Suppression of cytotoxic T-lymphocyte activity during human pregnancy

Journal of Reproductive Immunology, 23 (1993) 119-130

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Elsevier Scientific Publishers Ireland Ltd.

JRI 00811

Suppression of cytotoxic T-lymphocyte activity during human pregnancy N a o k i N a k a m u r a a, Kohjbi M i y a z a k i a, Y o i c h i r o K i t a n o a, Syunichi Fujisaki and Hitoshi Okamura a aDepartment of Obstetrics and Gynecology, Kumamoto University School of Medicine, Kumamoto and bNichinan Prefectural Hospital, Miyazaki (Japan) (Accepted for publication 10 November 1992)

Summary We have investigated alterations in Epstein-Barr virus antigen specific cytotoxic T-lymphocyte (EBV-CTL) activity during human pregnancy. EBV-CTL activity was determined by a modified EBV induced B-cell focus regression assay and was expressed in terms of a regression index (IR50), i.e. the initial cell concentration required to achieve a 50%-incidence of regression in EBV-infected cell culture. Increased values of IR50 indicate the suppression of EBV-CTL activity. In 113 human female T-cell leukemia type-I (HTLV-I) non-carriers, the IR50 values (mean 4- S.E.) in non-pregnant, pregnant (the first trimester, second trimester and third trimester of pregnancy) and puerperal women were 10.6 4- 1.4, 16.1 4- 1.1 (20.1 4- 2.0, 14.8 4- 2.0, 14.6 4- 1.6), and 12.1 4- 1.9 respectively. Among HTLV-I carriers, the IR50 values (mean 4- S.E.) were likewise 34.6 4- 8.0, 87.4 4- 5.2 (101.7 4- 6.3, 88.3 4- 8.4 and 79.5 4- 9.2) and 39.2 4- 7.1, respectively. This data demonstrate: 1) EBV-CTL activity was suppressed during pregnancy (P < 0.05), especially in the first trimester (P = 0.0003). 2). In HTLV-I carriers, this suppression was shown in the first trimester (P = 0.0002), in the second trimester (P = 0.0002) and in the third trimester of pregnancy (P = 0.0014) and 3). One month after delivery, this suppression had returned to the non-pregnant level in both HTLV-I non-carriers and HTLV-I carriers. Pregnancy therefore has a suppressive effect on antigen specific cytotoxic T-lymphocyte activity and this effect is amplified in HTLV-I carriers. Correspondence to: Naoki Nakamura, Department of Obstetrics and Gynecology, Kumamoto University School of Medicine, 1-1-1 Honjo, Kumamoto 860, Japan. 0165-0378/93/$06.00 © 1993 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland

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Key words: EBV antigen specific cytotoxic T-lymphocyte activity; human pregnancy; HTL V-I carrier

Introduction

Studies on peripheral blood mononuclear cells (PBMC) of pregnant women, have focussed on the changes in lymphocyte activity to non-specific mitogens (Purtilo et al., 1972; Metcalf et al., t972), the changes in natural killer (NK) cell activity (Lee et al., 1987; Hidaka et al., 1991) and more recently, changes in lymphocyte subsets, using monoclonal antibodies (Glassman et al., 1987: Castilla et al., 1989). However, little information is available on the effect of pregnancy on antigen specific cytotoxic T-lymphocyte (CTL) activity. To provide such data we have studied CTL activity using Epstein-Barr virus (EBV) as an antigen in women during pregnancy and in the puerperium. Katsuki et al. (1987), using a modified EBV-induced B cell focus regression assay, have reported that human T-cell leukemia type I (HTLV-I) suppressed Epstein-Barr virus antigen specific cytotoxic T-lymphocyte (EBV-CTL) function among HTLV-I carriers above 40 years of age. We have therefore also evaluated EBV-CTL of pregnant women who are HTLV-I carriers. Simultaneously, we examined lymphocyte subsets in peripheral blood samples (CD4+, CD8+, CD4+/DR+, CD8+/DR+, C D 8 + / C D l l b + and CD8+/CD 11b-lymphocytes). Materials and Methods

