Modulation of the immune response to teratocarcinoma in mice sensitized by sperm antigens

Journal of Reproductive Immunology, 4 (1982) 67-78 Elsevier Biomedical Press

67

Modulation of the immune response to teratocarcinoma in mice sensitized by sperm antigens Julita A. Teodorczyk-Injeyan, Michael A. Jewett and Rudolf E. Falk Department of Surgery, University of Toronto, Medical Sciences Building, Room 7344, Toronto, Ont., Canada M5S 1AS

Received 15 December 1980; accepted after revision 12 February 1982)

Mouse primitive teratocarcinoma cells share a common surface antigen with morulae, preimplantation embryo cells and murine and human spermatozoa. 129/Sv mice were immunized with either spermatozoa and subsequently inoculated with various doses of teratocarcinoma 6050. A significant inhibition or acceleration of tumor growth was observed when compared with controls immunized with compatible fibroblasts. These effects were sex-dependent, both the incidence and tumor growth being suppressed in sperm-immunizedmales. The opposite effects were observed in sperm-presensitizedfemales. Immune sera obtained from both male and female 129/Sv mice exhibited a high binding activity to human spermatozoa when tested in a cellular radioimmunoassay. Thus, immunization with sperm antigens provides immunotherapeutic and/or enhancing effects in male and female 129/Sv mice, respectively.

Introduction M u r i n e t e r a t o c a r c i n o m a cell lines have been extensively studied as a c o n v e n i e n t a n i m a l model of a t u m o r of germinal origin (Stevens, 1967, 1970a,b). Mouse a n d h u m a n teratoma cell lines share several p h e n o t y p i c a n d biochemical characteristics i n c l u d i n g expression of a c o m m o n teratoma-defined antigen(s) ( H o l d e n et al., 1977; O s t r a n d - R o s e n b e r g et al., 1977). Antigenically similar products were f o u n d o n m u r i n e spermatozoa, p r e i m p l a n t a t i o n mouse embryos, as well as on sperm a n d m o r u l a e of other m a m m a l i a n species (Artzt et al., 1973; Buc-Caron et al., 1974; G o o d i n g et al., 1976). E m b r y o n a l carcinoma-associated antigen(s) (F9) could be coded by genes located at or lined to the T / t complex in the m u r i n e system ( G l u e c k s o h n - W a e l s c h et al., 1970; Artzt a n d Bennett, 1975) a n d p r o b a b l y b y its h u m a n equivalent in m a n (De Wolf et aL, 1979). However, the F9 antigen appears to be associated only with certain haplotypes of the T / t locus, whereas others are F9 negative ( K e m l e r et al., 1976). It has been suggested that the T / t locus may represent a n early d e v e l o p m e n t a l precursor of the H-2 ( H L A ) system. Recent observations ( A v n e r et al., 1981) indicate that h u m a n t e r a t o m a lines Tera I a n d SuSa express both/~z-microglobulin a n d H L A antigens. M a j o r histocompatibility complex ( M H C ) products have not been detected o n m u r i n e t e r a t o m a cells ( F o r m a n et al., 0165-0378/82/0000-0000/$02.75 © 1982 Elsevier Biomedical Press

68 1975; Stern et al., 1975). The absence of H-2 antigens on embryonal carcinoma (EC) cell lines raises the question of potential immuno-defensive mechanisms against such tumors. Since M H C products are essential for the generation of cytotoxic T-cell response (Zinkernagel and Olstone, 1976a; Doherty et al., 1977) it is not surprising that demonstration of cellular immunity against teratocarcinoma cells is particularly difficult to achieve. Teratocarcinomas have been shown to be transplantable across histocompatibility barrier in several mouse strains. On the other hand these tumors are rejected by other aliogeneic hosts (Avner et al., 1978; Siegler et al., 1979). This suggests that rejection of EC cells may be based on antigens other than H-2 (Ostrand-Rosenberg et al., 1980). Recent results (Levine and Teresky, 1981; Shedlousky et al., 1981) indicate that teratocarcinoma transplantation loci Gt-1 and Gt-2 flank the H-2 locus on chromosome 17. It has been shown that administration of EC cells into syngeneic recipients induces the production of antibodies against teratoma-defined antigen (Artzt et al., 1973; Gachelin et al., 1976). Antibodies reacting specifically with the murine teratoma line were detected in sera from patients with teratocarcinoma (Ostrand-Rosenberg et al., 1977). Recently, circulating antibodies to teratocarcinoma associated antigens have been demonstrated in sera from patients with testicular tumors using human and murine sperm and murine teratocarcinoma 402 AX line as target cells (Teodorczyk-Injeyan et al., 1980.) Considering the above findings, the possibility exists that antibody raised against sperm-associated antigens may influence the immune reaction to teratocarcinoma. In this report we present evidence that both the incidence and growth rate of murine teratocarcinoma 6050 (OTT 6050) were altered in sperm-sensitized 129/Sv mice in a sex-dependent manner.

