Human natural killer cells fail to kill herpes simplex virus type 1-infected term villus trophoblast cells

Trophoblast Research 8:137-149, 1994

H U M A N N A T U R A L KILLER CELLS FAIL TO KILL HERPES SIMPLEX V I R U S TYPE 1-INFECTED TERM VILLUS T R O P H O B L A S T CELLS Peter Mosborg-Petersen, George Aboagye-Mathiesen, Vladimir Zachar2, Ferenc Toth 3, Niels Norskov-Lauritsen, Henrik Hager, Milan Zdravkovic, Jan A. Viladsen, and Peter Ebbesen 1 Department of Virus and Cancer The Danish Cancer Society Gustav Wieds Vej 10 8000 Aarhus C, Denmark 2Institute of Virology Slovak Academy of Sciences Bratislava, Czechoslovakia 3Institute of Microbiology University Medical School Debrecen, Hungary

INTRODUCTION In the placenta, the villus trophoblast forms the outer, continuous, fetusderived cell layer across which all the interactions between mother and fetus are accomplished. During pregnancy the fetus and its placenta can be considered as a semiallograft, and it is, therefore, a potential target for maternal immune rejection (Faulk and Hunt, 1989). However, it has been shown previously that uninfected trophoblast cells resist cell-mediated lysis (Head, 1989; Pross et al., 1985). NK cells represent a subset of lymphocytes having the ability to lyse a variety of syngeneic and allogeneic virus-infected target and tumor cells in an MHCnonrestricted manner as well as in the absence of prior sensitization (Jondal, 1987). The majority of the peripheral blood l y m p h o c y t e s mediating MHC-nonrestricted cytotoxicity express the CD56 antigen (Lanier et al., 1986). Results from several laboratories attest to the ability of human NK cells to lyse preferentially target cells infected with HSV-1 when compared with uninfected targets (Chin et al., 1979; Bishop et al., 1983a). Neonatal HSV infection is a serious condition frequently resulting in extensive damage to the offspring (Vistine et al., 1981). It is generally accepted that the fetus may become infected when passing the birth canal during delivery, but it has become increasingly evident that HSV may also infect the fetus in utero and may be a cause of spontaneous abortion (Hutto et al., 1987; Robb et al., 1986). Recently, the human term villus trophoblast cells have been found susceptible in vitro to HSV-1 infection

1To Whom Corresoondence Should Be Add~ssed

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(Nerskov-Lautitsen et al., 1992). The villus trophoblast layer is exposed to maternal blood throughout pregnancy. A maternal cellular cytotoxic response against virusinfected villus trophoblast cells seems an obvious possibility, and the NK cells would seem to be the most likely effector mechanism. The present investigation was undertaken to elucidate the in vitro susceptibility of HSV-infected term villus trophoblast cells to NK cell-mediated cytotoxic activity. The present results serve to further explore the inherited immune unresponsiveness of the trophoblast and provide a better understanding of the immune response at the maternal-fetal interface of the human placenta during virus infection. MATERIALS AND METHODS Target Cells And Infection Term placentae from normal pregnancies were obtained from spontaneous vaginal delivery. Villus trophoblast cells were isolated as described by Douglas and King (1989) using negative immunomagnetic sorting. Briefly, villus tissue was dissected from the placenta, enzymatically dissociated, and fractionated on a gradient of Percoll (Pharmacia, Uppsala, Sweden). The cells were then negatively sorted on a magnetic concentrator using immunomagnetic microspheres (Dyna-beads; Dynal A,S., Oslo, Norway) coupled to anti-HLA-ABC mAb (Dakopatts, Copenhagen, Denmark). Fetal placental fibroblasts were separated as described previously by Wilson et al. (1983). Briefly, fibroblasts were harvested from the 45-50% Percoll band after 30 minutes of enzymatic treatment. Cells were allowed to adhere to plastic dishes and the attached monolayers were then detached by treatment with 0.1% trypsin. Cells were washed and then propagated in complete medium. The positive NK cell target control, K562 (human erythroleukemia cell line) was obtained from the American Type Culture Collection, Rockville, MD, USA (ATCC). Placental target cells were plated in 96-well fiat-bottomed microtiter plates (Nunc, Roskilde, Denmark) at 104 cells per well in 100 ILl and incubated for 24 hours or 72 hours before infection. Human herpes simplex virus type 1 (strain McIntyre) was obtained from ATCC. Virus infection of target cells was conducted at a multiplicity of infection of 1 as previously described (Norskov-Lautitsen et al., 1992). Isolation Of Peripheral Blood Lymphocyte Effector Cells For preparation of effector cells, buffy coats from healthy adult blood donors were used. The cells were centrifugated on a Ficoll-Hypaque gradient (Pharmacia). The crude preparation of PBMC was washed and then used in a cellular cytotoxic assay at appropriate concentrations. For isolation of CD56 § NK cells with magnetic beads, the MACS separation system (Miitenyi Biotec, Sunnyvale, CA, USA) was used (Pflueger et al., 1990). The cells were reacted with mouse anti-human CD56 mAb (Becton Dickinson, Monoclonal Center, Mountain View, CA, USA) for 15 minutes at 4~ After washing, magnetic microbeads with conjugated antimouse IgG were added and incubated for 15 minutes. Finally, the cells were recovered using a magnetic separator. Isolated populations were viable in excess of 95% as determined by trypan blue supravital staining. All cell cultures were grown in RPMI-1640 containing 10% FCS, penicillin (100 U/ml), streptomycin (100 fag/ml), gentamycin (40 ~g/ml), and 10 mM HEPES.