Subjects The subjects were 113 female HTLV-I non-carriers and 55 female HTLV-I carriers. HTLV-I non-carriers and carriers were each divided into three groups; the non-pregnant women as a control, pregnant women and puerperal women (1 month after delivery) groups. All peripheral blood samples were collected at Kumamoto University Hospital between January 1989 and December 1991. The blood samples of the pregnant HTLV-I noncarriers and carriers were obtained at the outpatient clinic of our obstetric service with the written consent of the patients. The blood samples of the non-pregnant HTLV-I carriers were provided by the Blood Transfusion Service in the hospital. The blood samples of the non-pregnant HTLV-I noncarriers were obtained from healthy volunteers. All subjects of each group were matched for age and locality. Most of the women were born and are living in Kumamoto Prefecture, an adult T-cell leukemia-endemic area in

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southwestern Japan. Patients who had gynecological or obstetrical disorders were excluded. The pregnant groups were further divided into three categories; the first trimester (7-13 weeks of gestation), the second trimester (14-27 weeks) and the third trimester (28-41 weeks). The 113 HTLV-I non-carriers consisted of 23 non-pregnant women (mean age: 27.7 years of age, ranging from 21 to 36 years of age); 72 pregnant women (30.3 years, 22-41 years), of whom 19 were in the first trimester, 22 in the second trimester and 31 in the third trimester; and 18 puerperal women (30.7 years, 24-42 years). The 55 HTLV-I carriers consisted of 15 non-pregnant women (32.1 years, 22-40 years); 24 pregnant women (30.2 years, 23-39 years), of whom five were in the first trimester, nine in the second trimester and 10 in the third trimester; and 16 puerperal women (29.1 years, 23-37 years).

Screening of sera for HTL V-I antibodies Sera were surveyed for HTLV-I specific antibodies by an indirect immunofluorescence assay (Hinuma et al., 1982) and an enzyme-linked immunosorbent assay (ELISA) (Taguchi et al. 1983). The serum was considered positive for HTLV-I antibody when both assays were positive.

Preparation of peripheral blood mononuclear cells (PBMC) and lymphocytes (PBL) PBMC were obtained from 10 ml of heparinized (10 units/ml heparin) peripheral blood diluted in the same volume of phosphate buffered saline (PBS)(Ca 2÷, Mg 2÷ free) by means of Ficoll- (Pharmacia, Sweden) Conray (Daiichiseiyaku, Japan) density gradient centrifugation (400 × g for 30 min). PBMC were washed three times in PBS and were then used for analysis of cell populations. PBMC were resuspended in 7 ml of culture medium RPMI 1640 (GIBCO, USA) containing 10% heat inactivated fetal bovine serum, penicillin G 100 IU/ml, streptomycin 100 #g/ml and sodium bicarbonate 2 mg/ml - - in 25 cm 2 plastic culture dishes and were then incubated overnight in a 5% CO2, 95% air incubator in order to remove monocytes. Staining with Giemsa showed that almost all non-adherent cells were lymphocytes. Cell viability was examined by staining using trypan blue dye. Viability was found to be more than 95%. PBL were used for evaluation of EBV-CTL activity.

Evaluation of EBV-CTL activity The EBV used was a B95-8 derived strain provided by Dr. Katsuki. EBVCTL activity was determined by a modified EBV-induced B-cell focus regression assay described by Katsuki et al. (1987). This is a modified version of the method of Moss et al. (1978). Isolated PBL were incubated with EBV for

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60 min in a water bath at 37°C. Following this procedure all B cells in PBL were infected with EBV. EBV-infected PBL were cultured in 0.15-ml volumes at cell concentrations of 80 x 106/ml, 40 x 106/ml, 20 x 106/ml, 10 x 106 ml, 5 x 106/ml, 2.5 x 106/ml, 1.25 x 106/ml and 0.625 x 106/ml. Six replicate cultures were set up for each cell concentration in a microtiter plate (96-well U-bottom microplate, Nunc., Denmark) and incubated in a 5% CO2, 95% air incubator with a half dose medium change every 3 or 4 days. After 5 weeks, transformation or regression of EBV-infected PBL could be clearly determined by observation of the morphological features. Transformation was indicated by a morphological change to lymphoblastoid-like cells and by clumping of cells. The incidence of regression was scored as the number of culture wells among the six wells at initial cell concentration that showed no evidence of lymphoblastoid-like cells and clumping of cells. IR50 was expressed in terms of the initial cell concentrations ( x 106/ml), determined by the method of Reed and Muench proportional allotment, required to achieve a 50% incidence of regression in EBV-infected cells. Increased values of IR50 indicate the suppression of EBV-CTL activity.

