1 INSERM U 563, Centre de Physiopathologie de Toulouse Purpan, Hôpital Purpan, 31059 Toulouse cedex 3, France
2 Program in Molecular Immunology, Institute of Molecular Medicine and Genetics, Medical College of Georgia, 1120 15th Street, Augusta, GA 30912-2600, USA
Correspondence
Françoise Lenfant
lenfant{at}toulouse.inserm.fr
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ABSTRACT |
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Introduction |
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Human cytomegalovirus (HCMV) infection remains the most common congenital virus infection and is the cause of neurological defects that affect from 0·4 to 2·3 % of live-born infants (Plotkin, 1994). Several reports on infection of placental cytotrophoblasts in vitro and in utero demonstrated that trophoblast cells are permissive to HCMV infection (Hemmings et al., 1998
; Fisher et al., 2000
). The rate of mother-to-child transmission, around 40 %, is consistent with the existence of placental mechanisms preventing virus transmission. During the clearance of HCMV-infected cells by the immune system, CD8+ T lymphocytes are directed mainly against the matrix protein pp65 (UL83) (Kern et al., 1999
; McLaughlin-Taylor et al., 1994
). These CTLs play a prominent role in anti-HCMV defence in vivo, as the protein pp65 has been shown to be internalized immediately after virus input and then is rapidly available for presentation to specific CD8+ CTLs (Walter et al., 1995
; Wills et al., 1996
; Arrode et al., 2000
). In this context, we investigated whether HLA-G is able to present pp65-derived peptides and elicit a CTL response directed against HCMV infection.
In this study, we identified six pp65-derived peptides having the HLA-G consensus motif and analysed their binding capacity to HLA-G molecules. We then used the triple transgenic mice (HLA-G, human 2m, human CD8
) and HLA-G tetrameric complexes refolded with a HCMV pp65 epitope to analyse the HLA-G-restricted CTL response against pp65-derived peptides or the pp65 protein. Our data showed a limited induction of an HLA-G-restricted CTL response in vivo directed against HCMV infection.
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Methods |
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EL4 (H-2b), ELA/G [EL4 cells transfected with the HLA-G gene expressed under the control of H-2Kb promoter and human 2-microglobin (h
2m)], EL4/G-pp65 (EL4/G cells transiently transfected with pcDNA3-pp65 plasmid) cell lines were maintained in IMDM medium (Life Technologies) supplemented with 10 % FCS, 2 mM glutamine, 100 U penicillin ml-1 and 100 µg streptomycin ml-1.
U-373MG(U373) human astrocytoma cells (ATCC), U373/G [U373 cells stably transfected with pcDNA-G1 (Blaschitz et al., 1997)] and U373/G-IE1-pp65 [U373/G cells stably transfected with IE1-pp65 cDNA (Vaz-Santiago et al., 2001)] were used as HCMV-infected target cells.
Peptide-binding assay.
Six pp65-derived peptides were designed based on the HLA-G-binding motif (Diehl et al., 1996; Lee et al., 1995
). We also used peptides eluted from HLA-G molecules (Lee et al., 1995
) and an HLA-B27-binding peptide (Solache et al., 1999
) as positive and negative controls, respectively. Sequences of the peptides are described in Table 1
. Peptides were synthesized at the Molecular Biology Core Facility of the Medical College of Georgia (Augusta, GA, USA) or at the Peptide Synthesis Core Facility of the IFR30 (Toulouse, France). The peptides were dissolved in PBS at a concentration of 2 mg ml-1 and stored at -70 °C before use.
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Generation of peptide-specific CTLs.
HLA-G transgenic mice (CBA/Ca, H-2k, H-2Kb/HLA-G; h2m, hCD8
) (Horuzsko et al., 1997
) at 68 weeks of age were used for experiments. Mice were bred and maintained in a specific-pathogen-free facility at the Medical College of Georgia, GA, USA.
Dendritic cells (DCs) from triple transgenic mice were generated from bone marrow, according to Inaba et al. (1992). In brief, bone marrow was flushed from femurs and tibias with air-buffered wash medium (IMDM) (Life Technologies) and centrifuged. Cell suspensions were adjusted to 5x105 cells ml-1 in culture medium containing 25 ng murine granulocyte/macrophage colony-stimulating factor (GM-CSF) ml-1. IMDM medium supplemented with 10 % (v/v) foetal calf serum (FCS), 2 mM glutamine, 100 U penicillin ml-1 and 100 µg streptomycin ml-1 was used for cell culture. On day 3 of culture, non-adherent cells, which were mostly granulocytes, were gently removed and fresh medium containing GM-CSF was added. On day 10, non-adherent cells (DCs) were collected for immunizations. More than 95 % of cells produced in this way stained positively with the murine pan-DC marker, anti-CD11c monoclonal antibody (mAb) (Pharmingen). DCs (10x106) were washed twice in IMDM without FCS and incubated with 200 µM pp65-derived peptides. After 3 h of incubation at 37 °C, peptide-pulsed DCs were washed twice and 5x105106 cells per mouse were injected subcutaneously (s.c.) into the hind footpads. To increase the maturation process of the DCs, DCs were subsequently incubated overnight with an anti-CD40 mAb (Pharmingen) and then washed twice before injection. After 4 days, splenocytes were removed and restimulated in vitro once or twice with peptide-loaded DCs for 6 days in complete IMDM containing 5 U recombinant IL-2 ml-1.