Materials and Methods Animals

Female and male inbred 129/Sv mice, 8-12 weeks old, bred and maintained in the local animal facilities, were used throughout all experiments. Tumor cells

Teratocarcinoma 6050 (OTT 6050, originally obtained by Stevens, 1970a,b) was maintained as an ascites tumor in 129/Sv male mice. It was routinely passaged every 18-20 days. The ascites form of O T T 6050 is composed of spherical aggregates of cells designated embryoid bodies (EB) and consisting of a single layer of endoderm surrounding embryonal carcinoma cells. Solid tumors were induced by subcutaneous (s.c.) injection of l 0 4 o r 10 5 EB into 129/Sv mice. Starting 2 weeks after tumor inoculation animals were checked daily to detect the presence of palpable tumors. T u m o r dimensions were measured on alternate days with calipers. The growth of O T T 6050 was estimated by assessment of its volume. A volume of 1 cm 3 was considered as critical to terminate monitoring of the tumor growth.

69 A n tigens Freshly ejaculated sperm samples with pretested motility, viability and cell counts were obtained from healthy volunteers. The same donors provided fibroblasts, which were maintained in vitro and subcultured routinely in minimal essential alpha medium (MEM) supplemented with 15% fetal calf serum (Gibco, Grand Island, N.Y.). Mouse sperm was obtained from phosphate-buffered saline (PBS)-flushed vasa deferentia of 129/Sv males, aged 8-12 weeks. Both human and murine spermatozoa were gauze filtered, washed three times in PBS and adjusted to a desired concentration before use. Immunization Immunization schedules differed in frequency, quantity and time of antigen application in relation to inoculation with O T T 6050. (a) Mice were immunized with 1-1.5 × 107 human or murine sperm cells in Freund's complete adjuvant (CFA) given subcutaneously, and then by three intraperitoneal (i.p.) injections of 2.5-5 × 10 6 spermatozoa from the same donor administered every 2 weeks. Control animals were injected with cultured fibroblasts instead of spermatozoa. 10 days after the last immunization all animals were inoculated s.c. with 10 4 o r 10 5 EB from ascites tumor. (b) Animals previously inoculated with O T T 6050 and showing first palpable tumors received a single i.p. injection of 1.5-2.0 X 107 sperm cells. (c) Spermatozoa at a concentration of 1.0-1.5 × 107 per animal were administered i.p. simultaneously with s.c. injection of tumor cells. (d) Adult male mice were autoimmunized by bilateral vasectomy. The vas deferens was isolated through an inguinal incision by gentle traction on the testis and was then ligated using 00 silk. A second ligature was tied to ensure closure of the lumen. Sham-operated animals were used as parallel controls. All groups were inoculated with 105 EB 8 weeks later. Assessment of anti-sperm antibodies Immune and control sera from sperm- and fibroblast-sensitized mice were assessed by indirect immunofluorescence staining technique and cellular radioimmunoassay (CRIA). H u m a n spermatozoa were stained as previously described (Teodorczyk-Injeyan et al., 1980). The C R I A assay was performed in microtiter plates using glutaraldehyde-fixed spermatozoa as target cells. Sera collected from sperm-immunized animals (25 ~1) were added to 5 X 10 4 target cells and incubated overnight at 4°C. After the incubation, cells were spun and washed several times in 5% bovine serum albumin (BSA) in PBS. A second incubation with 20 ng of ~25I-labelled rabbit anti-mouse Fc was performed for 2 h at 4°C and was followed by washing in 5% BSA-PBS. Cells were collected onto fiberglass filters by an automatic cell harvester and radioactivity was measured in a g a m m a counter. Statistics Results are expressed as means of values per group ± S.D. ( n - - 10-30) and a

7O

two-way analysis of variance was used to assess the significance of differences between the groups. The value of P < 0.05 was considered significant.