Monoclonal antibodies ( MoAbs) MoAbs were used in dual immunofluorescence; these included LeuLeu-2a FITC (CD8), L e u - 2 a pE(phyc°erthrin) (CD8), HLA-DR PE and Leu-15 PE ( C D l l b ) (Becton Dickinson, Mountain View, CA). 3aFITC(fluorescein isothiocyanate) (CD4),

Flow-cytometric analysis of lymphocyte subsets The lymphocyte subsets we determined were Leu3a ÷ (helper/inducer), Leu2a ÷ (suppressor/cytotoxic), Leu3a+DR ÷ (activated helper/inducer), Leu2a ÷ DR + (activated suppressor/cytotoxic), Leu2a+Leul5 - (cytotoxic), Leu2a+Leul5 ÷ (suppressor) T lymphocytes. Flow-cytometric analysis was performed using an Ortho Spectrum III automated flow cytometry (Ortho Diagnostic System).

Statistical analysis Statistical analysis of the data was performed using the Student's t-test or the Mann-Whitney U-test under the analysis of variances. When the MannWhitney U-test was used with over 20 numbers, a P-value was decided by the Wilcoxon-Mann-Whitney U-test. Statistical analysis of the data in serial samples of 12 HTLV-I carriers was performed by the paired t-test. In all analyses, a P-value below 0.05 was considered to be statistically significant. Results

EBV-CTL activity in HTLV-I non-carriers The IR50 (mean 4- SE) of 23 non-pregnant women, 19 pregnant women in the first, 22 in the second and 31 in the third trimesters and 18 puerperal

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w o m e n w e r e 10.6 4- 1.4,20.1 4- 2.0, 14.8 4- 2.0, 14.6 4- 1.6, and 12.1 4- 1.9, respectively (Fig. 1). The IR50 of the pregnant women in the first trimester was significantly higher than those of non-pregnant women (P = 0.0003), pregnant women in the second trimester (P = 0.0429) and the puerperal women (P = 0.0059). The IR50 (mean 4- S.E.) of the 72 pregnant women as a whole was 16.1 4- 1.1, significantly higher than that of the non-pregnant women (P = 0.006). Lymphocyte subsets in H T L V-I non-carriers The results are shown in Table 1. The percentage of CD4+ T lymphocytes in the pregnant women in the first trimester and in the pregnant women overall was significantly lower as compared with those of non-pregnant women (P < 0.05). There were no other significant differences observed between groups in the other lymphocyte subsets. E B V - C T L activity in H T L V-I carriers The IR50 (mean 4- S.E.) of 15 non-pregnant women, five pregnant women in the first, nine in the second and 10 in the third trimesters, and 16 puerperal women were 34.6 4- 8.0, 101.7 ± 6.3, 88.3 4- 8.4, 79.5 4- 9.2, and 39.2 4- 7.1, respectively (Fig. 2). The IR50 of the pregnant women in first, second and third trimesters were significantly higher (P = 0.0002, 0.0002 and 0.0014, respectively) as compared with that of the non-pregnant women. For 24 pregnant women as a whole, the IR50 (mean 4- S.E.) was 87.5 4- 5.2, significantly higher (P < 0.0001) than that of the non-pregnant women. Likewise, the IR50 values of the pregnant women in the first, second and third trimesters, and of the pregnant w o m e n as a whole,, were significantly higher ( P = 0.0003, 0.0004, 0.0026 and <0.0001, respectively) compared with that of the puerperal women. Lymphocyte subsets in H T L V-I carriers The results are shown in Table 2. There were no other significant differences among these groups in any of the lymphocyte subsets. EBV-CTL activity in serial samples among 12 H T L V - I carriers The results are shown in Fig. 3. In 12 HTLV-I carriers, the IR50 values were serially determined during pregnancy and one m o n t h after delivery. The IR50 values (mean 4- S.E.) of HTLV-I carriers during pregnancy and one m o n t h after delivery were 90.9 4- 4.6 and 36.2 4- 7.0, respectively. This difference was significant (P = 0.0002). Discussion A variety of studies on maternal cell-mediated i m m u n e responsiveness have yielded conflicting results. Some investigators have reported decreased

