-NOTE- SELECTIVE EXPRESSION OF PATERNAL HUMAN MAJOR HISTOCOMPATI- BILITY ANTIGENS ON THE SURFACE OF HYDATIDIFORM MOLE CELLS*1

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1 -NOTE- SELECTIVE EXPRESSION OF PATERNAL HUMAN MAJOR HISTOCOMPATI- BILITY ANTIGENS ON THE SURFACE OF HYDATIDIFORM MOLE CELLS*1 Yoshiyuki TSUJI,*2 Shuichi MATSUDA,*3 Tsugukiyo HIRAI,*3 Hiromi FUJIWARA,*2 and Toshiyuki HAMAOKA*2 Department of Oncogenesis, Institute for Cancer Research, Osaka University Medical School,*2 and Renal Transplantation Center, Hyogo Prefectural Nishinomiya Hospital*3 The typing of human major histocompatibility antigens (HLA) of two cases of invasive hydatidiform mole showed that the fibroblast-like cells as well as trophoblasts from the mole selectively expressed paternal HLA haplotype specificity but not maternal HLA on the surface. This result was completely in agreement with the notion of androgenetic origin of hydatidiform mole from the aspects of HLA specificity. Immunological implication of this finding and development of chorionic tumor were discussed in the light of fetomaternal relationship and host immune surveillance against the tumor. Chorionic tumor is one of the neoplasms which have relatively higher incidence in Asian countries as well as in Latin American countries of the tropical zone than in European or North American countries.9) This tumor is classified into three entities; hydatidiform mole, invasive hydatidiform mole, and choriocarcinoma.9) These chorionic tumors are immunologically very peculiar tumor system with respect to the host-tumor relationship. These tumors are generated in regard to the role of the major histocompatibility complex (MHC) in the implementation of the T cell-mediated immunity. It has been suggested that TATA are closely associated with the MHC.2) All or most of TATA are recognized by T cells in conjunction with MHC of the self, and compatibility at the MHC locus leads to stronger or more effective target cell destruction. Because chorionic tumor may have alloantigen of the paternal side, it is conceivable that host immunity to chorionic tumor, if from fertilized ovum, and theoretically considered to be semiallogeneic to the host. any, may be directed not only to putative There has been a considerable body of TATA, but also to paternal alloantigens. evidence which indicates the presence of In actual clinical course, however, chorionic tumor-associated transplantation antigens tumor is potentially malignant, and causes (TATA) on the surface of tumor cells which frequent invasion or wide-spread metastasis provide the host with effective immune in the whole body. Thus, it is very important resistance against neoplasms. Furthermore, to study why such a theoretically semiallogeneic tumor grows progressively in the there has been a rapid progress recently in the understanding of mechanisms involved host. In order to understand this paradoxical in various immune responses, especially phenomenon, it may be important to know *1 This work was supported in part by a Grant-in-Aid for Cancer Research from the Ministry of Educa - tion, Science and Culture. *2 Fukushima , Fukushima-ku, Osaka 553 *3 13-9, Rokutanji-cho, Nishinomiya (6)

