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The Journal of Immunology, 2001, 166: 7244-7249.
Copyright © 2001 by The American Association of Immunologists

Extrathymic Development of V{alpha}11 T Cells in Placenta During Pregnancy and Their Possible Physiological Role

Masahiko Yamasaki*, Takahisa Sasho*,{dagger}, Hideshige Moriya{dagger}, Masamoto Kanno{ddagger}, Michishige Harada*, Noriaki Kamada*, Eiko Shimizu*, Toshinori Nakayama* and Masaru Taniguchi1,*

* Core Research for Evolutional Science and Technology, Department of Molecular Immunology, Graduate School of Medicine, and {dagger} Department of Orthopedics, School of Medicine, Chiba University, Chiba, Japan; and {ddagger} Department of Immunology and Parasitology, School of Medicine, Hiroshima University, Hiroshima, Japan


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The molecular and cellular mechanisms of the feto-maternal immune responses in the placenta in connection with natural abortion remain unclear. In this report we provide evidence that V{alpha}11 T cells developed in the placenta may be responsible for the induction of natural abortion. The majority of V{alpha}11 TCRs detected during pregnancy showed a consensus motif in the CDR3 region, similar to that of anti-GM3 TCR clones, and were of maternal origin. V{alpha}11 TCRs were found in the middle to late stages of gestation due to de novo generation in the placenta, not to migration from the maternal side, as evidenced by the significant increases in the out-of-frame V{alpha}11 TCR mRNA and the copy number of circular DNA generated by V{alpha}11 gene rearrangements. Furthermore, administration of anti-V{alpha}11 Ab to pregnant mice resulted in a significant decrease in the incidence of fetal demise, suggesting that V{alpha}11 T cells detected in the placenta develop extrathymically and are involved in natural abortion.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
In the pregnant uterus, fetuses are allowed to survive and develop regardless of their semiallogeneic or syngeneic situations. Extensive analyses of the mechanisms that allow this phenomenon have been performed by several investigators using (DBA/2 x CBA/J)F1 mice that show a high rate of spontaneous abortion. It has been found that the administration of GM-CSF, anti-CD8, or anti-asialo GM1 Ab prevents spontaneous abortion, suggesting the possible involvement of immune mechanisms in natural abortion and the maintenance of pregnancy. Several possibilities have been proposed, including various cytokines (i.e., TGF-{beta}, GM-CSF, CSF-1, IL-10, IFN-{gamma}, and TNF-{alpha}) (1, 2, 3, 4, 5, 6, 7, 8, 9, 10), suppressor cells (5, 11, 12, 13), {gamma}{delta} T cells (14, 15, 16), and NK cells (10, 17, 18). In addition, recent findings by Munn et al. suggest that indoleamine 2,3-deoxygenase, an enzyme that catabolizes tryptophan, plays a critical role in the maintenance of pregnancy by suppressing maternal allospecific {alpha}{beta} T cell activation in the decidua (19, 20). Thus, it is conceivable that lymphocytes with different functional activities and their cytokines contribute to pregnancy and abortion in different pathological situations. Despite the above experimental findings, immunological mechanisms involved in the feto-maternal interaction remain controversial. This might be due to the lack of an appropriate experimental model system for addressing critical questions about the maintenance of pregnancy and the induction of abortion.

However, important questions remain to be answered. For example, it is of great interest to know whether feto-maternal immune responses are mediated by cells of maternal or fetal origin, because recent studies have demonstrated that V{alpha}14 NK T cells, recently defined as a novel lymphocyte (21, 22, 23, 24, 25), develop in the fetus at an early stage of embryogenesis (26). Moreover, V{alpha}14 NK T cells that reside in the placenta have been shown to provoke abortion after they are activated by their specific ligand, {alpha}-galactosylceramide (27). It is thus important to determine whether lymphocytes detected during pregnancy develop in the placenta in situ or migrate from the maternal lymphoid reservoir.

