly5.1 or to RAG–/– males and revealed with an anti-mouse Ig Ab. As shown in C, two of seven RAG–/– mice mated to wild-type male had detectable Igs after three gestations.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
In this study, we report for the first time the capacity of fetal lymphoid progenitor cells acquired during pregnancy to develop in maternal thymus and bone marrow into functional T and B cells in wild-type as well as immunodeficient hosts. The cells of fetal origin consistently harbored a paternal genetic marker that was absent in the mother in a variety of study models. Thymic development of T cells has long been described and is characterized by a combination of cell surface markers at each stage (20). In our study, we found immature DP T cells of fetal origin in maternal thymus harboring characteristic IL-7R and TCR expression patterns (21). In addition, SP CD4 or CD8+ lymphocytes of fetal origin with high IL-7R and TCR expression patterns were also described. Altogether, the presence of fetal thymocytes at various T cell differentiation stages strongly implies that fetal T cell progenitors enter and differentiate in the maternal thymus. In accordance, morphologically, the fetal-derived thymocytes were observed in clusters, suggesting intense cell division. In addition, when harboring a TCR restricted to class II MHC molecules, fetal SP thymocytes displayed only CD4+ phenotype, suggesting that the positive selection of fetal cells in maternal thymus follows a similar course compared with potential maternal cells. Many studies have pointed on the presence of fetal T cells in the circulation or tissues of mothers after pregnancy (3, 4). Our results strongly suggest that these cells result at least in part from the differentiation of fetal T lymphocyte progenitors in the maternal thymus.

The number of mice displaying fetal-derived T cells in maternal thymus proved higher with real-time quantitative PCR than flow cytometry. This may be due to the difficulty of detecting cells with a frequency below 0.1% using FACS. The development of fetal progenitor cells in maternal thymus may therefore be a more widespread phenomenon underestimated by our flow cytometry assay. In addition, it has also been shown that T cell progenitors do not enter the thymus continuously, but have a gated importation time set (22). Given the very low frequency of microchimeric cells in the bone marrow (based on our PCR results), we believe that the odds of a fetal T lymphocyte progenitor entering the maternal thymus at a given time point before analysis of the thymus may explain the observed low frequency. In fact, in most mice, fetal lymphoid progenitor cells may either be absent, be present but unable to have a viable TCR β- and {alpha}-chain rearrangement, or be present with a viable TCR but at a stage before amplification. It seems reasonable to consider that the only stage in which fetal cells can be vizualized using flow cytometry is when TCR rearrangement has been successful, resulting in the amplification of the DP population. We also observed the presence of fetal CD19+IgM+ B cells in the bone marrow, suggesting again the development of fetal B lymphocytes in this maternal primary lymphoid tissue. However, these cells could be pre-B, immature as well as mature B lymphocytes, and our assay could not conclusively address this matter. We could not directly demonstrate the presence of fetal progenitors expressing c-kit in the bone marrow. However, such progenitors are expected to be present at a very low level, presumably below the detection threshold of flow cytometry. Our results overall strongly argue toward the transfer of lymphoid progenitor cells from the fetus to the mother.

The fetal lymphoid progenitor cells acquired by mothers during pregnancy have been previously described. CD34+ cells and clonogenic cells of fetal origin have been isolated from maternal blood during pregnancy (11, 15). In particular, fetal cells with surface markers of lymphoid progenitors have been reported decades after delivery (2). These cells may enter the maternal circulation from the placenta because this tissue has been shown to contain hemopoietic stem cells able to reconstitute an irradiated host (23). Our study demonstrates for the first time that fetal lymphoid progenitor cells acquired naturally during gestation are effective in terms of development in maternal thymus. In addition to lymphocytes, based on their size, fetal cells could also display a monocytic morphology, suggesting that the progenitors acquired by the mother during gestation may not be restricted to the lymphoid lineage. However, in our RAG–/– model, lymphocytes had a competitive advantage in the empty maternal host. Therefore, the capability of the fetal progenitor cells has been correctly explored within the lymphoid lineage only. Indeed, our results demonstrate a higher frequency of fetal-derived cells in RAG–/– compared with wild-type mice. Bonney and Matzinger (24) have previously reported the presence of higher numbers of microchimeric cells in immunodeficient scid mice. They argued that their observation was due to the lack of antifetal immune response by maternal cells. Our results suggest that this increased frequency may be in part due to a lack of homeostatic competition encountered by fetal T and B cells developing in maternal immunodeficient tissues.