Results In normal 129/Sv mice the time of appearance of the tumor is sex and dose-dependent (Fig. 1). When EB were administered at a dose of 105 per animal 90-100% of mice developed tumors, regardless of sex, within 16-23 days post-inoculation (Fig. la). T u m o r growth was usually slower in female mice (Fig. lb). Smaller doses of EB ( 1 0 4 per mouse) are sufficient to induce solid tumors in 60-80% of males 22-30 days postinoculation, but 60-90% of female mice did not develop tumors. Preimmunization with human sperm

The incidence and development of teratocarcinoma 6050 were studied in several experiments in which male and female 129/Sv mice were presensitized with human spermatozoa or cultured fibroblasts from compatible donors. Cumulative data from 3 experiments are illustrated in Fig. 2. Susceptibility to the effective dose of O T T 6050 (105 EB) was not changed by pre-immunization with human fibroblasts (Fig. 2a). However, the incidence of tumors and their growth were altered in animals sensitized with human sperm, prior to tumor challenge. Palpable tumors were observed on days 23 and 16 in the control groups of females and males, respectively. Experimental females appeared to be less resistant to tumor and palpable tumors

b

a

100

80 70

1.0

AJ , , f o J 1l I

90

v

s/r

.~_79_--..-_'2_-= - - 2

/

/

.9 .8

./

.7

E

"5 oj ro. .

60 50

/

40

"0 e. m

E v

0

30 20

/

.6

.4 .g

O~Q/0-0

E

~ E I---

.2

.1

10 0'

~

.5 ~

I

20

2'.

2'8

3'0

I

24

I

28

3'0

Days after tumor innoculation Fig. 1. The incidence and growth of teratocarcinoma 6050 in normal 129/Sv mice. Animals inoculated with 1X l05 EB: O . . . . . . O, female mice; /x . . . . . . / x male mice. Animals inoculated with l × l0 a EB: • 0 , female mice; • • , male mice.

71

';;//

100 a

I~

II

8o 70-

E ~= ~6

60

o - -

•

e~

•

7

b •

1,0

.9

i I,

.8 .7

•

E

.6

/ A

50

.5

// r;

,.4

~z

40

-

30

J.3

20

-;2

10' I

I

f

J

I

I

;""'l

16

18

21

23

25

30

16

18

I

~,/-

L

21

23

25

I

E

1

30

Days after tumor innoculation (1 x 10 s E.B., S.C.)

Fig. 2. Effects of preimmunization with human sperm on the incidence and growth of teratocarcinoma 6050 (1 × 105 EB s.c.) in 129/Sv mice. Compatible human fibroblast immunized: O . . . . . . O, female mice; A . . . . . . A, male mice. Sperm immunized: • 0, female mice; • • , male mice.

were found 5 days sooner in 60% ( 2 4 / 3 0 ) of female mice. Sperm immunization had an opposite effect on the incidence and time of tumor appearance in male mice. N o developing tumors could be found before day 23; moreover, 20% ( 6 / 3 0 ) of animals injected with a tumorogenic doge of EB never developed tumors. These effects were correlated with the increase in t u m o r volume in female and male groups. G r o w t h of O T T 6050 in sperm-immunized females was significantly ( P < 0.001) accelerated c o m p a r e d with fibroblast-immunized controls as well as sperm-immunized males. In contrast, tumors developed significantly slower ( P < 0 . 0 0 1 ) in h u m a n sperm-sensitized 1 2 9 / S v males c o m p a r e d with the fibroblast-immunized control group. The effects of the same type of immunization were then studied in animals inoculated with 104 EB where, as demonstrated above (Fig. 1), some natural resistance to teratocarcinoma could be observed. Neither female nor male fibroblast-immunized controls demonstrated any significant alteration in tumor incidence and its development when c o m p a r e d with normal animals inoculated with the same dose of O T T 6050 (Fig. 3). I m m u n i z a t i o n with h u m a n sperm, however, abolished resistance to tumor in all of the female mice and prevented tumor development in over 60% (13/20) of the male mice (Fig. 3a). T u m o r growth was significantly accelerated in the experimental female mice and greatly suppressed ( P < 0.001) in the corresponding male group (Fig. 3b). Palpable tumors were still observed in sperm-treated male mice when parallel control animals had to be eliminated from further studies. In subsequent experiments h u m a n spermatozoa were replaced by the murine sperm and used in identical experimental protocols. In both sexes, immunization with syngeneic sperm had a similar effect on tumor occurrence and development as

72

././"

a.