Alternatively, DCs (10x106) were washed twice in IMDM without FCS and loaded with 10 µg IE1pp65 fusion protein ml-1. After 3 h, DCs were injected s.c. into the hind footpads. Splenocytes were removed 4 days later and restimulated in vitro with peptide-loaded DCs for 5 days. Cells were then used as effectors in T cell cytotoxicity assays. The production of the IE1pp65 fusion protein in Sf9 cells infected with recombinant baculovirus and IE1pp65 fusion protein purification were performed as described previously (Vaz-Santiago et al., 2001).
Viruses and mice immunizations for generation of CTLs.
Recombinant canarypox virus, expressing the HCMV pp65 protein (ALVAC-pp65, vCP260), and vaccinia virus, either parental (vacwt, Lvar) or recombinant for pp65 (vac-pp65, vP1214), were a kind gift from J. Tartaglia (Virogenetics Corporation, NY, USA). Their construction has been described elsewhere (Gonczol et al., 1995). HCMV Towne strain (ATCC) was propagated in MRC5 human fibroblasts (BioMérieux).
HLA-G transgenic mice were immunized through intraperitoneal (i.p.) injection with 2x106 p.f.u. ALVAC-pp65 and were then transferred to the infectious quarters of the animal facility.
At 2 weeks after immunization, spleen cell cultures were removed and subpopulations of CD8+ cells were enriched using the Murine T cell CD8 Subset Column kit (R&D Systems). After purification, cells were grown in RPMI supplemented with 10 % FCS, 1 % sodium pyruvate, 1 % non-essential amino acids, 0·5 % L-glutamine, 0·5 % penicillin, 0·5 % streptomycin, 10 mM N-acetyl cysteine, pH adjusted to 7·2 (Sigma), 20 U human IL-2 ml-1, 20 ng recombinant murine IL-12 ml-1 and 20 ng human IL-7 ml-1 (Fowler et al., 1996). Cells were restimulated in vitro in 24-well plates, once with autologous irradiated and UV-inactivated splenocytes infected previously with vac-pp65 at an m.o.i. of 0·5 for 5 days, and a second time with DCs loaded with pp65-derived peptides for 5 additional days. Alternatively, splenocytes were used directly for a 51Cr-release assay.
Cytotoxicity assays.
Effector cells were tested for cytotoxicity in a standard 51Cr-release assay. Peptide-pulsed targets were prepared by incubating the T2/G cell line or the EL4/G cell line for 3 h at 37 °C with peptides (200 µg ml-1). EL4 and EL4/G target cells were uninfected or infected with vacwt or vac-pp65 vaccinia viruses at an m.o.i. of 2·5 for 3 h on a minimal volume. U373 and U373/G cells were infected with HCMV Towne strain at an m.o.i. of 3 for 2 h on a minimal volume of RPMI medium without FCS, and further incubated 4 h with complete RPMI medium. For 106 cells, 100 µCi (1 MBq) 51Cr was added for 1 h, with one drop of FCS. Labelled target cells were washed three times and mixed with effector cells at various effector : target (E : T) cell ratios in triplicate using 96-well U-bottomed microtitre plates. Cells were incubated for 6 h. The percentage of specific 51Cr release was calculated as follows: [(c.p.m. experimental release-c.p.m. spontaneous release)/(c.p.m. maximal release-c.p.m. spontaneous release)]x100. Spontaneous 51Cr release (<20 %) and maximal release (100 %) were determined in the presence of either medium or 5 % Triton X-100, respectively. In blocking experiments, target cells were pre-incubated with 5 µg mAb 87G ml-1 for 20 min and then washed before mixing with effector cells.
HLA-G tetramers and labelling.
HLA-G tetramers were produced essentially as described previously (Allan et al., 1999), using synthetic self-peptide RIIPRHLQL (RI9L) derived from histone H2A, which is conserved in mice and humans and shown previously to bind HLA-G (Lee et al., 1995
), and pp65-derived peptide VFPTKDVAL (VF9L). Tetramerization was performed by the addition of streptavidinphycoerythrin (PE) (Molecular Probe). Tetramers were named HLA-G-control-tet/PE and HLA-G-pp65-tet/PE, respectively.