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blastogenic responses of maternal PBL during pregnancy (Purtilo et al., 1972; Fujisaki et al., 1979), whilst other workers have found no difference (Metcalf et al., 1972). In NK cell activity, increased, normal and decreased activities have all been reported during pregnancy (Hidaka et al., 1991; Lee et al., 1987; Baley and Schacter, 1985; Tartof et al., 1984). Similarly, in the changes of lymphocyte subsets during normal pregnancy, discrepant results have been reported (Sridama et al., 1982; Tallon et al., 1984; Glassman et al., 1985; Degenne et al., 1988; Castilla et al., 1989). The regression phenomenon of EBV-transformed B cells in the EBVinduced B cell focus regression assay (Katsuki et al. 1987) may represent a highly complex interaction between soluble and cell-mediated cytotoxic and suppressive factors whose overall effect is to inhibit continued outgrowth of EBV-transformed B cells. Moss et al. (1979) and Rickinson et al. (1979) have presented evidence that EBV-specific memory T cells were responding to EBV-infected B cells in the culture firstly by proliferation and secondly by the development of cytotoxic activity against the autologous EBVtransformed cell line. Our present study, the first in vitro study on the activity of virus antigen specific CTL during human pregnancy, shows that EBV-CTL activity was suppressed, especially in the first trimester of pregnancy, and that it recovered to the non-pregnant level one month after delivery. This result is in agreement with previous reports for suppression of maternal cell mediated immunity (CMI) (Purtilo et al., 1972; Fujisaki et al., 1979; Baley and Schacter, 1985; Gill, 1985; Clark et al., 1987). Pregnancy may increase vulnerability to certain viral infections such as hepatitis, rubella and herpes simplex (Waterson, 1979). Increased prevalence of human papilloma virus and a high replication rate of viral DNA in the lower genital tract have also been demonstrated during pregnancy (Schneider et al., 1987). Sakamoto et al., (1982) reported that EBV was reactivated in many of the pregnant women. Our data demonstrating EBV-CTL activity decrease during pregnancy also supports these findings. Since Medawar's proposed a hypothesis in 1956 that maternal CMI is lowered in pregnancy, many investigators have studied not only PBL function but also feto-maternal local immune responses. Wegmann (1987) has presented the theory of immunotrophism, whereby maternal T cells are activated to enhance placental growth and function, in which immunostimulatory factors and non-specific immuno-suppressive factors may be produced and cooperate in the maintenance of pregnancy. As immunostimulatory factors, interleukin (IL)-I, IL-6 (Masuhiro et al., 1991), granulocyte macrophage colony stimulating factor (Robertson et al., 1992) and tumor necrosis factor (Li et al., 1992), and as immuno-suppressive factors, progesterone, estrogen (Clemens et al., 1979), human chorionic