100 9O

b. ,,s

/

8o

/

s

1.0

/

.9 .8

I

.7

~E

60

.6

E

50

.5 ~

-~

40

.4

E

-

30

.3

I.--

o

70

•

E

/

.2

20 10

o--.o

o ....

....

~/ I I

23 25

J

28

l

30

J

J

32

; ~ /

_ - - o 2-

- - o

.1

J

35

38

23

25

28

30 32

35

38

Days after tumor innoculation (1 x 104 E.B., S.C.)

Fig. 3. Effects of preimmunization with human sperm on the incidence and growth of teratocarcinoma 6050 (1 × 104 EB, s.c.) in 129/Sv mice. Compatible human fibroblast immunized: (3 . . . . . . (D, female mice; A . . . . . . /x, male mice. Sperm immunized: • • , female mice; • A, male mice.

d e s c r i b e d a b o v e in a n i m a l s treated with xenogeneic antigen. Increase in the resistance to t u m o r following i m m u n i z a t i o n with s p e r m in males, a n d suppression of i m m u n i t y o b s e r v e d in female mice, were d e m o n s t r a t e d m o r e readily in a n i m a l s injected with smaller doses of t e r a t o c a r c i n o m a cells as well.

Sperm administration into O T T 6050-inoculated mice F u r t h e r studies dealt with the influence of s p e r m a d m i n i s t r a t i o n on a l r e a d y d e v e l o p i n g tumors. Mice were i n o c u l a t e d s.c. with 105 EB. W h e n first p a l p a b l e t u m o r s were noted, male a n d female mice were injected with either 2 × 107 h u m a n s p e r m a t o z o a or fibroblasts. This t r e a t m e n t did not inhibit the d e v e l o p m e n t of the t u m o r in e x p e r i m e n t a l females, a n d only slightly repressed t u m o r growth (Fig. 4b). A d m i n i s t r a t i o n o f h u m a n s p e r m to male mice p r e v e n t e d further d e v e l o p m e n t of the t u m o r in 40% ( 6 / 1 5 ) of animals. In the r e m a i n i n g males t u m o r s d e v e l o p e d signific a n t l y slower ( P < 0.05) (Fig. 4b). Simultaneous administration of O T T 6050 and human spermatozoa N o effect on the incidence of t u m o r s or their growth was o b s e r v e d when i n o c u l a t i o n with the effective (105) or s u b o p t i m a l doses (104) of O T T 6050 was a c c o m p a n i e d b y i.p. s p e r m a d m i n i s t r a t i o n . The influence of autoantibodies on the immune response to teratocarcinoma V a s e c t o m y was p e r f o r m e d in 20 1 2 9 / S v males a n d the same n u m b e r of animals was s h a m - o p e r a t e d . T h e o p t i m a l dose of EB i n d u c e d t u m o r growth in all s h a m - o p e r a t e d controls, b u t 8 v a s e c t o m i z e d mice never d e v e l o p e d tumors. However, the g r o w t h rate of solid t e r a t o c a r c i n o m a was identical in b o t h groups.

73

°

a 100

o.~

/

90

-o . . . .

o

1 7,;--~--o

~/'

.~

E 80 O

"6 60

8

i<

'e

E 70

"! I /

,

.7

I,' ;,)t

50

I

o 40

I

o

.4 ~

30 20

.2

10

/t/'li I

22

//

I

I

I

~l~

A//

I/j" t

A/I

28 30 34 22 28 30 Days after t0mor innoculation/,1 x 10~E.B., S.C.)

-1 I

36

Fig. 4. The influence of the human sperm administration (2× 107 cells, i.p.) on the development of teratocarcinoma 6050 in 129/Sv mice inoculated with 1 x 105 EB. Compatible human fibroblast immunized: © . . . . . . ©, female mice; ~ . . . . . . ~ , male mice. Sperm immunized: • 0, female mice; • A, male mice.

o x r,

o

nn

4/1~

I

7

I

8

I

9

I

10

1=1

1~2

Dilution of antiserum [log2] Fig. 5. Specific binding of sera from human sperm-immunized 129/Sv mice to glutaraldehyde-fixed human spermatozoa. Compatible fibroblast immunized: O . . . . . . O, female mice; /x . . . . . . A male mice. Sperm immunized: • • , female mice; • A, male mice.