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Cytotoxicity Assays NK activity was measured using a 12-hour 51Cr release assay as described by Degliantoni et ai. (1985). The placental target cells, 104 cells per well, were infected with HSV-1 or mock-infected and then labeled with 5 I~Ci of 51Cr-sodium chromate (Amersham, England) for 2 hours in total volume of 40 ~fl of RPMI 1640. Cells were then washed five times and effector cells were added to achieve varying E:T ratios to a final volume of 0.2 ml. Purified CD56 § cells were employed at E:T ratios ranging from 25:1 to 3:1, while the crude unsorted PBMC were used at ratios ranging from 100:1 to 12:1. The positive target control for NK cells, the K562 cells were labeled with 100 I.tCi of 51Crsodium chromate/106 cells and plated in 96-well tissue culture plate (Nunc) at 104 cells/well in 100 ~tl of medium before addition of effector cells. After 12 hours of incubation the plates were centrifuged and 100 ~l of supernatant was harvested from each well. Radioactivity was measured in a gamma counter. All experiments were performed in triplicate. The standard deviation never exceeded 10%. Spontaneous release measured in the absence of effector cells was less than 15% of maximum release. Maximum release was estimated by lysing the 51Cr-labeled target cells with 5% Triton X-100. The percentage of specific release was calculated as follows: 100 x (experimental-spontaneous release cpm)/(maximum-spontaneous release cpm).

Cold Target Inhibition Assay In cold target inhibition experiments, the cytotoxicity assays were carried out for 51Cr labeled K562 ceils with the addition of unlabeled competitive target cells in the ratio of 10:1 and 1:1. CD56 § cells were then added to the wells to achieve an overall E:T ratio of 12:1 to a total volume of 0.2 ml. The percentage of inhibition was calculated as 100 x [1 - (% specific release in the presence of competitor cells/% specific release in the absence of competitor cells)]. FACS Analysis The purity of the target cell populations was assayed using F1TC-conjugated cytokeratin, by phycoerythrin-conjugated vimentin and anti-CD14 mAb (all from Dakopatts). Effector cell populations were analyzed for CD56 expression using Leu-19 mAb (Becton Dickinson). HSV-l-infected and mock-infected target cells were analyzed for the following cell membrane antigens at I2 hours after infection; HSV-1, transferrin receptor and MHC-I. Briefly, subconfluent cultures were dislodged from cultivation flasks by a 1 minute trypsinization, resuspended in complete medium, and washed. Aliquots of cells were then incubated for 30 minutes at 4~ with saturating amounts of FITC-conjugated anti-HSV-1 (McIntyre), anti-human HLA-ABC (W6/32), and antihuman transferrin (CD71) mAb (all from Dakopatts). FITC-conjugated mouse IgG1 (Dakopatts) was used as negative isotope control antibody. Effector cell populations were analyzed for CD56 expression using Leu-19 mAb (Becton Dickinson). After staining the cells were fixed with 2% paraformaldehyde and analyzed by flow cytometry using FACStar Plus cytometer (Becton Dickinson). IFN Assay IFN production during the 12 hour cytotoxic assay period was assessed as previously described (Toth et al., 1991) by inhibition of vesicular stomatitis virus (Indiana strain) plaque formation on the human amniotic cell line WISH (ATCC). The