Splenocytes from RAG–/– mice that were mated to wild-type males displayed cells that were able to react to alloantigens when compared with RAG–/– mice that were mated with RAG–/– males. In both groups of mice, maternal NK cells could be responsible for the observed IFN-{gamma} secretion. It has indeed been shown that murine NK cells could recognize the absence of syngeneic histocompatibility molecules on neighboring cells, resulting in their activation (25). However, we believe the differences seen between the two groups of mice are also due to the presence of T cells of fetal origin. This is further proven by the experiments on RAG–/– mice mated to Marilyn males. We observed Ag-specific and dose-dependent T cell response to the H-Y peptide only in mice that were bred to Marilyn males and that could have acquired transgenic fetal cells capable of developing anti-H-Y TCR-bearing T lymphocytes. In addition, spontaneous Ab production was found in two of seven tested RAG–/– mice after gestation.

Our results add to a strain of previous findings suggesting that during pregnancy, women acquire a population of multipotent stem cells able to adopt various phenotypes in maternal injured tissues (26). Fetal-derived cells can adopt epithelial, hepatocytic, hemopoietic, cardiomyocytic phenotypes in human studies (27, 28, 29, 30). Murine models have also clearly shown the capacity of fetal cells acquired during pregnancy to home to damaged maternal tissues and to acquire different phenotypes, such as hepatocytes, kidney tubular cells, or various CNS phenotypes (31, 32, 33). However, none of these studies could demonstrate a functional activity of the differentiated fetal cells. In addition, it was not clear whether the microchimeric fetal cells had an impact on maternal health. In this study, we demonstrate for the first time that fetal microchimeric cells can help restore a function in a genetically deficient mother. RAG–/– thymus is devoid of any CD4+CD8+ DP cell. It has been shown that in genetically T cell-deficient humans, the very few wild-type progenitors in the thymus can amplify into DP T cells and give rise to an oligoclonal T cell population (34). In accordance, the Igs identified had an electrophoretic migration profile suggesting monoclonality. These results show for the first time that in some mice with a history of prior pregnancies, a genetic deficiency can be partially corrected by microchimeric fetal cells.

Fetal T cell microchimerism is a frequent phenomenon in women with a prior history of pregnancy (3, 4, 5). These cells were described in the circulation of healthy women, as well as women affected with scleroderma. It is therefore striking that the presence of allogeneic fetal T cells does not result in a graft-vs-host-type reaction among the group of healthy women. Our results imply that because fetal T cells that develop in maternal thymus may undergo an educational process in terms of positive and negative selection in maternal thymic environment, they cannot display antimaternal allogenic responses. In fact, alloreactive T cells of fetal origin developing in the maternal thymus are most probably deleted, and some level of histocompatibility may be necessary for positive selection and the development of microchimerism, as previously described (5, 35). This may explain the high frequency of women with microchimeric fetal T cells without any sign of immune disorder.

In conclusion, our results strongly suggest that fetal cells with lymphoid progenitor capacity are transferred to the mother during pregnancy and develop into T cells in maternal thymus as well as B cells in RAG–/– mice. We believe this implies the following: 1) fetal microchimeric cells can partially correct a maternal genetic deficiency, and 2) because they develop in the maternal thymus, fetal T cells alloreactive to maternal Ags may be subjected to negative selection and deleted.


   Acknowledgments
 
We are grateful to Dr. Claude Carnaud for helpful discussion. We thank Dr. Pierre Aucouturier for his contribution to identifying serum Igs, and Isabelle Renault for her help in animal husbandry.


   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 Université Pierre et Marie Curie (Paris VI) BQR 2005 Grant, the Fondation pour la Recherche Médicale INE20050303543, the Société Française de Dermatologie, the Assistance Publique-Hôpitaux de Paris CRC04026, and the Association Française contre les Myopathies G12670A/S01975, Paris, France. M.L. was supported by the French Ministry of Education. Back

2 K.K. and M.L. contributed equally and should be considered as first authors. Back

3 Address correspondence and reprint requests to Dr. Kiarash Khosrotehrani, Service de Dermatologie, Hôpital Tenon, 4 rue de la Chine, 75020 Paris, France. E-mail address: kiarash.khosrotehrani{at}tnn.aphp.fr Back

4 Abbreviations used in this paper: EGFP, enhanced GFP; DP, double positive; SP, single positive. Back

Received for publication January 18, 2007. Accepted for publication November 8, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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