74 The studies described thus far demonstrate the sex-dependent effects of the stimulation of the immune system with sperm antigens on neoplastic cells of germinal origin. It was of interest to examine if such stimulation itself could induce different antibody response to sperm-associated antigens. Sera collected from human sperm-immunized mice prior to O T T 6050 inoculation were absorbed 1:10 ( v / v ) with compatible human fibroblasts and tested by indirect immunofluorescence. All the immune sera showed a strong positive fluorescence on human sperm smears without a preponderance of any defined staining pattern or noticeable differences in intensity between samples obtained from female or male mice. The same sera were then tested in CRIA. Both female and male sera exhibited high binding activity (Fig. 5). In several batches tested on the same target cells, sera from sperm-sensitized females consistently showed much stronger binding to target cells. Control sera demonstrated weak binding activity, no different from that observed when fibroblast-absorbed normal 129/Sv serum was tested in the assay.

Discussion

Sperm-immunized male and female strain 129/Sv mice, subsequently challenged with effective doses of embryonal carcinoma cells from ascites teratocarcinoma 6050, demonstrated altered immunity to the tumor. Increased resistance and inhibition of the tumor growth were observed in males, whereas the opposite effect was seen in female mice. Similar responses occurred when mice were sensitized with syngeneic sperm cells. Administration of spermatozoa to animals already developing tumors significantly suppressed a n d / o r prevented teratocarcinoma growth in male groups. The presence of anti-sperm antibodies in both female and male sperm-immunized mice was demonstrated by immunofluorescence technique and in CRIA. It can be postulated therefore that humoral response to sperm-associated antigens provides an immunotherapeutic effect in males of 129/Sv strain. On the other hand, immunization of females seems to induce an enhancing effect manifested by the suppression of natural immunity to testicular tumor observed in this sex. Differences in susceptibility to teratocarcinoma challenge between males and females, were observed by Isa and Sanders (1975). These authors described enhancement of the tumor growth in animals treated with anti-macrophage serum and suggested .that adherent cells had a significant role in resistance to testicular carcinoma regardless of hormonal control from male and female endocrine systems. Sex-related differences in the immune response to fetal antigens were first reported by Coggin et al. (1971); immunization with irradiated cells from 10-day-old fetuses effectively inhibited tumorigenesis in adenovirus-infected male hamsters. Fetal tissue-sensitized female hamsters, however, did not demonstrate any increase in immunity to either autologous or transplanted tumors. Further studies have shown (Dierlam et al., 1971) that cell-mediated immunity could be demonstrated only in males immunized with syngeneic fetal tissue, whereas cytotoxic antibody to Sv 40 tumor were present in both sexes. Embryonal carcinoma-associated antigen(s) have been detected on the whole male