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highest dilution resulting in a 50% reduction of the number of plaques was considered as the end point titer. An internal laboratory human IFN standard was included with each titration. All IFN titers were standardized to the National Institute of Health standard for h u m a n IFN-alpha. Neutralization of antiviral activity was performed with 10-fold equivalent excess of horse immune sera against human!FN-alpha, -beta and -gamma (Boehringer Mannheim, Germany). RESULTS

NK Activity Against HSV-infected Villus Cytotrophoblast Cells The purity of the isolated trophoblast and fibroblast cell preparations were phenotypic characterized by FACS analysis. Villus trophoblast cell preparations were found to be >95% positive for cytokeratin expression and <5% for both vimetin, CD14 and HLA-ABC expression. Fibroblast cell preparations were always >98% positive for both vimetin and HLA-ABC expression (data not shown). Both placental trophoblast and fibroblast cells are susceptible to lytic infection with HSV in vitro (Norskov-Lauritsen et al., 1992). The spontaneous release of 51Cr from infected cells began to increase at 18-20 hours after addition of virus, indicating the appearance of virus-induced cell lysis. The measurement of NK activity against HSV-infected cells were therefore restricted to 12 hours after infection, at which time the spontaneous release from infected cells was identical to that obtained with uninfected cells (results not shown). Both HSV-infected and uninfected trophoblast cells were resistant to the effect of enriched CD56 § NK cells (Figure 1). Contrary to this, a preferential lysis of HSV-l-infected placental fibroblast cells was noted compared to their matched uninfected controls. The high level of the inherent NK activity of CD56 § lymphocytes is clear from the highly efficient killing of the NKsusceptible cell line K562. According to previous investigations (Paya et al., 1990; Howell and Fitzgerald-Bocarsly, 1991), different NK cell populations could be involved in the differential lysis of the targets cells. Alternatively, the presence of accessory cell populations of the PBMC could be necessary for NK cell killing of the HSV-infected targets. In order to determine if any complementary effectors in addition to cytotoxic CD56 § NK cells could be operative in the killing of HSV-infected villus trophoblasts, the entire heterogeneous population of PBMC was employed in the cytotoxic assay. However, similar results as with CD56 § lymphocytes were found. The crude PBMC population did exhibit NK activity against both HSV-infected fibroblasts and K562 cells, while HSV-infected trophoblast cells were found to be resistant to the NK activity of PBMC (Figure 2). Flow cytometric analysis demonstrated that 10-15% of PBMC consisted of CD56 § cells, while MACS-enriched CD56 § NK cells were more than 96% pure. The trophoblast cells were cultured for one or three days before infection in order to determine if the stage of in vitro differentiation of the trophoblast cells before the infection would influence the susceptibility to NK cell lysis. During in vitro cultivation, the mononuclear trophoblast cultures differentiated into morphological syncytia-like cultures. However, no effect of in vitro morphological differentiation of trophoblasts was observed: The cultures remained uniformly resistant to NK cell killing whether cultured for 1 or 3 days (data not shown).

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Figure 2. The susceptibility of HSV-infected and mock-infected trophoblast and fibroblast cells to NK lysis mediated by the crude population of PBMC from healthy blood donors. Effector cells were incubated for 12 hours with HSV-l-infected trophoblast cells ( . . . . . ), mock-infected trophoblast cells (--o--), HSV-l-infected fibroblast cells (--T--), mock-infected fibroblast cells (--T--), or with cell line K562 (-D--). Results represent six similar experiments.

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Cold Target Inhibition Experiments An indirect measure of the possible inhibition for triggering of NK cells or for binding of NK cells to target cells is provided by the ability of target cells to competitively inhibit NK cell lysis of known susceptible cells (Collins et al., 1981). The results of cold target inhibition assays demonstrated that neither HSV-infected nor uninfected term villus trophoblast cells were able to inhibit the lysis of K562 cells. In contrast, placental fibroblast cells, whether HSV-l-infected or not, are found to inhibit competitively the lysis of 51Cr labeled K562 cells (Table I).