2 how the HLA haplotype specificity of the maternal and paternal origin is expressed on the chorionic tumor. We examined two cases of invasive hydatidiform mole and found that paternal HLA specificity was selectively expressed on the surface of the cells derived from the mole, and that to our surprise, no maternal HLA was present on the cells. Materials and Methods Hydatidiform Mole: Two cases of invasive hydatidiform mole from patients M. O. and M. I. were examined. Both of them were in the first trimester when the therapy was begun. Definitive diagnosis was made before operation by ultrasonic echography and pelvic angiography. Maximum urinary human chorionic gonadotropin (HCG) titers were 500,000IU/liter in M. O. and 1,000,000IU/ liter in M. I. M. O. showed invasion of mole cells into the endometrium and was hysterectomized one month after artificial mole delivery. M. I. had vaginal metastasis and was operated at the time of uterine curettage but not hysterectomized yet. Now, she is still receiving chemotherapy because of high urinary HCG excretion. The mole tissues were obtained aseptically by curettage of uterine cavity, and stored in a sterilized cold Eagle's minimum essential medium (MEM) containing Y. TSUJI, ET AL. HLA Typing: We used a series of HLA typing panel antisera comprising 14 type-specificities for HLA-A locus and 22 type-specificities for HLA-B locus. HLA specificity of the patient and her husband was determined by complement-dependent microcytotoxicity assay using standard anti-hla antisera and peripheral blood lymphocytes as targets.11) For the HLA typing of mole tissues, the single target cells were obtained by the following two ways: (a) Discreted single cells from M. O. mole for HLA typing: M. O. mole tissue was sufficiently washed with cold MEM to avoid contamination of patient's cell, and cultured in a Falcon tissue culture dish (No. 3002) in MEM containing 20% heat-inactivated fetal calf serum (FCS), 100U/ml dium was changed weekly and the cells were continuously cultured for 40 days. The growing cells were all fibroblast-like cells on microscopic observation, and no HCG was detected in the medium by the immunoassay using anti-hcg hemaggultinin assay kit (High Gonavis Kit, Mochida Pharm. Co., Tokyo). Cultured cells which had grown on almost entire surface of the plate were harvested by treatment with 0.25% trypsin and resuspended in MEM containing 20% FCS. 5%CO2 atmosphere. All the cells adhered to the bottom of the well and formed spindle-like antiserum of relevant specificity was added to the well. The relevant anti-hla antiserum to be added was formerly determined by the HLA specificity of peripheral blood lymphocytes of the patient and her husband. After incubation for 30 were fixed with 10% neutral Formalin. Viability of the target cells was determined by counting Eosin-stained cells under a phase-contrast microscope. (b) Cells from M. I. mole for HLA typing: Approximately 200ml of mole tissue was homogenized gently with loosely fitted glass homogenizer in a cold sterilized MEM, washed once with 0.02% EDTA, and then treated with 0.25% trypsin solution for 4hr at room temperature, with gentle rotation. After passing through stainless steel mesh to remove tissue debris, a single-cell suspension was prepared in sterile MEM containing ml, and cytotoxicity was assayed in the same manner as above. Over 90% of the cells obtained by this method were viable at the end of assay in the control well containing complement alone. Immunoenzymic Detection of HCG in Hydatidiform Mole Cells: M. I. cells (103) separated as above were cultured in a Falcon tissue culture submitted to anti-hcg immunoenzymic assay according to Nakane's method.8) Briefly, the M. I. cells were fixed with 10% neutral Formalin for antiserum, and then incubated with peroxidaselabeled goat anti-rabbit IgG. Cultured endometrial fibroblast cell line established from normal woman was used as control cells for this assay. Results HLA Specificity of M. O. Hydatidiform Mole Cells: The HLA types of peripheral blood lymphocytes of M. O. and her husband were found to be HLA-A 2, w24; B5, 40, and HLA-A 26; B7, 12. The percent- 850 Gann

3 HLA ON HYDATIDIFORM MOLE ages of complement-dependent cytotoxicity of the cultured fibroblast-like cells derived from M. O. hydatidiform mole to the corresponding anti-hla antiserum were 10% to anti-a2, 10% to anti-b5, 10% to Aw24, 10% to anti-b40, 100% to anti-a26, 80% to anti-b7, and 10% to anti-b12 antiserum. Thus, only HLA-A26 and HLA-B7 were detected on the surface of M. O. mole cells. These specificities seemed to correspond to the haplotype of her husband, and patient's haplotype specificity was not detected. In order to exclude the possibility that this preferential expression of paternal HLA on the surface of mole-derived fibroblast-like cells is simply due to the change in properties after cultured in vitro for a certain period, the endometrial fibroblasts were obtained from a normal subject and cultured in a similar manner as above before HLA typing. The HLA type of peripheral lymphocytes of this control subject was HLA-A2, w24; B40, w22. The reactivities of this cultured endometrial fibroblast to the corresponding HLA type-specific antisera were 100% to anti-a2, 100% to anti-aw24, 80% to anti-b40, and 70% to anti-bw22. No other positive reaction to any other HLA-type specific antisera was found. Thus, the specificity of cultured endometrial fibroblast was completely identical to that of peripheral lymphocytes. HLA Specificities of M. I. Hydatidiform Mole Cells: In order to ascertain further the above peculiar HLA expression on the surface of hydatidiform mole cells, fresh single-cell suspension was obtained from M. I. hydatidiform mole, and HLA-typing was performed in the same manner as above. Fresh M. I. mole cells obtained were relatively large and round, and did not adhere to the bottom of wells within 12-hr proximately 99% of these cells were still round, and only 1% of the cells formed spindle-like fibroblastic pattern. By the immunoenzymic staining of HCG in these cells, approximately 50% of the cells were HCG positive, and active production of HCG was proved by the presence of HCG (more than 1.0IU/mi) in the second changed medium of long-cultured cells. HLA types of M. I. patient and her husband were HLA-A w24, w31; B5, 40 and HLA-A2, 11; B15, 40. On the other hand, percentages of the HLA-positive cells from M. I. mole reactive with each corresponding HLA type-specific serum were 10% to anti-aw24, 10% to anti-aw31, 10% to anti-b5, 10% to anti-b40, 10% to anti-a2, 70% to anti-a11, 10% to anti-b15, and 80% to anti-b40. These results clearly indicated that only paternal haplotype specificity was expressed on the surface of M. I. mole cells. The HLA specificities of these two cases of hydatidiform mole are summarized in Table I, with respective haplotype specificities of paternal and maternal HLA. From this result, it can be unequivocally concluded that the hydatidiform mole cells selectively express paternal HLA haplotype specificity and not any maternal HLA. Table I. Selective Expression of Paternal HLA on the Surface of Hydatidiform Mole 69(6)