75 germ line cells (Gachelin et al., 1976) but not on any other cell line derived from the adult 129 mouse (Artzt et al., 1973). In addition several other antigenic determinants such as Ia (Hammerling, 1975), H-2 (Goldberg et al., 1970; Vojtiskova and Pokorna, 1972) or HLA-associated fl2-microglobulin (Fellous et al., 1976) have been detected on mammalian spermatozoa. None of the above structures, however, is present on murine embryonal carcinoma cells. It is, therefore, not likely that antibodies directed against such antigens could influence the immune response to teratocarcinoma 6050 in our model. Moreover, the response of fibroblast-immunized control groups did not differ from that observed in untreated animals. There is little doubt that sera raised against sperm contain antibodies representing a rich repertoire of specificities. Absorption of the anti-sperm serum with testicular carcinoma cells does not remove anti-sperm activity completely (Goldberg and Takuda, 1977). However, spermatozoa from rabbit, pig, bull and man remove anti-F9 activity from murine sera prepared in a syngeneic system (Buc-Caron et al., 1974; Jacob, 1977). The expression of teratocarcinoma-associated antigens on human and murine spermatozoa has been shown directly using immunocytochemical techniques (Fellows et al., 1975). Immunofluorescence studies have also shown that anti-sperm antibodies (Beck et al., 1962), sera from vasectomized men (Tung, 1975) and patients with testicular tumors (Teodorczyk-Injeyan, 1980) demonstrate similar patterns of staining involving the post-arosomal segment and the sperm tail. Since a direct cytotoxic effect of specific anti-EC antibodies has also been demonstrated (Jacob, 1977) preimmunization with spermatozoa in our studies could result in the production of antibodies with anti-EC activity and provide an immunoprotective effect at least in male 129/Sv mice. The observation that administration of spermatozoa to teratocarcinoma-inoculated mice exerts a similar effect is not fortuitous considering the successful results obtained by using immunotherapeutic tumor cell vaccines in human and animal systems (McKhann and Gunnarsson, 1974; Peters et al., 1979). Further studies involving adoptive transfer experiments and determination of an activity of antisperm antibodies directly on OTT 6050 are required to prove that the effects of sperm-immunization described herein are, indeed, due to the presence of antibodies directed against embryonal carcinoma cells. If so, similar products possibly of high specificity (monoclonal) might eventually find an application in the therapy of testicular tumors. We cannot exclude that other defense mechanisms can also be triggered as a result of stimulation by fetal antigens. These could include activation of natural killer (NK) cells, since recent studies of Stern et al. (1980) indicate that N K cells are primarily involved in elimination of undifferentiated EC cells. The immunosuppressive effects of preimmunization with spermatozoa found in females are more difficult to explain. Frequently, F9 teratocarcinoma cells are considered an experimental analogue of the cleavage stage embryo (Delovitch et al., 1978). The immunological mechanisms involved in the protection of the conceptus particularly in the early phase of pregnancy are not clear. However, a specific enhancement of paternal type tumors has been shown (Kaliss and Dagg, 1964) and antibody activity towards paternal strain antigens has been demonstrated in the maternal strain bearing tumor allografts (Voisin et al., 1964). It has been suggested

76

that allograft rejection is counterbalanced in pregnant animals by a reaction involving the production of enhancing antibodies and suppressor cells (Voisin, 1976). Cell surface antigens associated with early embryonic development disappear before immunological self-tolerance can be achieved (Alexander, 1972). Therefore, teratocarcinoma-associated antigens present on adult sperm, normally located within the seminiferous tubes, may act as autoantigens in males. The female reaction to sperm antigens is certainly more complex and may induce infertility (Tung et al., 1979). Similarly, female infertility of 129 mice was demonstrated in animals immunized against the F9 murine teratocarcinoma antigen (Hamilton et al., 1978). It cannot be determined how far the enhancing effect of preimmunization with spermatozoa, observed in our studies in females inoculated later with EC, mimics physiological consequences of pregnancy, since the subcutaneous inoculation of the tumor eliminates the protective effect provided by the placental barrier. However, humoral response to fetal antigens in females could result in the production of antibodies masking or blocking teratocarcinoma-associated antigens. Preliminary data obtained in our laboratory (unpublished) and by others (Isa and Sander, 1975) indicate that natural resistance to teratocarcinoma is eliminated in pregnant 129/Sv females. This suggests that similar mechanisms may indeed be involved. If so, preimmunization with sperm and subsequent challenge with embryonal carcinoma cells may provide a useful model to study pregnancy-related immune phenomena.

Acknowledgements This work was supported by grants from the Ontario Cancer Treatment and Research Foundation, the National Cancer Institute and the Medical Research Council of Canada.

References Alexander, P. (1972) Foetal antigens in cancer. Nature (London) 235, 137-140. Artzt', K. and Bennett, D. (1975) Analogies between embryonic (T/t) antigens and adults major histocompatibility (H-2) antigens. Nature (London) 256, 545-547. Artzt, K., Dubois, P., Bennet, D., Condamine, H., Babinet, C. and Jacob, F. (1973) Surface antigens common to mouse cleavage embryos and primitive teratocarcinoma cells in culture. Proc. Natl. Acad. Sci. U.S.A. 60, 2988-2992. Avner, P.R., Dove, W.F., Dubois, P., Galliard, J.A., Gu~net, J.-L., Jacob, F., Jacob, H. and Shedlovsky, A. (1978) The genetics of teratocarcinoma transplanatation: tumor formation in allogeneic hosts by the embryonal carcinoma cell lines F9 and PCC3. Immunogenetics 7, 103-115. Avner, P., Bono, R., Berger, R. and Fellous, M. (1981) Characterization of human teratoma cell lines for their in vitro developmental properties and expression of embryonic and major histocompatibility loizus-associated antigens. J. Immunogenet. 8, 151-162. Beck, J.S., Edwards, R.G. and Young, M.R. (1962) Immune fluorescence technique and the isoantigenicity of mammalian spermatozoa. J. Reprod. Fertil. 4, 103-110. Buc-Caron, M.H., Gachelin, G., Hofnung, M. and Jacob, F. (1974) Presence of a mouse embryonic antigen on human spermatozoa. Proc. Natl. Acad. Sci. U.S.A. 71, 1730 1733.