Analysis of Target Structures Implicated in NK Cell Recognitions Bishop et al. (1983b) demonstrated that an efficient expression of HSV-1 glycoproteins was essential for recognition and subsequent killing by NK cells. Therefore, investigation of possible variations in the level of HSV-antigen expression on the cell membrane of infected trophoblast and fibroblast cells were initiated to explain the observed differences in NK susceptibility. Kinetic studies showed that both trophoblast and fibroblast cells expressed similar amounts of HSV-1 glycoproteins 12 h after infection (Figure 3). Accordingly, the resistance of villus trophoblast cells to NK cell killing was not attributed to insufficient presentation of virus-specific determinants on the cell surface.

Table I Cold Target Inhibition of NK Cell Activity Against K562 Cells

% inhibition of cytotoxicity Competitor Cells

A

B

Trophoblast

2 _+3

3+2

Trophoblast-HSV

0 -+2

2 +_2

Fibroblast

67_+ 15

35 + 8

Fibroblast-HSV

71 + 18

38 + 9

Radiolabeled K562 cells were incubated with unlabeled competitor cells at a ratio of 10:1 (A) and 1:1 (B). Purified blood CD56 § lymphocytes were added at an E:T ratio of 12:1. The results are the medians of four experiments. Percentage of inhibition was calculated as described in Materials and Methods.

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Previously reported data (Carbone et al., 1991) suggested that MHC class I expression correlated inversely with the susceptibility to NK cell lysis. However, in contrast to these observations, the present study demonstrated that MHC-I positive fibroblast cells infected with HSV were susceptible to NK cells and, on the other hand, virus-infected trophoblast cells were MHC-I negative and yet resisted NK lysis (Figure 3). The transferrin receptor was another determinant which has been proposed to be instrumental in NK cell cytotoxicity (Borysiewicz et al., 1986). As with MHC-I, no significant difference in the expression of transferrin receptor between HSV-infected trophoblast and fibroblast cells was found (Figure 3).

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Table II

IFN Production During Coculture of Target Cells with CD56 § Lymphocytes

Target Cells

IFN Induced (U/ml)

Trophoblast

<4

Trophoblast-HSV

1640 + 180

Fibroblast

<4

Fibroblast-HSV

1880 + 220

IFN production was measured during cocuitivation of effector and target cells at the ratio of 25:1. Data represent the mean of four experiments using CD56§ lymphocytes as effector cells. Infection with HSV-1 and |FN quantitation were performed as described in Materials and Methods.

Relationship Between IFN Production and Susceptibility to NK Cell Lysis In order to determine if any possible variations in the production of IFN would affect the outcome of the cytotoxic reaction, the levels of IFN were measured during the cocultivation of NK cells with infected targets. As shown in Table II, IFN was detected in the supernatant of all cocultures with HSV-l-infected targets, while in control cocultures with uninfected targets <4 U/ml of IFN was found. The effector cells appeared to be the principal producer of IFN because <4 U/ml of IFN was produced in the control cultures of HSV-infected trOphoblast and fibroblast cells. Moreover, the amounts of IFN induced by HSV-l-infected trophoblast and fibroblast cells were comparable. Consequently, IFN released during in vitro culture did not seem to provide a clue as to the mechanisms underlying the differential susceptibility of HSV-infected placental trophoblast and fibroblast cells to NK cytotoxicity. DISCUSSION Virus infection of the trophoblast cell layer of the placenta is a potentially dangerous situation for the mother as well as for the fetus and an adequate cellular immune reaction against virus-infected trophoblasts could conceivably protect both the mother and the fetus/placenta. The aim of our work has been to test for one cellular immune mechanism which possible could cope with infection of trophoblast cells caused by an exogenous cytopathic virus. No experimental data of such kind have been available to date. In the present study, for the first time the in vitro activity of allogeneic peripheral blood NK cells against HSV-infected term villus trophoblast cells has been described. Further it has been demonstrated that virus infection does not alter the resistance of trophoblast to cytotoxicity mediated by NK effectors.