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gonadotropin, and transforming growth factor beta (Frolic et al., 1983; Clark et al., 1990; Matsuzaki et al., 1992) have been reported. We have added progesterone, estrogen or human chorionic gonadotropin in the culture medium in our assay to investigate the potential suppressive effect against EBV-CTL, but without any positive influence (data not shown). As IL-6, one of the proposed immuno-stimulatory factors during pregnancy, has been reported to be an autocrine growth factor in EBV transformed B lymphocytes (Taga et al., 1987; Tosato et al., 1988), it is possible that the present observation of suppressed EBV-CTL activity during pregnancy may be due to increased IL-6 secretion (Kameda et al., 1990). HTLV-I induced immunosuppression has been demonstrated in the in vivo study of suppression of the tuberculin skin reaction (Tachibana et al. 1988) and in reports of increased spontaneous proliferation and decreased mitogenic responses of PBL in vitro (Nakasone et al., 1992). Suppressed EBV-CTL activity in HTLV-I carriers (Katsuki et al., 1987) has provided information on functional impairment of T lymphocyte activity. In this study, decreases of EBV-CTL activity during pregnancy in HTLV-I carriers were significant in comparison with that in the HTLV-I non-carriers. This assay in HTLV-I carriers may be useful in understanding the role of immunomodulators in the maternal immune response. On the other hand, HTLV-I carriers seem to be susceptible to infection, especially during pregnancy (Ohba et al., 1992). The close association of various opportunistic infections such as Pneumocystis carinii pneumonia (Taguchi and Miyoshi, 1989) or strongyloidiasis (Nakada et al., 1984) have already been reported in HTLV-I carriers. It is therefore important to survey the frequency of virus infection (Herpes simplex, Cytomegalovirus, etc.) in HTLV-I carriers during pregnancy. In HTLV-I non-carriers, we demonstrated a decrease in CD4+ T lymphocytes in the first trimester of pregnancy. These data conform well with the report of Castilla et al. (1989). However, in HTLV-I carriers, no differences were detected in lymphocyte subsets between the non-pregnant, pregnant and puerperal women. Between HTLV-I non-carriers and carriers there was no difference except in the percentage of activated CD4+ T lymphocytes. This suggests that immunosuppression in HTLV-I carriers may arise from dysfunction rather than changes in lymphocyte cell surface antigens. Biggar et al. (1989) showed that the percentage of CD4+ cells in human immunodeficiency virus (HIV) seropositive women fell during pregnancy and did not recover in the postpartum period. It is of interest that there exists a difference between impairment of immunity in HTLV-I carriers and HIV carriers. Our data provide evidence that T-cell function is suppressed during pregnancy, especially in HTLV-I carriers, and additive suppressive effects seem to exist between pregnancy and HTLV-I infection. Decreased EBV-CTL

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activity may well be a useful parameter to determine the presence of immunological modulators in the maternal immune response. The mechanism of immunosuppression enhanced by pregnancy and HTLV-I infection has not been clarified. A further study of the role of cytokines, such as IL-6, which suppressed EBV-CTL activity, may provide useful information in this direction. Acknowledgements We are grateful to Dr. Takato Katsuki at the Department of Medical Technology, Kumamoto University College of Medical Science, for providing the EBV and giving expert technical assistance, to Dr. Kazunari Yamaguchi at the Blood Transfusion Service, Kumamo to University School of Medicine for providing the blood samples of the non-pregnant HTLV-I non-carriers. References Baley, J.E. and Schacter, B.Z. (1985) Mechanisms of diminished natural killer cell activity in pregnant women and neonates. J. Immunol. 47, 3042-3048. Biggar, R.J., Pahwa, S., Minkoff, H., Mendes, H., W~lloughby, A., Landesman, S. and Gordert, J.J. (1989) Immunosuppression in pregnant women infected with human immunodeficiency virus. Am. J. Obstet. Gynecol. 161, 1239-1244. Castilla, J.A., Rueda, R., Vargas, L., Gonzalez-Gomez, F. and Garcia-Olivares, E. (1989) Decreased levels of circulating CD4 ÷ T lymphocytes during normal human pregnancy. J. Reprod. Immunol. 15, 103-111. Clark, D.A., Croy, B.A., Wegrnann, T.G. and Chaouat, G. (1987) Immunological and paraimmunological mechanisms in spontaneous abortion: recent insights and future directions. J. Reprod. Immunol. 12, 1-12. Clark, D.A., Flanders, K.C., Banwatt, D., Millar-Book, W., Manuel, J., Stedronska-Clark, J. and Rowley, B. (1990) Murine pregnancy decidua produces a unique immunosuppressive molecule related to transforming growth factor beta-2. J. Immunol. 144, 3008-3014. Clemens, L.E., Siiteri, P.K. and Stites, D.P. (1979) Mechanism of immunosuppression of progesterone on maternal lymphocyte activation during pregnancy. J. Immunol. 122, 1978-1985. Degenne, D., Canepa, S., Lecomte, C., Renoux, M. and Bardos, P. (1988) Serial study of T-lymphocytes subsets in women during very early pregnancy. Clin. Immunol. lmmunopathol. 48, 187-191. Frolik, C.A., Dart, L.L., Meyers, C.A., Smith, D.M. and Sporn, M.B. (1983) Purification 'and initial characterization of a type beta transforming growth factor from human placenta. Proc. Natl. Acad. Sci. U.S.A. 80, 3676-3680. Fujisaki, S., Mori, N., Sasaki, T. and Maeyama, M. (1979) Cell-mediated immunity in human pregnancy: changes in lymphocyte reactivity during pregnancy and postpartum. Microbiol. Immunol. 23~ 899-907. Gill, T.J. (1985) Immunity and pregnancy. CRC Crit. Rev. Immunol. 5, 201-227. Glassman, A.B., Bennett, C.E., Christopher, J.B. and Self, S. (1985) Immunity during pregnancy: Lymphocyte subpopulations and survival and mitogen responsiveness. Ann. Clin. Lab, Sci. 15, 357-362. Hidaka, Y., Amino, N., lwatani, Y., Kaneda, T., Mitsuda, N., Morimoto, Y., Tanizawa, O. and Miyai, K. (1991) Changes in natural killer cell activity in normal pregnant and postpartum period and decrease in late pregnancy. J. Reprod. Immunol. 20, 73-83. Hinuma, Y., Komoda, H., Chosa, T., Kondo, T., Kohakura, M., Takenaka, T., Kikuchi, M., lchimaru,