77 Coggin, J.H. Jr., Ambrose, K.R., and Anderson, N.G. (1971) Immunization against tumors with fetal antigens. In: Proceedings of the First Conference and Workshops on Embryonic and Fetal Antigens in Cancer (Anderson, N.G. and Coggin, J.H. Jr., eds.), pp. 185-198. Oak Ridge National Laboratory, Oak Ridge. DeWolf, W.C., Lange, P.H., Einarson, M.E. and Yunis, E.J. (1979) HLA and testicular cancer. Nature (London) 227, 216-217. Delovitch, T.L., Press, J.L. and McDevitt, H.O. (1978) Expression of murine Ia antigens during embryonic development. J. Immunol. 120, 818-824. Doherty, P.C., Solter, D. and Knowles, B.B. (1977) H-2 gene expression is required for T cell-mediated lysis of virus infected target cells. Nature (London) 266, 361-362. Dierlam, P., Anderson, N.G. and Coggin, J.H. Jr. (1971) Immunization against tumors with fetal antigens: detection of immunity by the colony inhibition test and by adoptive transfer. In: Proceedings of the First Conference and Workshop on Embryonic and Fetal Antigens in Cancer (Anders, N.G. and Coggin, J.H. Jr., eds.), pp. 203-214. Oak Ridge National Laboratory, Oak Ridge. Fellous, M., Gachelin, G., Buc, M.H., Dubois, P. and Jacob, F. (1975) Similar location of an early embryonic antigen on mouse and human spermatozoa. Dev. Biol. 41, 331-337. Fellous, M., Colle, A. and Tonelle, C. (1976) The expression of beta 2-microglobulin on human spermatozoa. Eur. J. lmmunol. 6, 21-24. Forman, J. and Vitetta, E. (1975) Absence of H-2 antigens capable of reacting with cytotoxic T cells on a teratoma line expressing T / t locus antigen. Proc. Natl. Acad. Sci. U.S.A. 72, 3661-3665. Gachelin, G., Fellous, M., Guenet, J.-L. and Jacob, F. (1976) Developmental expression of an early embryonic antigen common to mouse spermatozoa and cleavage embryos, and to human spermatozoa: its expression during spermatogenesis. Dev. Biol. 50, 310-320. Goldberg, E.H. and Tokuda, S. (1977) Evidence for related antigens on sperm, tumor, and fetal cells in the mouse. Transplant. Proc. IX, 1363-1366. Goldberg, E.H., Aoki, T., Boyse, E.A. and Bennett, D. (1970) Detection of H-2 antigens on mouse spermatozoa by the cytotoxicity test. Nature (London) 228, 570-572. Gooding, L.R., Hsu, Y.C. and Edidin, M. (1976) Expression of teratoma associated antigens on murine ova and early embryos. Dev. Biol. 49, 479-486. Gluecksohn-Waelsch, S. and Erickson, R.P. (1970) The T-locus of the mouse: implications for mechanism of development. Curr. Top. Dev. Biol. 5, 281-315. Hamilton, M.S., Vitetta, E.S. and Beer, A.E. (1978) The effect of immunizing female mice to F9 teratocarcinoma cells on subsequent conception. Fed. Proc. 37, 1474. Hammerling, G.J., Mauve, G., Goldberg, C. and McDevitt, H.O. (1975) Tissue distribution of la antigens: la on spermatozoa, macrophage and epidermal cells. Immunogenetics 1,428-437. Holden, S., Bernard, O., Artzt, K., Whitmore, W.F. and Bennett, D. (1977) Human and mouse embryonal carcinoma cells in culture share an embryonic antigen (F9). Nature (London) 270, 518-520. lsa, A.M. and Sanders, B.R. (1975) Enhancement of tumor growth in allogeneic mice following impairment of macrophage function. Transplantation 29, 296-302. Jacob, F. (1977) Mouse teratocarcinoma and embryonic antigens. Immunol. Rev. 33, 3-32. Kaliss, N. and Dagg, M.K. (1964) Immune response engendered in mice by multiparity. Transplantation 2, 416-425. Kemler, R., Babinet, C., Condamine, H., Gachelin, G., Gu6net, J.-L. and Jacob, F. (1976) Embryonal carcinoma antigen and the T / t locus of the mouse. Proc. Natl. Acad. Sci. U.S.A. 73, 4080-4084. Levine, A.J. and Teresky, A.K. (1981) Teratocarcinoma transplantation rejection loci: genetic localization of the Gt-I locus on chromosome 17 and the expression of alternate alleles. Immunogenetics 13, 405-412. McKann, C.F. and Gunnarsson, A. (1976) Approaches to immunotherapy. Cancer 34, 1521-1531. Ostrand-Rosenberg, S., Edidin, M. and Jewett, M.A.S. (1977) Human teratoma cells share antigens with mouse teratoma cells. Dev. Biol. 61, 11-19. Ostrand-Rosenberg, S., Rider, T.M. and Twarowski, A. (1980) Susceptibility of allogeneic mice to teratocarcinoma 402 A X . Immunogenetics 10, 607-612. Peters, L.C., Brandhorst, J.S. and Hanna, M.G. Jr. (1979) Preparation of immunotherapeutic autologous tumor cell vaccines from solid tumors. Cancer Res. 39, 1353-1360.