4 Y. TSUJI, ET AL. Discussion The present study demonstrated that invasive hydatidiform mole selectively expressed paternal HLA haplotype-specificity and not any maternal HLA. Kajii and Ohama4) and Wake et al.13) analyzed chromosomal polymorphism of hydatidiform mole by the Q-banding technique, and claimed its androgenetic origin. The present result is completely in agreement with the notion of androgenetic origin of hydatidiform mole from the aspects of HLA specificity which is encoded by the MHC locus of the VIth chromosome. Fukushima and Makino7) analyzed the chromosome number of hydatidiform mole and concluded that the mole cells were composed of 46 chromosomes of XX type. Subsequently, this was also confirmed by other investigators.3,12) Therefore, the HLA haplotype of the tumors in the present study must be HLA-A26, B7/A26, B7 in M. O. mole, and HLA-A11, B40/A11, B40 in M. I. mole. Androgenesis of tumor may be interpreted as the development of a fertilized ovum under the condition of the egg nucleus being either absent or inactivated. Androgenetic zygoids are usually haploid. Diploid androgenesis, as in the mole, may be caused by duplication of sperm chromosomes in the process of fertilization. The selective expression of paternal HLA on the hydatidiform mole gives rise to an intriguing question with respect to the host-tumor relationship, as to why such completely histoincompatible tumor grows progressively in the maternal host. The high malignancy rate of the cells themselves could be explained by the recessive mutation of a gene (or genes) which controls cell growth. The mutation in chromosome of the mole is homozygous and such a cell may escape from normal intracellular growth control. If this is the case, all of the cells comprising the mole such as fibroblasts, trophoblasts, and other cells would have an equal potency for malignant growth. However, clinically most metastatic choriocarcinoma cells in the brain or the lung were typical trophoblasts, and no other cell types such as fibroblastic cells have been detected. One of the reasons for this may be that the allogeneic paternal HLA is not expressed on the trophoblasts, thereby enabling the cells to escape from the host immune surveillance mechanisms, whereas the fibroblasts and other cells were effectively eliminated by the host allotransplantation immunity. However, this possibility may not be the case in the present study. In the case of M. I. mole, the cells were morphologically composed of 99% of large, round trophoblasts, over 50% of them containing HCG by immunoenzymic assay. The positivity of HCG-producing cells in this M. I. mole cell population was comparable to that of the established cultured cell line of trophoblasts as Kenjo et al.5,6) reported. reacted with anti-hla sera may imply that HLA are also expressed on the trophoblast as much as other cellular components of the mole. This notion, however, disagrees with other reports which failed to demonstrate histocompatibility antigens on the trophoblast component of normally fertilized blastocyst.1,10) Although from the present study it cannot be concluded whether such a discrepancy of the presence or absence of MHC is due to the difference in properties of normal or potentially malignant trophoblasts, one of the possibilities worthwhile considering may be that the trophoblasts may secrete some components which easily mask the histocompatibility antigens, and an appropriate treatment with an enzyme such as done in this study may expose such antigens on the surface. At any rate, a more precise comparative study of the mode of expression of HLA on the trophoblasts of normal and malignant sources may be required to explain such a discrepancy, and this is of vital importance for understanding the mechanism of pregnancy Gann

5 ALA ON HYDATIDIFORM MOLE and development of chorionic tumor from the aspects of fetomaternal relationship and immune surveillance. Finally, apart from the discussion of such immunological implications of peculiar expression of HLA on the surface of mole cells, it may be also relevant to mention that the in vitro cultured mole cells such as established in the present study may be a useful target system to study the D-locus of HLA, since the homozygous cells in HLA haplotype is of very low incidence in human target cell population. The authors are greatly indebted to Drs. Yoshio Minagawa and Kazutaka Maeyama, Department of Gynecology, Osaka University Medical School, and Dr. Kazutaka Hamada, Department of Gynecology, Osaka City University Medical School, for providing the clinical materials. They also thank Dr. Kenichi Takahashi, Department of Anatomy, Osaka City University Medical School, for his valuable advices for immunoenzymic assay. (Received July 20, 1978) REFERENCES 853