Mice were immunized s.c. with 5x105106 VF9L-pulsed DCs and the lymph nodes were removed 6 days after immunization. Labelling of lymph nodes was performed at room temperature for 1 h using PE-labelled HLA-G tetramers, washed and incubated with the FITC-conjugated murine anti-CD8 (Ly 2 clone, Pharmingen) for 30 min at 4 °C in staining buffer (PBS plus 0·1 % NaN3 and 1 % BSA). Samples were analysed on a FACSCalibur using the CELLQUEST software (Becton Dickinson).
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Results |
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Thus, using the T2/G-binding assay, five HCMV-pp65-derived peptides that stabilize the HLA-G molecule have been identified and will be used for subsequent experiments.
Induction of HLA-G-restricted CTLs directed against pp65 peptides
To determine whether pp65-derived peptides that bind to HLA-G were immunogenic, we assessed their capacity to induce an HCMV-specific CTL response in vivo.
Injection of peptide-pulsed DCs in vivo has been shown to elicit virus-specific CTLs (Ludewig et al., 1998). Consequently, HLA-G transgenic mice were injected s.c. with DCs loaded either with synthetic pp65-derived peptides VF9L, VL9L or HL9I alone or with the three peptides together. To assess specific responses against the inoculated peptides, splenocytes were removed and after one in vitro restimulation, T2/G cells loaded with the same peptides were used as targets. Specific lysis against peptides-pulsed T2/G cells (1520 % specific lysis) was obtained as opposed to unpulsed T2/G cells (5 % specific lysis), demonstrating a specific cytotoxicity against viral peptides (Fig. 2
A). The CTL response against the peptide VF9L, which exhibits the strongest affinity for HLA-G, gave a CTL response identical to that obtained with the peptide VL9L. Immunization with DCs pulsed with the three peptides generated a similar range of lysis (around 20 % specific lysis) (Fig. 2B
). Addition of the anti-HLA-G mAb 87G abolished cytotoxicity, thus demonstrating that the anti-pp65 cytotoxic activity was HLA-G restricted and specific (Fig. 2B
).
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Altogether, these results provide evidence that HLA-G-restricted CTLs specific to pp65-derived peptides that are processed endogenously can be selected in HLA-G transgenic mice, although the specific CTL response was weak.
Induction of HLA-G-restricted, pp65-specific CTLs using virus infection of transgenic mice
Besides the use of professional antigen-presenting molecules (DCs), we also used viruses for immunizations in order to generate and increase the specific HLA-G-restricted CTL response. Since HCMV is not infectious in mice, we used recombinant canarypox virus expressing the pp65 protein (ALVAC-pp65), which has been used already for classical MHC class I molecules (Gonczol et al., 1995). HLA-G transgenic mice were inoculated first i.p. with a single dose of ALVAC-pp65. Splenocytes were removed, enriched in CD8 cells and then restimulated in vitro with autologous splenocytes infected with vac-pp65 for 5 days. In order to increase the specific CTL response against pp65, a second in vitro stimulation was also performed with DCs loaded with synthetic pp65-derived peptides. Under these conditions, a significant lysis of EL4/G target cells infected with vac-pp65 (50 % at 30 : 1) was obtained as compared to EL4/G target cells infected with vacwt (25 % at 30 : 1) (Fig. 4
). The specificity of the anti-pp65 CTL response was also confirmed using the EL4/G-pp65 transfectant cell line. No killing of EL4-vac-pp65 target cells, which do not express HLA-G, was observed, demonstrating that the reactivity of pp65-specific CTLs was HLA-G restricted.
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HLA-G-restricted, pp65-specific CTLs are able to kill human cell lines infected with HCMV
The reactivity of the HLA-G-restricted, pp65-specific CTLs was tested finally on human cell lines infected with HCMV to assess their function in the control of HCMV infection. The HLA-G transgenic mice were immunized with the canarypox virus expressing pp65 and boosted twice with the same dose to increase CTL response (Gonczol et al., 1995). Mice were sacrificed 1 week after the third inoculation and the lysis efficiency of HLA-G-restricted CTLs specific for pp65 protein was then measured using the U373 cell line transfected with the HLA-G cDNA following infection with the HCMV Towne strain. The U373 astrocytoma cell line was used as the target cell line, as this human cell line is permissive to HCMV infection (Duclos et al., 1989
). The pp65 structural protein is delivered rapidly to the cytosol by the infecting virion and is presented to CD8+ cells by MHC class I molecules.