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NK cells represent an important cytotoxic effector in the lysis of other HSVinfected cells (Welsh, 1986; Lopez and Fitzgerald-Bocarsly, 1989). The effector population of peripheral blood lymphocytes which have been correlated with NK cell activity are the subpopulation of CD56 § lymphocytes. The present investigation demonstrates that the CD56 § fraction of PBMC are unable to lyse HSV-infected term trophoblast ceils, while HSV-infected placental fibroblast cells are preferentially lysed (Figures 1 and 2). The fact that the crude population of PBMC even in high E:T ratios have no cytotoxic effect on HSV-infected or uninfected trophoblast cells, argues against the possibility that the failure of CD56 § cells to lyse HSV-infected trophoblast cells is due to the lack of auxiliary functions provided by complementary cell populations. It is the syncytiotrophoblast, that is in direct contact with maternal blood in the villus areas of the h u m a n placenta. In order to investigate if in vitro differentiation of the trophoblast cultures, would affect their susceptibility to NK cell killing, cultures have been HSV-infected after 3 days of culture. However, in the present system, neither HSV-infected mononuclear trophoblast cultures or syncytio-like trophoblast cultures were susceptible to NK cell lysis. There is some controversy concerning the role of IFN in NK-mediated lysis of virus-infected cells (Bishop et al., 1983b; Fitzgerald-Bocarsly et al., 1989; Canessa et al., 1990). The present investigation showed that the NK cell subpopulation was able to produce IFN when cocultured with both HSV-infected targets. No significant differences in the IFN production induced by HSV-l-infected trophoblast and fibroblast cells were observed (Table II). These results obviously indicate that IFN is not a discriminating factor that accounts singularly for the differential susceptibility to NK cell-mediated killing of HSV-l-infected targets. The structural determinants which are net:essary for NK cell recognition and binding to HSV-infected cells are not well understood (Lopez-Guerrero et al., 1988; Storkus and Dawson, 1991). A previous work by Pross et al. (1986) suggests that uninfected trophoblast cells do not express receptors that can be recognized by NK cells. The present results are in line with these observations: Using HSV-infected villus trophoblast cells as competitive target, no inhibitory effect on NK lysis of K562 cells have been observed. In contrast, placental fibroblast cells, whether HSV-l-infected or not, inhibit competitively the lysis of 51Cr labeled K562 cells (Table 1). Furthermore, in an attempt to elucidate the role of possible receptors relevant for NK cytotoxicity, this study has focused on the expression of MHC class I antigens, transferrin receptors, and virus-specific glycoproteins on HSV-infected trophoblast and fibroblast cells. Previous findings have indicated that the susceptibility to NK cell lysis is inversely proportional to the surface expression of MHC class I antigens (Carbone et al., 1991). However, the current results do not confirm these observations: The villus trophoblast cultures show a low level of MHC-I expression after infection (Figure 3) and are not susceptible to NK lysis. Inversely, HSV-infected placental fibroblast cells show high levels of MHC-I expression and were lysed efficiently. Other determinants which have been correlated with the susceptibility to NK cells include viral determinants (Bishop et al., 1983a) and transferrin receptors (Borysiewicz et al., 1986). FACS analysis, however, reveals that HSV-1 glycoproteins and transferrin receptors are expressed on the surface of HSV-infected trophoblasts and fibroblasts almost to the same degree (Figure 3). Thus, the expression of viral antigens on the surface of trophoblast cells is not sufficient for recognition by the NK cells. A plausible

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explanation would seem to be offered either by the presence on the trophoblast cells of structures that interfere with the recognition process or by the lack of accessory recognition structures otherwise expressed by placental fibroblast cells. The presented results suggest that the resistance of trophoblast cells to NK cell-mediated lysis apparently is not changed by HSV-1 infection. None of the investigated parameters, previously reported to be relevant for NK cell susceptibility, could explain the observed differences in NK cell-mediated lysis between HSVinfected term placental villus trophoblast cellss and placental fibroblasts. The mechanisms by which HSV-infected trophoblast cells resist NK cell iysis remains to be elucidated. The ability of trophoblast cells to resist lysis by various cellular immune effectors may play an important role in protecting the placenta from maternal immune rejection. The immune resistant quality of the trophoblast cells may have such a high biological priority, that it explains why trophoblast cells differs from other cell types in being resistant to NK cell lysis even after HSV infection. The current results, however, do not exclude the possibility that HSV-infected trophoblast cells may be susceptible to other forms of cell-mediated lysis based on different mechanisms. When considering the accumulation of CD56 § large granular lymphocytes in the early maternal decidua (King et al., 1989; Ferry et al., 1991), one can still hypothesize their participation in the antiviral defense of the placenta. The resistance of HSV-infected term villus trophoblast cells to the immune attack of allogeneic peripheral blood NK cells raises further questions which is currently being addressed: 1) What are the implications of the observed resistance of HSV-infected trophoblasts to NK cytotoxicity for other viruses? 2) Are virus-infected trophoblasts from a different gestational period (first trimester) susceptibility to MHC-nonrestricted cytotoxicity? 3) Are other alternative effector mechanisms (maternal a n d / o r fetal) involved in the killing of virus-infected trophoblast cells? SUMMARY The study was undertaken to determine if HSV infection of trophoblast cells would alter the general resistant state of trophoblast cells to MHC non-restricted cellmediated cytotoxicity. The NK cell activity against HSV-infected human term placental trophoblast cells was investigated. In a 12-hour SlCr release assay, PBMC, or immunosorted peripheral blood CD56 § NK cells from healthy donors were found not to lyse HSV-l-infected trophoblast cells, while HSV-l-infected placental fibroblast cells were preferentially lysed when using the same experimental set-up. Furthermore, using cold target competition assay, no inhibition of NK cell activity against the susceptible target cell K562 was noted when using HSV-infected or uninfected trophoblast cells. Inversely, both HSV-infected and uninfected fibroblast cells demonstrated a competitive inhibition of NK cell lysis of K562 cells. The observed differences between the susceptibility of HSV-infected placental trophoblast and fibroblast cells to NK lysis could not be explained by a difference in the surface expression of HSV-antigens or transferrin receptors. Additionally, no significant differences were observed for de novo production of IFN by effector cells during the cocultivation with HSV-infected trophoblast and fibroblast cells that would explain the observed discrepancies in NK susceptibility. Consequently, these in vitro results suggest that the observed resistance of trophoblast cells to maternal cell-mediated