The Ags involved in the feto-maternal immune responses have not yet been fully characterized. A ganglioside, GM3, is a major component, and its level increases during pregnancy (28, 29). Changes in ganglioside expression in the rat placenta have shown that GM3 is predominant during the middle stage of pregnancy, while GD3 is expressed in the late stage (30). Moreover, GM3 serves as a shedding immunosuppressive molecule that can be found in the amniotic fluid with suppressive properties in the pregnant host (31, 32). It has been also reported that human placental gangliosides, including GM3, suppress the cytotoxic activity of human NK cells and/or stimulate the Con A-induced T-suppressor activity of human lymphocytes (33).

As GM3 has been demonstrated to be immunogenic and to stimulate T cells under some conditions (34, 35, 36, 37), it is possible that GM3 is an important Ag in the feto-maternal immune responses. In fact, our previous studies have shown that a certain density of GM3 generates strong antigenicity (34) and activates anti-GM3 T cells in a density-dependent fashion (35, 36, 37). In addition, several independently established anti-GM3 T cell clones have been found to use an invariant TCR {alpha}-chain encoded by V{alpha}11 and J{alpha}281 gene segments with a one-nucleotide N-region (37). Therefore, it is possible that some V{alpha}11 T cells react with GM3 and play a decisive role in vivo in the regulation of feto-maternal immune responses.

In the present study we demonstrate that V{alpha}11 T cells develop in situ in the placenta during the middle and late stages of gestation, because a high frequency of out-of-frame V{alpha}11 sequences and a high copy number of circular DNA generated by V{alpha}11 and J{alpha}281 rearrangement events are detected in placenta compared with PBMC. Furthermore, the elimination of V{alpha}11 T cells from the pregnant hosts results in a significant decrease in the frequency of fetal demise, suggesting the active contribution of V{alpha}11 T cells to natural abortion.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals

Specific pathogen-free BALB/c and C57BL/6 mice were purchased from Japan SLC (Shizuoka, Japan). Recombination-activating gene (RAG)-1-deficient mice originally produced by P. Mombearts (Massachusetts Institute of Technology, Boston, MA) were maintained in our animal facility under specific pathogen-free conditions.

Semiquantitative RT-PCR

RT-PCR was conducted using 10 µg total RNA from placenta or PBL at different days of gestation as previously described (24). The amounts of cDNA obtained from RNA samples were first measured by semiquantitative PCR with C{alpha}/C{alpha} primers. The normalized samples were subsequently used for PCR to measure the frequency of TCR transcripts of V{alpha}11J{alpha}281 and control V{alpha}4J{alpha}281. For amplification of V{alpha}11J{alpha}281, V{alpha}4J{alpha}281, and the C{alpha}/C{alpha} regions of mRNA, the materials were first incubated at 94°C for 5 min and then subjected to 35 cycles of PCR at 94°C for 1 min, 60°C for 1 min, and 72°C for 1 min, followed by a final extension at 72°C for 5 min. Oligonucleotide primers used for PCR amplifications were 5'-TTCAGGAACAAAGGAGAATGGGA-3' and 5'-CAATCAGCTGAGTCCCAGCT-3' for V{alpha}11J{alpha}281, 5'-TCGAATTCCTGTCCTGAGACCGAGGATC-3' and 5'-CAATCAGCTGAGTCCCAGCT-3' for V{alpha}4J{alpha}281, and 5'-CCTCTGCCTGTTCACCGACT-3' and 5'-CAGGAGGATTCGGAGTCCCA-3' for C{alpha}. PCR products were subjected to electrophoresis in a 1.5% agarose gel and hybridized with the following probes: the oligomer 5'-GAAGTAAGTGCCTGAGTCC-3' for V{alpha}11J{alpha}281 and V{alpha}4J{alpha}281 products, and 5'-TTCAAAGAGACCAACGCCAC-3' for C{alpha} products. Radioactivity was measured with Bio Image Analyzer (Fujix BAS2000; Fuji Film, Tokyo, Japan) as photo-stimulated luminescence (PSL).2 Amounts of V{alpha}11J{alpha}281 and V{alpha}4J{alpha}281 TCR mRNA are expressed as arbitrary units of PSL estimated as the quotient of the PSL of the V{alpha}11J{alpha}281 or V{alpha}4J{alpha}281 products divided by the PSL of the C{alpha} products.