Fig. 5 showed that only targets expressing HLA-G infected with HCMV or expressing the IE1pp65 fusion protein were killed significantly. The anti-HCMV CTL response restricted by HLA-G increases by 16 % at an E : T ratio of 30 : 1, while no significant killing of HCMV-infected U373 target cells (negative for HLA-G expression) was observed (4 %). This result indicated that HLA-G functions as a restriction element to control HCMV infection. This specific lysis assessed in vivo CTL responses, since it was measured directly after immunization using bulk splenocytes without any restimulation procedures.
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HLA-G tetramers containing pp65-derived peptides bind to specific CD8+ T lymphocytes in transgenic mice
The in vivo selection of anti-pp65 HLA-G-restricted CTLs was also measured using HLA-Gpeptide tetrameric complexes. The following HLA-G tetramers were constructed: one was refolded with the VF9L peptide derived from pp65 (HLA-G-pp65-tet/PE), while the second was refolded with a control RI9L peptide that had been eluted from HLA-G (HLA-G-control-tet/PE) (Lee et al., 1995). The percentage of peptide-specific CD8+ T cells was then determined in lymph node cells from mice injected with VF9L-loaded DCs. At 6 days after the injection, these cells were evaluated by flow cytometry using PE-labelled HLA-G tetramers. As shown in Fig. 6
(A), tetramer staining revealed a significant frequency of HLA-G-restricted, CD8+ cell population specific for the pp65 peptide in the immunized mice (1 %). In contrast, the levels of tetramer positive CD8+ T cells in normal lymph nodes from non-immunized mice were below the limits of detection (0·2 % of CD8+ T cells). However, labelling with the control tetramer showed some binding capacity, although the intensity was lower (0·5 % for HLA-G-control-tet/PE versus 1 % for the specific HLA-G-pp65-tet/PE). The percentage of HLA-G-pp65-positive cells within the CD8+ cell population represents 4 versus 2 % for the control HLA-G tetrameric complex. Repeat experiments showed a similar range of results and tetramer staining was always higher with HLA-G-pp65-tet/PE than with the control tetramer. We also observed that both HLA-G tetramers stained some CD8- cells, especially on immunized mice. A similar observation was reported by others (Allan et al., 1999
; Horuzsko et al., 2001
): i.e. HLA-G tetramers are able to recognize both myelomonocytic cells and DCs. Consequently, this labelling of CD8- T cells on immunized mice is likely to be associated to the DCs that we have injected.
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The use of HLA-G tetramers directly detected HLA-G-restricted, pp65-specific CD8+ T cells generated in vivo in the transgenic mice.
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Discussion |
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We have also used HLA-G tetrameric complexes to detect the HLA-G-restricted CTL response specific for pp65. Although labelling of lymph node cells from immunized mice with the HLA-Gpeptide tetramer showed non-specific staining with the control tetramer, a higher percentage of positive cells was always obtained with the HLA-Gpp65 tetramer. These data indicated selection of specific anti-pp65 CD8+ T cells restricted by HLA-G. Cross-reactions during tetramer labelling have been reported already for other MHC molecules, due to binding to non-specific T cells by interacting with the co-receptor CD8 (Moris et al., 2001).
Immunizations of transgenic mice with recombinant canarypox virus expressing pp65 also demonstrated that an anti-pp65 CTL response restricted by HLA-G can be selected. Furthermore, the use of human astrocytoma HLA-G transfected U373 cells that could be infected by HCMV provide the first evidence that HLA-G is able to trigger a virus-specific CD8+ CTL response, in particular against HCMV, which is known to infect trophoblasts, suggesting that they might play a role in controlling HCMV infection. Indeed, trophoblasts are capable of presenting endogenously derived peptides, and a significant cytolysis of trophoblast cells has been observed (Gobin et al., 1997). Unfortunately, the use of HCMV-infected trophoblasts as target cells was complicated in our cytotoxicity assays, since the level of ex vivo infection remained low (10 %) (Hemmings et al., 1998
).
Despite the use of different immunization schedules, one main concern was that the antigen-specific cytotoxic T cell response was consistent but relatively weak (2025 %). The low level of HLA-G-restricted anti-HCMV CTLs in mice should be explained by poor immunogenicity of the pp65 epitopes presented by HLA-G or by the influence of HLA-G on the development of the CTL response. Using murine MHC molecules as antigen-presenting cells and the lymphocytic choriomeningitis virus (LCMV), comparison of the antiviral H-2k-restricted CTL response in HLA-G transgenic mice to wild-type CBA mice demonstrated that the anti-LCMV CTL response was also limited and less potent in the HLA-G mice than in wild-type mice (50 % specific lysis in wild-type mice as compared to 20 % in HLA-G mice at an E : T ratio of 30 : 1) (Horuzsko et al., 2001). This reduced immune responsiveness was attributed to a defect in DC functions in HLA-G mice (Horuzsko et al., 2001
). Using the CD40 mAb to increase DC maturation, we showed an increase in the number of anti-pp65 CD8+ T cells using HLA-Gpp65 tetramer labelling. These data suggest that the reduction in HLA-G-specific CTLs is probably not specific for the pp65 response but is due to inhibitory triggering signals on the induction of antiviral CTL responses in transgenic mice. Further analysis showed that HLA-G modifies the function of murine DCs via interactions with the PIR-B inhibitory receptor, a homologue of the human inhibitory receptor ILT4 (Liang et al., 2002
). This is consistent with results obtained in humans, where specific binding of HLA-G tetramers was associated with ILT4 on myelomonocytic cells (Allan et al., 1999
).