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130 Sridama, V., Pacini, F., Yang, S.L., Moawad, A., Reilly, M. and DeGroot, L.J. (1982) Decreased levels of helper T cells. A possible cause of immunodeficiency in pregnancy. N. Engl. J. Med. 307, 352-356. Tachibana, N., Okayama, A., Ishizaki, J., Yokota, Tsutomu., Shishme, E., Murai, K., Shiori, S., Tsuda, K., Essex, M. and Mueller, N. (1989) Suppression of tuberculin skin reaction in healthy HTLV-I carriers from Japan. Int. J. Cancer 42, 829-831 Taga, T., Kawasaki, Y., Hardy, R.R., Hirano, T. and Kishimoto T. (1987) Receptors for B cell stimulatory factor 2. Quantitation specificity, distribution, and regulation of their expression. J. Exp. Med. 166, 967-981. Taguchi, H. and Miyoshi, I. (1989) Immune suppression in HTLV-1 carriers: a predictive sign of adult T-cell leukemia. Acta. Med. Okayama 43, 317-321. Taguchi, H., Sawada, T., Fujishita, M., Morimoto, T., Niiya, K. and Miyoshi, I. (1983) enzyme-linked immunosorbent assay of antibodies to adult T-cell leukemia-associated antigens. Jpn J. Cancer Res. (Gann) 74, 185-187. Tallon, D.F., ArachCorcoran, D.J. O'Dwyer, E.M. and Greally, J.F. (1984) Circulating lymphocyte subpopulations in pregnancy: a longitudinal study. J. Immunol. 132, 1784-1787. Tartof, D., Curran, J.J., Yang, S.L. and Livingston, C (1984) NK cell activity and skin test antigen stimulation of NK like CMC in vitro are decreased to different degrees in pregnancy and sarcoidosis. Clin. Exp. Immunol. 57, 502-510. Tosato, G., Gerrard, T.L., Goldman, N.G. and Pike, S.E. (1988) Stimulation of EBV-activated human B cells by monocytes and monocyte products. Role of IFN-beta2/B cell stimulatory factor 2/1L-6. J. lmmunol. 140, 4329-4336. Waterson, A.P. (1979) Virus infections (other than rubella) during pregnancy. Br. Med. J. 2, 564-566. Wegmann, T.G. (1987) Placental immunotrophism: Maternal T cells enhance placental growth and function. Am. J. Reprod. Immunol. Microbiol. 15, 67-70.