Preparation of nuclear DNA

The methods for the preparation of nuclear DNA have been described previously (22). In brief, cells (1–5 x 106) were suspended in 500 µl of 0.5% Nonidet P-40 buffer containing 10 mM Tris-HCl (pH 7.5), 10 mM NaCl, and 5 mM MgCl2; homogenized; and centrifuged at 10,000 rpm for 10 min. The pellet containing the nuclei was resuspended in 500 µl buffer (300 mM NaCl, 10 mM Tris-HCl (pH 7.5), 5 mM EDTA, and 0.5% SDS) and subsequently treated with 1.6 U protease K. The mixture was treated with phenol and chloroform isoamyl alcohol, and the supernatant was dialyzed against buffer (10 mM Tris-HCl, pH 8.0, and 1 mM EDTA) and used for experiments. For the measurement of DNA, quantitative PCR was conducted with RAG-2 primers on the genomic DNA from different sample materials to normalize the amounts of DNA in samples on the basis of copy numbers of RAG-2 genes. The PCR products, amplified at various concentrations of RAG-2 cDNA, were used as a standard. The oligonucleotides used for the amplifications for RAG-2 were 5'-CACAGTCTTGCCAGGAGGAA-3' and 5'-GGGGGTTTCTTTTGGGAGTTT-3'. PstI fragments (852 bp) of RAG-2 cDNA were used as the specific probe for DNA blots.

Quantitative analysis of recombination signal sequences in the circular DNA

The copy number of recombination signal sequences in the circular DNA generated by gene rearrangement events was measured in a quantitative fashion as described previously (24). The primers were prepared for PCR in opposite outward orientations at the unrearranged germline TCR locus in such a way that no DNA amplification is possible. When circular episomal products are created by the formation of a signal joint, the PCR primers will amplify fragments with a signal sequence. This sequence includes signal heptamer repeats, two nonamers, and a 12/23 spacer in flanking sequences of V{alpha}11/V{alpha}4 and J{alpha}281 segments. The primers used were 5'-TCCCTGTGACTGGTAGAAATC-3' and 5'-CTGGCGGTGGAAAGACTATTG-3' for V{alpha}11-J{alpha}281, and 5'-CTGCAGTTTCTGCCACTG-3' and 5'-CTGGCGGTGGAAAGACTATTG-3' for V{alpha}4-J{alpha}281. PCR was conducted by preincubating the samples at 94°C for 5 min, followed by 35 cycles of PCR amplification at 94°C for 1 min, 60°C for 1 min, 72°C for 1 min, and 72°C for 5 min. The PCR products were subjected to electrophoresis in a 1.5% agarose gel, transferred onto Hybond N+, and hybridized with a 32P-labeled 288-bp XhoI- and HindIII-digested V{alpha}14J{alpha}281 fragment cloned into pCR vector (Invitrogen, San Diego, CA). The amounts of circular DNA are expressed as arbitrary units, estimated as the quotient of the PSL of the PCR products of circular DNA divided by the PSL of the PCR products of RAG-2 genes. PCR amplifications were confirmed not to have reached the plateau level in all experiments.

Ab treatment

Anti-V{alpha}11 (RR8-1) (38) and anti-V{alpha}3 (39) mAbs were obtained from ascites produced by hybridoma cells provided by O. Kanagawa (Washington University, St. Louis, MO). Abs were injected into pregnant C57BL/6 or RAG-1-/- mice i.v. one time at a dose of 2 mg between days 6.5 and 8.5 of pregnancy.