In addition, the low CTL responses can be explained by specific functions of membrane-bound and soluble isoforms of HLA-G, expressed both in HLA-G mice and in target cells (Ishitani & Geraghty, 1992). In humans, transfection of membrane-bound HLA-G1 in target cells reduced the lytic activity of antigen-specific CTLs (Le Gal et al., 1999
). Besides, soluble HLA-G1 has been found to induce apoptosis of activated CD8+ T cells through the engagement of CD8 (Fournel et al., 2000
). While the production of this soluble HLA-G isoform was thought to contribute to the local elimination of CD8+ alloreactive maternal T cells, it could also participate in the elimination of antiviral-activated T cells in mice, where soluble HLA-G1 molecules have been detected in sera (data not shown). Although these different mechanisms might have turned off the immune system locally, generation of antiviral HLA-G-restricted CD8+ T cells still occurred, supporting the idea that activation of an antiviral response restricted by HLA-G molecules was initiated. The data obtained from the transgenic mice study probably reflect what might happen in humans, due to the similarity between the two organisms in the balance between activation of antiviral CTLs and HLA-G inhibitory triggering signals. However, the detection of such antiviral HLA-G-restricted CD8+ T cells still remains to be determined in humans during pregnancy.
In HLA-G transgenic mice, the HLA-G gene was expressed under the H2-Kb promoter. HLA-G was thus expressed in all tissues. We do not exclude the possibility that the absence of a wider distribution of HLA-G in humans prevents generation of the HLA-G-restricted CD8+ T cells. It remains to be determined what would occur if HLA-G expression was restricted to placental tissues and consequently, how HLA-G could regulate a peripheral cellular immune response. Several reports suggested that HLA-G has been found on antigen-presenting cells following infection. During HCMV infection, Fisher et al. (2000) demonstrated that virus is often transmitted from infected trophoblasts to fibroblasts, placental macrophages and endothelial cells in the villous cores. Infected placental macrophages namely Hofbauer cells that express HLA-G seem to be good candidates to enter the venous circulation of the placenta. Moreover, induction of HLA-G antigens was observed upon reactivation of latent peripheral blood monocytes infected by HCMV (Onno et al., 2000
) following treatment with recombinant HCMV IL-10 (Spencer et al., 2002
) and on monocytes obtained from human immunodeficiency virus type 1 (HIV-1)-infected patients (Lozano et al., 2002
), suggesting that HCMV or HIV-1 infection upregulates HLA-G expression on monocytes. Elucidation of these mechanisms could facilitate our understanding of induction of an antiviral peripheral immune response restricted by HLA-G during pregnancy.
Finally, our data agree with the fact that, in most uterine virus infections, maternal infection may spread to the placenta but fails to progress to the foetus, suggesting that trophoblast cells have established some mechanisms of antiviral defence (Lew & Fowler, 1998). The correlation between spontaneous pregnancy loss and HCMV infection during early pregnancy may also argue in favour of a strong maternal immune response against viruses (Kriel et al., 1970
; Naib et al., 1970
). A mixed villous infiltrate of lymphocytes has been reported in some virus infections and is associated commonly with tissue destruction (Fox, 1993
).
The detection of antiviral HLA-G-restricted CD8+ T cells in humans will definitively prove this physiological function of HLA-G during pregnancy. Staining of human peripheral blood mononuclear cells (PBMCs) has been done already using HLA-G tetramers refolded with self-peptides (histone H2A) (Allan et al., 1999). They revealed an interaction of HLA-G tetramers with blood monocytes and failed to detect labelling of the CD8+ population. Moreover, these experiments have been done using PBMCs from healthy donors and HLA-G tetramers refolded with self-peptides, which would not be expected to interact with antiviral-specific CD8+ T cells. The constructed HLA-G tetramer containing the pp65 peptide may be a useful tool to examine the development of anti-HCMV-specific, HLA-G-restricted CTLs using PBMCs from an HCMV-infected pregnant women.