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lysis is not modified by HSV-1 infection and that the peripheral blood NK cells are not involved in the prevention of a possible vertical HSV spread through infected trophoblast cells. ACKNOWLEDGEMENTS This work was supported by the Danish Medical Research Council and by Erik and Knudsine Leiions Mindelegat. REFERENCES Bishop, G.A., Glairiest, J.C., and Schwartz, S.A. (1983a) Relationship between expression of HSV glycoprotein and susceptibility of target cells to human natural killer activity. J. Exp. Med. 157, 544-1561. Bishop, G.A., Glairiest, J.C., and Schwartz, S.A. (1983b) Role of interferon in human natural killer activity against target cells infected with HSV-1. J. Immunol. 131, 1849-1853. Borysiewicz, L.K., Graham, S., and Patrick-Sissons, J.G. (1986) Human natural killer cell lysis of virus-infected cells. Relationship to expression of the transferrin receptor. Eur. J. Immunol. 16, 405-411. Canessa, A., Chatterjee, S., Whitley, S., Prasthofer, R.J., Grossi, E.F., and Tilden, A.B. (1990) Individual NK cell clones lyse both tumor cell target and herpes simplex virus-infected fibroblasts in the absence of interferon. Viral Immunol. 3, 217224. Carbone, E., Racioppi, L., La Cava, A., Portella, G., Velotti, F., Zappacosta, S., and Fontana, S. (1991) NK and LAK susceptibility varies inversely with target cell MHC Class I antigen expression in a rat epithelial tumour system. Scand. J. Immunol. 33, 185-195. Chin, C. and Lopez, C. (1979) Natural killing of HSV-1 infected target cells: Normal human responses and influence of antiviral antibody. Infect. Immun. 26, 49-60. Collins, J.L., Patek, P.Q., and Cohn, M.J. (1981) Tumorigenicity and lysis by natural killers. J. Exp. Med. 153, 89. Degliantoni, G., Murphy, M., Kobayashi, M., Francis, B. Perussia, M.K., and Trinchieri, G. (1985) Natural killer (NK) cell-derived hematopoietic colonyinhibiting activity and NK cytotoxic factor. Relationship with tumor necrosis factor and synergism with immune interferon. J. Exp. Med. 162, 1512-1530. Douglas, G.C. and King, B.F. (1989) Isolation of pure villous cytotrophoblast from term human placenta using immunomagnetic microspheres. J. Immunol. Methods 119, 259-268. Faulk, W.P. and Hunt, J.S. (1989) Human placentae: View from an immunological bias. Am. J. Reprod. Immunol. 21,108-113.