Natural abortion rate

The morning of sighting the vaginal plaque was designated day 0.5 of pregnancy. On day 15 of gestation the mice were killed by cervical dislocation, and the total numbers of resorbing pups were recorded.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Increase in the number of V{alpha}11 TCR transcripts in the placenta during pregnancy

The preferential usage of a TCR {alpha}-chain encoded by V{alpha}11 and J{alpha}281 gene segments with a one-base N-region has been shown in several independently established anti-GM3 T cell clones (37). Furthermore, it has been reported that GM3 is a major ganglioside in the placenta and is predominant during the middle stage of pregnancy, while GD3 is expressed in the late stage of gestation (28, 29, 30). We assume that V{alpha}11 T cells generated in the placenta play a certain role in feto-maternal immune responses. Thus, we attempted to investigate V{alpha}11 TCR expression in the placenta at different stages of gestation by quantitative RT-PCR and compared the results with those in PBL. Southern blot analysis of PCR products revealed that the expression of V{alpha}11J{alpha}281 TCR transcripts in the placenta starts to appear on day 14 of gestation, reaches a maximum at a frequency of 0.2% of total TCR on day 15, and continues to the end of pregnancy, while the expression of control V{alpha}4 TCR mRNA shows no increase during pregnancy (Fig. 1GoA). V{alpha}11 TCR expression in the placenta seems to be specific and tightly correlated with pregnancy, because the increase in V{alpha}11 expression is detected only at a certain stage of pregnancy, preferentially in the placenta, but not in PBL, and also because the expression of other TCR transcripts (V{alpha}4J{alpha}281) does not change during pregnancy (Fig. 1GoB).



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  		FIGURE 1. Quantitative PCR analysis of TCR transcripts during pregnancy. The amounts of cDNA obtained from RNA samples of placenta or PBL on different days of gestation were first measured by quantitative PCR with C{alpha}/C{alpha} primers. Then, the normalized cDNA samples were used for PCR to measure the frequency of TCR transcripts of V{alpha}11.J{alpha}281 and control V{alpha}4J{alpha}281. A, DNA blot hybridization on the quantitative PCR products of TCR transcripts analyzed in placenta on different days of gestation. B, Graphic representation of the expression of TCR transcripts in placenta and PBL during pregnancy. Two other experiments produced similar results. C, Standard curves of copy numbers of C{alpha} and V{alpha}11J{alpha}281 sequences. Various concentrations of the standard cDNA (V{alpha}11J{alpha}281 or C{alpha} cDNA) were amplified and hybridized with the J{alpha}281 or C{alpha} probe. The radioactivity (copy number) of the sample materials is expressed in arbitrary units of PSL obtained from the standard curves.

 
Nucleotide sequences of V{alpha}11 TCR in placenta

V{alpha}11 TCR products amplified by PCR were sequenced on day 14 and days 15-17 of gestation and compared with those from anti-GM3 T cell clones. Fig. 2Go summarizes the sequence data. About 67% (50 of 75) of total V{alpha}11 TCR sequences detected in the placenta were out-of-frame, while only 33% (25 of 75) were in-frame, with invariant sequences in some cases (Fig. 2Go, A and B). Compared with the placenta, fewer than 30% (8 of 27) were out-of-frame in PBL and >70% (19 of 27) of V{alpha}11 TCR were in-frame with heterogeneous sequences (Fig. 2GoC). The high frequency of out-of-frame V{alpha}11 TCR sequences strongly suggests the extrathymic development of V{alpha}11 T cells in the placenta.



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  		FIGURE 2. J{alpha} usage and its frequency in V{alpha}11 TCR detected in the placenta on day 14 (A) or days 15–17 (B) of gestation and in peripheral blood lymphocytes on day 14 of gestation (C). The J{alpha} usage and its frequency in V{alpha}11 TCR are summarized on the basis of the nucleotide sequences. About 20 samples of placenta and two samples of PBL on day 14 of gestation or 26 samples of placenta on days 15–17 of gestation were obtained from four to six mice and used for analysis. mRNA from these samples were pooled and subdivided into three subsamples for experiments. A total of ~50–100 cDNA bacterial colonies of each sample were randomly picked up and sequenced. The results were expressed as a sum of V{alpha}11 sequences obtained from two or three experiments. The sequence patterns were similar in different experiments.