In conclusion, these data provide the first evidence that HLA-G molecules have the capacity to present HCMV peptides to CD8+ T cells and function as restriction elements to recognize and kill HCMV-infected astrocytoma cells. Further studies are needed to evaluate how and whether such induction of the HLA-G-restricted, anti-HCMV response exists in humans.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Arrode, G., Boccaccio, C., Lule, J., Allart, S., Moinard, N., Abastado, J. P., Alam, A. & Davrinche, C. (2000). Incoming human cytomegalovirus pp65 (UL83) contained in apoptotic infected fibroblasts is cross-presented to CD8+ T cells by dendritic cells. J Virol 74, 1001810024.
Blaschitz, A., Lenfant, F., Mallet, V., Hartmann, M., Bensussan, A., Geraghty, D. E., Le Bouteiller, P. & Dohr, G. (1997). Endothelial cells in chorionic fetal vessels of first trimester placenta express HLA-G. Eur J Immunol 27, 33803388.[Medline]
Braud, V. M. & McMichael, A. J. (1999). Regulation of NK cell functions through interaction of the CD94/NKG2 receptors with the nonclassical class I molecule HLA-E. Curr Top Microbiol Immunol 244, 8595.[Medline]
Braud, V. M., Allan, D. S., Wilson, D. & McMichael, A. J. (1998). TAP- and tapasin-dependent HLA-E surface expression correlates with the binding of an MHC class I leader peptide. Curr Biol 8, 110.[Medline]
Clover, L. M., Sargent, I. L., Townsend, A., Tampe, R. & Redman, C. W. (1995). Expression of TAP1 by human trophoblast. Eur J Immunol 25, 543553.[Medline]
Crisa, L., McMaster, M. T., Ishii, J. K., Fisher, S. J. & Salomon, D. R. (1997). Identification of a thymic epithelial cell subset sharing expression of the class Ib HLA-G molecule with fetal trophoblasts. J Exp Med 186, 289298.
DeMars, R., Chang, C. C., Shaw, S., Reitnauer, P. J. & Sondel, P. M. (1984). Homozygous deletions that simultaneously eliminate expressions of class I and class II antigens of EBV-transformed B-lymphoblastoid cells. I. Reduced proliferative responses of autologous and allogeneic T cells to mutant cells that have decreased expression of class II antigens. Hum Immunol 11, 7797.[CrossRef][Medline]
Diehl, M., Munz, C., Keilholz, W., Stevanovic, S., Holmes, N., Loke, Y. W. & Rammensee, H. G. (1996). Nonclassical HLA-G molecules are classical peptide presenters. Curr Biol 6, 305314.[CrossRef][Medline]
Duclos, H., Elfassi, E., Michelson, S., Arenzana-Seisdedos, F., Hazan, U., Munier, A. & Virelizier, J. L. (1989). Cytomegalovirus infection and trans-activation of HIV-1 and HIV-2 LTRs in human astrocytoma cells. AIDS Res Hum Retroviruses 5, 217224.[Medline]
Fisher, S., Genbacev, O., Maidji, E. & Pereira, L. (2000). Human cytomegalovirus infection of placental cytotrophoblasts in vitro and in utero: implications for transmission and pathogenesis. J Virol 74, 68086820.
Fournel, S., Aguerre-Girr, M., Huc, X., Lenfant, F., Alam, A., Toubert, A., Bensussan, A. & Le Bouteiller, P. (2000). Cutting edge: soluble HLA-G1 triggers CD95/CD95 ligand-mediated apoptosis in activated CD8+ cells by interacting with CD8. J Immunol 164, 61006104.
Fowler, D. H., Breglio, J., Nagel, G., Eckhaus, M. A. & Gress, R. E. (1996). Allospecific CD8+ Tc1 and Tc2 populations in graft-versus-leukemia effect and graft-versus-host disease. J Immunol 157, 48114821.[Abstract]
Fox, H. (1993). The placenta and infection. In The Human Placenta, pp. 313333. Edited by C. W. Redman, I. L. Sargent & P. M. Starkey. Oxford: Blackwell Science.
Gobin, S. J., Wilson, L., Keijsers, V. & Van den Elsen, P. J. (1997). Antigen processing and presentation by human trophoblast-derived cell lines. J Immunol 158, 35873592.[Abstract]
Gonczol, E., Berensci, K., Pincus, S., Endresz, V., Meric, C., Paoletti, E. & Plotkin, S. A. (1995). Preclinical evaluation of an ALVAC (canarypox): human cytomegalovirus glycoprotein B vaccine candidate. Vaccine 13, 10801085.[CrossRef][Medline]
Hemmings, D. G., Kilani, R., Nykiforuk, C., Preiksaitis, J. & Guilbert, L. J. (1998). Permissive cytomegalovirus infection of primary villous term and first trimester trophoblasts. J Virol 72, 49704979.