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Ferry, B.L., Sargent, I.L., Starkey, P.M., and Redman, C.W.G. (1991) Cytotoxic activity against trophoblast and choriocarcinoma cells of large granular lymphocytes from human early pregnancy decidua. Cell. Immunol. 132, 140-149. Fitzgerald-Bocarsly, P., Feldman, M., Curl, S., Schnell, J., and Denny, T. (1989) Positively selected Leu-lla (CD16 § cells require the presence Of accessory cells or factors for the lysis of herpes simplex virus-infected fibroblasts but not herpes simplex virus-infected Raji. J. Immunol. 143, 1318-1326. Head, J.R. (1989) Can trophoblast be killed by cytotoxic cells: In vitro evidence and in vivo possibilities. Am. J. Reprod. lmmunol. 20, 100-105. Howell, D.M. and Fitzgerald-Bocarsly, P. (1991) Natural killer-mediated lysis of some but not all HSV-I-or VSV-infected targets requires the participation of HLA-DR-positive accessory cells. Immunology 72, 443-447.

Hutto, C., Arvin, A., Jacobs, R., Steele, R., Stagno, S., Lyrene, R., Willett, L., Powell, D., Andersen, R., Werthammer, J., Ratcliff, G., Nahmias, A., Christy, C., and Whitley, R. (1987) Intrauterine herpes simplex virus infections. J. Pediatr. 110, 97-101. Jondal, M. (1987) The human NK cell: A short overview and a hypothesis on NK recognition. Clin. Exp. Immunol. 70, 255-262. King, A., Birkby, B., and Loke, Y.W. (1989) Early human decidual cells exhibit NK activity against the K-562 cell line but not against first trimester trophoblast. Cell. Immunol. 118, 337-344. Lanier, L.L., My L.E., A., Civin, C.T., Loken, M.R., and Phillips, J.H. (1986) The relationship of CD16 (leu-11) and leu-19 (NKH-1) antigen expression on human peripheral blood NK cells and cytotoxic lymphocytes. J. Immunol. 136, 44804486. Lopez, C. and Fitzgerald-Bocarsly, P. (1989) Natural host defense systems active against herpes simplex virus infections. In: Function of the Natural Immune System, (eds.) C.W. Reynolds and R.H. Wiltrout, Plenum Press, New York, pp. 85-110. Lopez-Guerrero, J.A., Alarcon, B., and Fresno, M. (1988) Mechanism of recognition of herpes simplex virus type 1-infected cells by natural killer cells. J. Gen. Virol. 69, 2859-2868. Norskov-Lauritsen, N., Aboagye-Mathiesen, G., Juhl, C.B., Petersen, P.M., Zachar, V., and Ebbesen, P. (1992) Herpes simplex virus infection of cultured human term trophoblast. J. Med. Virol. 36, 162-166. Paya, C.V., Schoon, R.A., and Leibson, P.J. (1990) Alternative mechanisms of natural killer cell activation during herpes simplex virus infection. J. lmmunol. 144, 4370-4375.

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Pflueger, E., Mueller, E.A., and Anderer, F.A. (1990) Preservation of cytotoxic function during multi-cycle immunomagnetic cell separation of human NK cells using a new type of magnetic beads. J. Immunol. Methods 129, 165-173. Pross, H., Mitchell, H., and Werkmeister, J. (1985) The sensitivity of placental trophoblast cells to intraplacental and allogeneic cytotoxic lymphocytes. Am. J. Reprod. lmmunol. Microbiol. 8, 1-9. Robb, J.A., Benirschke, K., and Barmeyer, J. (1986) Intrauterine latent herpes simplex virus infection: I. Spontaneous abortion. Hum. Pathol. 17, 1196-1209. Storkus, W.J. and Dawson, J.R. (1991) Target structures involved in natural killing (NK): Characteristics, distribution, and candidate molecules. Crit. Rev. Immunol. 10, 393-405. Toth F.D., Norskov-Lauritsen, N. Juhl, C., and Ebbesen, P. (1991) Human trophoblast interferon: Pattern of response to priming and superinduction of purified term trophoblast and choriocarcinoma cells. J. Reprod. Immunol. 19, 55-67. Vistine, A., Nahmias, A., Whitley, R., and Alford, C. (1981) The natural history and epidemiology of neonatal herpes simplex virus infection. In: The Human Herpes Viruses, (eds.) A.J. Nahmias, W.R. Dowdle, and R.E. Schinazi, Elsevier, New York, pp. 599-600. Welsh, R.M. (1986) Regulation of virus infections by natural killer cells. Nat. Immun. Cell. Growth Regul. 5, 169-199. Wilson, C.B., Hass, J.E., and Weaver, W.M. (1983) Isolation, purification and characteristics of mononuclear phagocytes from human placentas. J. Immunol. Methods 56, 305-317.