 
Among the in-frame V{alpha}11 TCR sequences detected in the placenta on day 14 of gestation, junctional amino acid sequences identical with those of anti-GM3 T cell clones (V{alpha}11J{alpha}281) were predominant at 33% of total V{alpha}11 TCR (4 of 12; ADRGSAL), and 58% (7 of 12) of V{alpha}11 T cells used the J{alpha}281 gene segment (Fig. 3GoA). Considering all junctional amino acid sequences, including other J{alpha}, we found a consensus sequence motif, i.e., A-D/E-X-(X)-G-S-A-L or A/V-polar-X-(X)-hydrophobic-small-hydrophobic-hydrophobic amino acids, in a broad sense. Junctional amino acid positions 3 and 4 correspond to the P/N region, and positions 5–8 corresponded to the J region. Junctional positions 1 and 2, corresponding to the end of the V region, were A–D or A–E (83%, 10 of 12), or at least polar amino acids (92%, 11 of 12, D/E/S). Positions 3 and 4 seemed to be random. Position 5 was G (75%, 9 of 12), or at least a hydrophobic amino acid (100%, 12 of 12, G/I/M/L). Position 6 was S (67%, 8 of 12) or a small amino acid (92%, 11 of 12; S/N/G); position 7 was A (75%, 9 of 12) or a hydrophobic amino acid (92%, 11 of 12; A/Y), and position 8 was L (58%, 7 of 12) or a hydrophobic amino acid (100%, 12 of 12: L/G/K/I).



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  		FIGURE 3. Deduced amino acid sequences of the VJ junctional regions of V{alpha}11 TCR detected in placenta on day 14 (A) and days 15–17 (B) of gestation as shown in Fig. 2Go. Position 1 of the VJ junctional amino acids corresponds to position 92 of the V{alpha}11 TCR variable region according to Kabat et al. (44 ). , Identical amino acids; {square}, similar amino acids. The J{alpha} usage and its frequency are shown in the right columns. Two experiments showed the similar frequencies of the clones isolated.

 
In contrast to the V{alpha}11 sequences on day 14 of gestation, the sequences on days 15–17 of gestation showed a different usage of J{alpha} segments (Fig. 3GoB). V{alpha}11 T cells using the J{alpha}281 gene segment comprised only 8% (1 of 13), while those using J{alpha} gene segments 11-2 (31%, 4 of 13) and TA72 (31%, 4 of 13) became dominant. Despite the different usage of J{alpha} segments, it is surprising that the consensus amino acids in the VJ junction are similar to those encoded by the J{alpha}281 segments detected on day 14 of gestation. Junctional position 1 was A or V, and position 2 was at least a polar amino acid (69%, 9 of 13, N/E/S). Position 3 was A or N (77%, 10 of 13) or a small amino acid (100%, 13 of 13). Position 5 was G or M (85%, 11 of 13) or a hydrophobic amino acid (100%, 13 of 13, G/M/A). Position 6 was G (85%, 11 of 13) or a small amino acid (100%, 13 of 13, G/S/T), position 7 was Y (62%, 8 of 13) or a hydrophobic amino acid (100%, 13 of 13, Y/A/G/K), and position 8 was K (77%, 10 of 13) or a hydrophobic amino acid (92%, 12 of 13, K/L). Thus, the consensus sequence motif represented nearly all V{alpha}11 TCR sequences isolated. This indicates that the majority of V{alpha}11 TCR sequences detected in the placenta have an amino acid composition similar to that of anti-GM3 TCR (V{alpha}11J{alpha}281 TCR with ADRGSAL in the VJ junction), suggesting the possible selection of V{alpha}11 T cells by GM3 in the placenta.