Horuzsko, A., Antoniou, J., Tomlinson, P., Portik-Dobos, V. & Mellor, A. L. (1997). HLA-G functions as a restriction element and a transplantation antigen in mice. Int Immunol 9, 645653.[Abstract]
Horuzsko, A., Portik-Dobos, V., Hansen, K. A., Markowitz, R. B., Helman, S. W. & Mellor, A. L. (1999). Induction of HLA-G-specific human CD8+ T cell lines by stimulation across a polymorphism of HLA-G. Transplant Proc 31, 18601863.[CrossRef][Medline]
Horuzsko, A., Lenfant, F., Munn, D. H. & Mellor, A. L. (2001). Maturation of antigen-presenting cells is compromised in HLA-G transgenic mice. Int Immunol 13, 385394.
Inaba, K., Inaba, M., Romani, N., Aya, H., Deguchi, M., Ikehara, S., Muramatsu, S. & Steinman, R. M. (1992). Generation of large numbers of dendritic cells from mouse bone marrow cultures supplemented with granulocyte/macrophage colony-stimulating factor. J Exp Med 176, 16931702.[Abstract]
Ishitani, A. & Geraghty, D. E. (1992). Alternative splicing of HLA-G transcripts yields proteins with primary structures resembling both class I and class II antigens. Proc Natl Acad Sci U S A 89, 39473951.[Abstract]
Kern, F., Surel, I. P., Faulhaber, N., Frommel, C., Schneider-Mergener, J., Schonemann, C., Reinke, P. & Volk, H. D. (1999). Target structures of the CD8+-T-cell response to human cytomegalovirus: the 72-kilodalton major immediate-early protein revisited. J Virol 73, 81798184.
King, A., Allan, D. S., Bowen, M., Powis, S. J., Joseph, S., Verma, S., Hiby, S. E., McMichael, A. J., Loke, Y. W. & Braud, V. M. (2000). HLA-E is expressed on trophoblast and interacts with CD94/NKG2 receptors on decidual NK cells. Eur J Immunol 30, 16231631.[CrossRef][Medline]
Kovats, S., Main, E. K., Librach, C., Stubblebine, M., Fisher, S. J. & DeMars, R. (1990). A class I antigen, HLA-G, expressed in human trophoblasts. Science 248, 220223.[Medline]
Kriel, R. L., Gates, G. A., Wulff, H., Powell, N., Poland, J. D. & Chin, T. D. (1970). Cytomegalovirus isolations associated with pregnancy wastage. Am J Obstet Gynecol 106, 885892.[Medline]
Lanier, L. L. (1999). Natural killer cells fertile with receptors for HLA-G? Proc Natl Acad Sci U S A 96, 53435345.
Le Bouteiller, P., Solier, C., Proll, J., Aguerre-Girr, M., Fournel, S. & Lenfant, F. (1999). Placental HLA-G protein expression in vivo: where and what for? Hum Reprod Update 5, 223233.
Lee, N., Malacko, A. R., Ishitani, A., Chen, M. C., Bajorath, J., Marquardt, H. & Geraghty, D. E. (1995). The membrane-bound and soluble forms of HLA-G bind identical sets of endogenous peptides but differ with respect to TAP association. Immunity 3, 591600.[Medline]
Lee, N., Goodlett, D. R., Ishitani, A., Marquardt, H. & Geraghty, D. E. (1998). HLA-E surface expression depends on binding of TAP-dependent peptides derived from certain HLA class I signal sequences. J Immunol 160, 49514960.
Le Gal, F. A., Riteau, B., Sedlik, C., Khalil-Daher, I., Menier, C., Dausset, J., Guillet, J. G., Carosella, E. D. & Rouas-Freiss, N. (1999). HLA-G-mediated inhibition of antigen-specific cytotoxic T lymphocytes. Int Immunol 11, 13511356.
Lew, J. F. & Fowler, M. G. (1998). Perinatal HIV-1 transmission in the United States and Internationally. Trophoblast Res 12, 85103.
Liang, S., Baibakov, B. & Horuzsko, A. (2002). HLA-G inhibits the functions of murine dendritic cells via the PIR-B immune inhibitory receptor. Eur J Immunol 32, 24182426.[CrossRef][Medline]
Lozano, J. M., Gonzalez, R., Kindelan, J. M., Rouas-Freiss, N., Caballos, R., Dausset, J., Carosella, E. D. & Pena, J. (2002). Monocytes and T lymphocytes in HIV-1-positive patients express HLA-G molecule. AIDS 16, 347351.[CrossRef][Medline]
Ludewig, B., Ehl, S., Karrer, U., Odermatt, B., Hengartner, H. & Zinkernagel, R. M. (1998). Dendritic cells efficiently induce protective antiviral immunity. J Virol 72, 38123818.