78 Shedlovsky, A., Clipson, L.J., VandeBerg, J.L. and Dove, W.F. (1981) Strong teratocarcinoma transplantation loci, Gt-1 and Gt-2, flank H-2. Immunogenetics 13, 413-419. Siegler, E.L., Tick, N., Teresky, A.K., Rosenstraus, M. and Levine, A.J., (1979) Teratocarcinoma transplantation rejection loci: an H-2-1inked tumor rejection locus. Immunogenetics 9, 207 220. Stern, P.L., Martin, G.R. and Evans, M.J. (1975) Cell surface antigens of clonal teratocarcinoma cells at various stages of differentiation. Cell 6, 455-465. Stern, P., Gidlund, M., Orn, A. and Wigzell, H. (1980) Natural killer cells mediate lysis of embryonal carcinoma cells lacking MHC. Nature (London) 285, 341-342. Stevens, L.C. (1967) Origin of testicular teratomas from primordial germ cells in mice. J. Natl. Cancer Inst. 38, 549-552. Stevens, L.C. (1970a) The development of transplantable teratocarcinomas from intratesticular grafts of pre- and post implantation mouse embryos. Dev. Biol. 21, 364-382. Stevens, L.C. (1970b) Experimental production of testicular teratomas in mice strain 129, A / H e and their F I hybrids. J. Natl. Cancer Inst. 44, 923-929. Teodorczyk-Injeyan, J.A., Jewett, M.A.S., Burke, C.A. and Ostrand-Rosenberg, S. (1980) Detection of the circulating antibodies to teratocarcinoma-defined antigens in patients with testicular tumors. Clin. Exp. Immunol. 40, 438-444. Tung, K.S.K. (1965) Human sperm antigens and antisperm antibodies. I. Studies on vasectomy patients. Clin. Exp. Immunol. 20, 93-104. Tung, K.S.K., Goldberg, E.H. and Goldberg, E. (1979) Immunological consequence of immunization of female mice with homologous spermatozoa: induction of infertility. J. Reprod. Immunol. 1, 145-158. Voisin, G.A. (1976) Suppressor cells and enhancing antibodies: immune agents of facilitation reaction. Adv. Exp. Med. Biol. 66, 645-651. Voisin, G.A. and Chaouat, G. (1974) Demonstration, nature and properties of maternal antibodies fixed on placenta and directed against parental antigens. J. Reprod. Fertil. Suppl. 21, 89-102. Vojtiskova, M. and Pokorna, L. (1972) Developmental expression of H-2 antigens in the spermatogenic cell series. Possible bearing on haploid gene action. Fol. Biol. (Prague) 18, 1-12. Zinkernagel, R.M. and Olstone, M.B. (1976) Cells that express viral antigens but lack H-2 determinants are not lysed by immune thymus-derived lymphocytes but are lysed by other antibiral immune attack mechanisms. Proc. Natl. Acad. Sci. U.S.A. 73, 3666-3670.