Extrathymic development of V{alpha}11 T cells in situ in the placenta

In an attempt to determine whether the V{alpha}11 TCRs detected in the placenta are migrants from outside the uterus or are generated extrathymically in situ in the placenta. PCR primers were prepared to amplify DNA fragments with signal sequences containing signal heptamer repeats, two nonamers, and a 12/23 spacer in the flanking sequences of V{alpha}11/V{alpha}4 and J{alpha}281 segments. DNA blot analysis of the PCR products was conducted using specific probes for the signal joints.

We detected a 300-bp band hybridizable with the signal sequence created by V{alpha}11-J{alpha}281 rearrangement in the placenta only in the middle stage of gestation (Fig. 4Go). By quantitative PCR, the relative amounts of V{alpha}11-J{alpha}281 signal sequences per DNA in the placenta were calculated to be 3.0–5.0, peaking on day 9 of gestation, gradually decreasing, and becoming undetectable by day 17 of gestation (Fig. 4Go). In contrast, the peak amounts of V{alpha}11-J{alpha}281-mediated signal sequences in PBL were significantly lower and delayed for ~2 days compared with those in placenta. The frequency of other TCR rearrangements, such as V{alpha}4-J{alpha}281 TCR, was very low and did not change during pregnancy. Therefore, it is likely that V{alpha}11 T cells are indeed generated in situ in the placenta, and the V{alpha}11 signal sequences detected in PBL are derived from migrants from the placenta.



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  		FIGURE 4. Quantitative PCR analysis of circular DNA generated by V{alpha}11J{alpha}281 gene rearrangements during pregnancy. The amounts of nuclear DNA isolated from placenta or PBL on various days of gestation were determined by quantitative PCR with RAG-2 PCR primers and normalized. The samples were subsequently used for PCR to measure the frequency of signal sequences in the circular DNA generated by V{alpha}11-J{alpha}281 and V{alpha}4-J{alpha}281 rearrangements. A, DNA blots of PCR products of circular DNA from placenta and PBL during pregnancy. B, Graphic representation of the results obtained from DNA blots. Two other experiments produced similar results. C, Standard curves of copy numbers of RAG-2 and signal sequences in the circular V{alpha}11-J{alpha}281 DNA. Various concentrations of standard DNA (RAG-2 or circular V{alpha}11-J{alpha}281 DNA) were amplified and hybridized with the RAG-2 or V{alpha}11-J{alpha}281 signal sequence probe. The radioactivity (copy number) of the sample materials is expressed in arbitrary units of PSL obtained from the standard curves.

 
Maternal origin of V{alpha}11 T cells in placenta

The origin of V{alpha}11 T cells in the placenta was investigated by the detecting V{alpha}11-J{alpha}281-mediated signal sequences in the placenta of (RAG-1-/- x BALB/c)F1 and normal mice. In (RAG-1-/- x BALB/c)F1 mice, only TCR rearrangements of fetal, not maternal, origin are expected to be detected, because the RAG-1-/- female mice have no T cells. As shown in Fig. 5Go, the signal sequence generated by V{alpha}11 and J{alpha}281 gene rearrangement events was undetectable in (RAG-1-/- x BALB/c)F1, while it was detected in wild-type BALB/c mice, indicating that V{alpha}11 T cells in the placenta are entirely maternally derived.



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  		FIGURE 5. Detection of signal sequences generated by V{alpha}11 and J{alpha}281 gene segments in placenta from (RAG-1-/- x BALB/c)F1 and normal BALB/c mice. PCR was performed with the set of primers used in Fig. 4Go.