Mallet, V., Blaschitz, A., Crisa, L., Schmitt, C., Fournel, S., King, A., Loke, Y. W., Dohr, G. & Le Bouteiller, P. (1999). HLA-G in the human thymus: a subpopulation of medullary epithelial but not CD83+ dendritic cells expresses HLA-G as a membrane-bound and soluble protein. Int Immunol 11, 889898.
McLaughlin-Taylor, E., Pande, H., Forman, S. J., Tanamachi, B., Li,C.R., Zaia, J. A., Greenberg, P. D. & Riddell, S. R. (1994). Identification of the major late human cytomegalovirus matrix protein pp65 as a target antigen for CD8+ virus-specific cytotoxic T lymphocytes. J Med Virol 43, 103110.[Medline]
Moris, A., Teichgraber, V., Gauthier, L., Buhring, H. J. & Rammensee, H. G. (2001). Cutting edge: characterization of allorestricted and peptide-selective alloreactive T cells using HLA-tetramer selection. J Immunol 166, 48184821.
Naib, Z. M., Nahmias, A. J., Josey, W. E. & Wheeler, J. H. (1970). Association of maternal genital herpetic infection with spontaneous abortion. Obstet Gynecol 35, 260263.[Medline]
Onno, M., Le Friec, G., Pangault, C., Amiot, L., Guilloux, V., Drenou, B., Caulet-Maugendre, S., Andre, P. & Fauchet, R. (2000). Modulation of HLA-G antigens expression in myelomonocytic cells. Hum Immunol 61, 10861094.[CrossRef][Medline]
Park, B., Lee, S., Kim, E., Chang, S., Jin, M. & Ahn, K. (2001). The truncated cytoplasmic tail of HLA-G serves a quality-control function in post-ER compartments. Immunity 15, 213224.[Medline]
Plotkin, S. A. (1994). Vaccines for varicella-zoster virus and cytomegalovirus: recent progress. Science 265, 13831385.[Medline]
Salter, R. D. & Cresswell, P. (1986). Impaired assembly and transport of HLA-A and -B antigens in a mutant TxB cell hybrid. EMBO J 5, 943949.[Abstract]
Sanders, S. K., Giblin, P. A. & Kavathas, P. (1991). Cellcell adhesion mediated by CD8 and human histocompatibility leukocyte antigen G, a nonclassical major histocompatibility complex class 1 molecule on cytotrophoblasts. J Exp Med 174, 737740.[Abstract]
Schmidt, C. M., Garrett, E. & Orr, H. T. (1997). Cytotoxic T lymphocyte recognition of HLA-G in mice. Hum Immunol 55, 127139.[CrossRef][Medline]
Solache, A., Morgan, C. L., Dodi, A. I., Morte, C., Scott, I., Baboonian, C., Zal, B., Goldman, J., Grundy, J. E. & Madrigal, J. A. (1999). Identification of three HLA-A*0201-restricted cytotoxic T cell epitopes in the cytomegalovirus protein pp65 that are conserved between eight strains of the virus. J Immunol 163, 55125518.
Spencer, J. V., Lockridge, K. M., Barry, P. A., Lin, G., Tsang, M., Penfold, M. E. & Schall, T. J. (2002). Potent immunosuppressive activities of cytomegalovirus-encoded interleukin-10. J Virol 76, 12851292.
Vaz-Santiago, J., Lule, J., Rohrlich, P., Jacquier, C., Gibert, N., Le Roy, E., Betbeder, D., Davignon, J. L. & Davrinche, C. (2001). Ex vivo stimulation and expansion of both CD4+ and CD8+ T cells from peripheral blood mononuclear cells of human cytomegalovirus-seropositive blood donors by using a soluble recombinant chimeric protein, IE1pp65. J Virol 75, 78407847.
Walter, E. A., Greenberg, P. D., Gilbert, M. J., Finch, R. J., Watanabe, K. S., Thomas, E. D. & Riddell, S. R. (1995). Reconstitution of cellular immunity against cytomegalovirus in recipients of allogeneic bone marrow by transfer of T-cell clones from the donor. N Engl J Med 333, 10381044.
Wills, M. R., Carmichael, A. J., Mynard, K., Jin, X., Weekes, M. P., Plachter, B. & Sissons, J. G. (1996). The human cytotoxic T-lymphocyte (CTL) response to cytomegalovirus is dominated by structural protein pp65: frequency, specificity, and T- cell receptor usage of pp65-specific CTL. J Virol 70, 75697579.[Abstract]
Received 29 July 2002;
accepted 18 October 2002.