 
Effects of anti-V{alpha}11 on pregnancy

As V{alpha}11 T cells are thought to be generated preferentially in the placenta during pregnancy, especially during the middle and late stages of gestation, it has been expected that they play either a positive or a negative role in feto-maternal immune responses. To study the functional role of V{alpha}11 T cells, anti-V{alpha}11 mAb was injected into pregnant C57BL/6 mice. PCR Southern blot analysis showed clearly that anti-V{alpha}11 treatment successfully removes V{alpha}11 T cells from both the placenta and PBL of the pregnant mother (Fig. 6GoA). Under these conditions, the numbers of total placentas and resorbing pups were counted on day 15 of gestation. As shown in Fig. 6GoB, the rate of resorption in C57BL/6 pregnant mice was about 13.4% in the PBS-injected control group. Similar results (10.5%) were obtained by the group receiving the control anti-V{alpha}3 Ab. In contrast, the administration of anti-V{alpha}11 Ab produced a significantly lower rate of resorption (3.1%) in C57BL/6, while no significant difference between anti-V{alpha}11- and anti-V{alpha}3-treated groups was observed in RAG-1-/- pregnant mice. These results suggest that V{alpha}11 T cells are involved in fetal demise.



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  		FIGURE 6. Effects of anti-V{alpha}11 on pregnancy. Pregnant C57BL/6 or RAG-1-/- mice were injected with monoclonal anti-V{alpha}11 (2 mg), anti-V{alpha}3 (2 mg), or PBS between days 6.5 and 8.5 of pregnancy, and their V{alpha}11 TCR expression in C57BL/6 mice (A) and the rate of resorption on day 15 of gestation in C57BL/6 and RAG-1-/- mice (B) were determined. Actual numbers of resorbing pups per litter are 2 of 11, 2 of 12, 2 of 11, 2 of 15, 1 of 12, 1 of 11, and 1 of 10 (13.4 ± 1.6%) in the PBS-treated group (); 2 of 12, 0 of 13, 0 of 11, 0 of 10, 0 of 12, 0 of 8, 1 of 12, and 0 of 10 (3.1 ± 2.2%) in the anti-V{alpha}11-treated group ({blacksquare}); and 2 of 11, 2 of 12, 1 of 11, 1 of 11, 1 of 11, 1 of 10, 1 of 10, 1 of 10, 1 of 9, 1 of 9, 1 of 9, and 0 of 10 (10.5 ± 1.3%) in the anti-V{alpha}3-treated group ({square}) in C57BL/6 mice and are 1 of 9, 1 of 8, 0 of 3, and 0 of 8 (5.9 ± 3.4%) in the anti-V{alpha}11-treated group ({blacksquare}) and 1 of 8, 1 of 8, 0 of 9, and 0 of 8 (6.3 ± 3.6%) in the anti-V{alpha}3-treated group ({square}) in RAG-1-/- mice. The data are expressed as the mean resorption rates (column) ± SE (bars).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In the present study we demonstrate that V{alpha}11 T cells of maternal origin are generated in the placenta at certain stages of pregnancy (Figs. 1Go and 5Go) and seem to play a role in the induction of natural abortion. About 33% of V{alpha}11 TCR detected in the placenta on day 14 of gestation show a junctional sequence (A-D-R-G-S-A-L) identical with those of anti-GM3 T cell clones, which have a unique TCR feature characterized by the expression of invariant TCR{alpha} encoded by V{alpha}11 and J{alpha}281 gene segments (37); most other V{alpha}11 TCR have a consensus motif, A-D/E-X-(X)-G-S-A-L, similar to an anti-GM3 TCR sequence motif (Figs. 2GoA and 3A). In contrast, on days 15–17 of gestation, the dominant V{alpha}11 TCR shows a consensus motif, A/V-E-X-(X)-G-G-Y-K, similar to but slightly different from that on day 14 of gestation (Figs. 2GoB and 3B). The changes in the amino acid sequences of V{alpha}11 TCR seem to occur in the placenta and to be associated with the stage of pregnancy, albeit both sequences have a consensus motif, A/V-polar-X-(X)-hydrophobic-small-hydrophobic-hydrophobic amino acid, in a broad sense (Fig. 3Go).

GM3 ganglioside is known to be a major component in the placenta, preferentially associated with pregnancy (28, 29, 30, 31, 32) and is also predominant during the middle stage of pregnancy on day 14, after which GD3 expression predominates during the late stage of pregnancy on day 16 in rat placenta (30). The changes in the V{alpha}11 TCR sequences seem to be associated with changes in the ganglioside composition according to the stage of pregnancy. It has been demonstrated that a certain density of GM3 (10–25 mol %) indeed serves as an immunogen and stimulates V{alpha}11 T cells (34, 37). Thus, it is likely that V{alpha}11 T cells in the placenta recognize GM3 or related oligosaccharides and mediate a specialized function. If this is the case, GM3 ganglioside with conserved molecular characteristics in nature might be more important in feto-maternal immune responses, regardless of the semiallogeneic and syngeneic situations, than the alloantigens proposed by other investigators (12, 40).

It is intriguing that the V{alpha}11 T cells detected here are, in fact, generated extrathymically in situ in the placenta, because we successfully detected signal sequences of the circular DNA generated by V{alpha}11 and J{alpha}281 gene rearrangement events in the placenta (Fig. 4Go). In addition, the majority (67%) of V{alpha}11 TCR sequences detected in placenta were out-of-frame (Fig. 2Go). Because approximately two-thirds of the TCR products generated by gene rearrangement events are theoretically out-of-frame, and only one-third are in-frame at the site of T cell development (24), the detection of out-of-frame V{alpha}11 TCR in the placenta at high frequency strongly suggests that the placenta is one of the extrathymic organs generating a subset of T cells.

Accumulative evidence have been demonstrated that T cells in the maternal immune system are tolerized to the fetal Ags and do not attack the fetus. In fact, it is reported that male (H-Y)-specific T cells of maternal origin were tolerized by Fas-mediated clonal deletion mechanisms. Moreover, the remaining H-Y-specific T cells are unresponsive to antigenic stimulation, although neither TCR nor coreceptor is down-regulated (41). It has also demonstrated in the H-2Kb-transgenic mouse system that maternal H-2Kb-specific T cells were tolerized by fetal Ag of paternal origin during pregnancy, but became responsive shortly after parturition (41, 42). Therefore, these studies in combination with our work presented here suggest that the maternal immune system prevents potential rejection of the fetus by peripheral tolerance mechanisms, such as clonal deletion, receptor down-regulation, anergy, and regulatory T cells.

Although molecular events of the V{alpha}11 T cell-mediated function in vivo remained unclear, we speculate that maternal V{alpha}11 T cells generated in the placenta function as pregnancy-associated regulatory T cells. This assumption may be correct because the elimination of V{alpha}11 T cells by the injection of anti-V{alpha}11 Ab resulted in a significant decrease in the rate of natural abortion in vivo (Fig. 6Go). A similar functional subset of lymphocytes has recently been reported, that is, V{alpha}14 NK T cells detected in the placenta provoke abortion only after stimulation with their specific glycolipid ligand, {alpha}-galactosylceramide, in the early stages of gestation (27). Although several mechanisms of natural abortion have been proposed, it is possible that V{alpha}11 T cells may provoke abortion after infection by either pathological or nonpathological bacteria in which gangliosides are expressed as normal components. This assumption appears possible, because it has been reported that bacterial GM1 induces anti-GM1 Ab that produces Guillain-Barré syndrome in the hosts (43). In any event, the present results strongly suggest that V{alpha}11 T cells in the placenta play an important role in the feto-maternal interaction during the pregnancy.


   Acknowledgments
 
We thank Hiroko Tanabe for her secretarial work.


   Footnotes
 
1 Address correspondence and reprint requests to Dr. Masaru Taniguchi, Department of Molecular Immunology, Graduate School of Medicine, Chiba University, 1-8-1 Inohana, Chuoku, Chiba 260-8670, Japan. E-mail address: taniguti{at}med.m.chiba-u.ac.jp Back

2 Abbreviations used in this paper: PSL, photo-stimulated luminescence; RAG, recombination-activating gene. Back

Received for publication November 14, 2000. Accepted for publication April 12, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Y. Uemura, M. Suzuki, T.-Y. Liu, Y. Narita, S. Hirata, H. Ohyama, O. Ishihara, and S. Matsushita
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