Immunology 1979 38 539

Inhibition of antigen-induced lymph node cell proliferation by murine amniotic fluid and its components

KOJI SUZUKI & T. B. TOMASI, JR Department of Immunology, Mayo Clinic, Rochester, Minnesota, U.S.A.

Acceptedfor publication 7 June 1979

Summary. Murine amniotic fluid (MAF), alpha-foetoprotein (AFP) and MAF depleted of AFP by affinity chromatography (MAF-AFP) inhibited the T-cell dependent in vitro proliferative responses of lymph node cells sensitized to a variety of soluble antigens. Variable degrees of inhibition were observed with the different antigens used in the assay. In general, the highertheproliferativeresponse inducedbyaparticular antigen, the less it was inhibited by the three inhibitors. Enhancement of proliferation was not infrequently observed at lower concentrations followed by a dosedependent inhibition as the concentration of the inhibitor was increased. Usually the order of inhibition was MAF > MAF-AFP > AFP although variations in inhibitory potency were noted between different preparations of AFP and MAF-AFP. The existence of inhibitors in preparations of MAF depleted of AFP raised the question as to whether MAF contains single or multiple inhibitory factors. The most facile explanation is that two inhibitors exist; AFP and the as yet uncharacterized non-AFP suppressor present in MAF-AFP. INTRODUCTION It is well known that AFP, one of several oncofoetal Correspondence: Dr T. B. Tomasi, Department of Immunology, Mayo Clinic, Rochester, Minnesota 55901, U.S.A. 0019-2805/79/1100-0539 $02.00

© 1979 Blackwell Scientific Publications 539

proteins, is present in high concentrations in foetal and neonatal sera as well as pregnant sera. AFP also appears in the sera of patients with certain malignant tumours, particularly primary liver cell cancer and teratoblastomas as well as several non-malignant diseases (Abelev, 1971). Although the biological significance of AFP in vivo remains unclear, recent work (Tomasi, 1977, 1978) has shown that purified mouse AFP has an immunosuppressive effect on mitogeninduced responses (PHA, Con A, LPS), the mixed lymphocyte reaction, the generation of cytotoxic T lymphocytes and on the in vitro primary and secondary antibody synthesis to sheep red blood cells. In some reports, however, (Sheppard, Sell, Trefts & Baku, 1977; Charpentier, Guttman, Shuster & Gold, 1977) the suppressive effects of MAF and AFP were minimal in certain in vitro systems while in others AFP was even enhancing. The possible origins of these discrepancies have been discussed in detail (Tomasi, 1978) and are in significant part due to technical details in the isolation of AFP and the culture conditions employed in the in vitro test systems as discussed in this paper. The present study was undertaken to examine more closely the immunosuppressive effects of mouse amniotic fluid (MAF), AFP and MAF depleted of AFP (MAF-AFP) on antigen-induced T-cell dependent proliferative responses of lymph node cells obtained from animals sensitized to a variety of soluble antigens. We show that MAF, AFP and an MAF-AFP preparation have an inhibitory effect on the antigen-

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Koji Suzuki & T. B. Tomasi

specific proliferative response and that the degree of inhibition varies with the antigen employed. This variation is most likely due to the magnitude of the proliferation induced by the antigen at optimal concentration. An immunoenhancing effect was not infrequently noted at low doses and was followed by suppression at increasing concentrations. MATERIALS AND METHODS Mice

CBA/J females, 8-12 weeks old, were purchased from Jackson Laboratories, Bar Harbor, Maine. Fifteen to eighteen day pregnant Ha/ICR mice, used as a source of amniotic fluid, were bred at the Mayo Clinic. Preparation of AFP and AFP depleted ofMAF MAF was collected as described previously (Murgita & Tomasi, 1975), repeatedly diafiltered (Amicon PM 10 positive pressure system, Amicon Corp., Lexington, Mass.), and flushed with 0-14 M phosphatebuffered saline (PBS) pH 7-2 until all of the dialysable material (by optical density) was removed. AFP was prepared as previously reported (Labib & Tomasi, 1978). Briefly, the dialysed MAF was applied to an anti-normal mouse serum (NMS) affinity column consisting of the gammaglobulin fraction of rabbit anti-NMS serum, coupled with cyanogen bromide to Sepharose 4B (Pharmacia Fine Chemicals, Inc., Piscataway, N.J.). The effluent was tested by gel diffusion against anti-AFP and anti-NMS. All AFP-positive fractions were pooled and the concentrated fractions re-applied to the column if they showed any reaction with anti-NMS on gel diffusion. The columns were regenerated by washing with 4 M KI and then 0 14 M PBS. During the course ofstudies designed to compare AFP preparations obtained from MAF by various methods, we have employed some six different isolation schemes. These include various combinations of the following procedures: preparative polyacrylamide electrophoresis, isoelectric focusing, DEAE chromatography with linear gradients, gradient sieve absorption chromatography, hydroxyapatite chromatography, reverse ammonium sulphate salting-out chromatography, gel filtration involving various sizes of molecular sieves and affinity chromatography employing oestradiol and anti-AFP affinity columns. We have also employed the identical procedures used by other workers (Sheppard et al., 1977; Yachnin, 1976). The technique employed in this paper is simple, rapid and

has an excellent yield (95 + %); and the preparations obtained by this procedure are as homogeneous as those prepared by most other, more complicated schemes. The key to this method is the use of a particularly potent antiserum to adult NMS which contains high titres of antibodies to the serum components present in MAF and the use of recycling when necessary. With certain anti-NMS we have found that preparations contained a small amount of a fast-moving band (anodal to AFP) seen when 100 ug of protein were applied to analytical gels. With such preparations an additional step such as preparative polyacrylamide gel electrophoresis was necessary to obtain homogeneous samples of AFP. The fast component is difficult to detect in MAF by Ouchterlony diffusion but can be seen with certain anti-NMS antisera by placing the unstained PAGE gel in agar and the antiserum in a trough close to the gel. MAF depleted of AFP (MAF-AFP) was prepared using an anti-AFP affinity column and collecting AFP negative fractions. The anti-AFP serum was prepared by injecting rabbits with AFP which had been purified from MAF by the polyacrylamide gel electrophoresis technique using a Gilson gel crusher. The gammaglobulin from the antisera (prepared by one-step elution from DEAE) was then passed over an affinity column prepared from the partially purified AFP (approximately 90% AFP) obtained by stripping the anti-AFP column with KI. This 'antigen column' was underlayed with Sephadex G-25 so that the AFP antibodies could be eluted and separated from the 4 M KI in one step. Thus, the anti-AFP antisera employed were enriched for specific antibody. The MAF-AFP preparations were re-run on the column if significant concentrations of AFP (> 200 ng/ml) were detected when analysed by double antibody radioimmunoassay (Gleich, Averbeck & Swedlund, 1971). AFP and MAF-AFP from the affinity columns were diafiltered with 0 14 M PBS and reconstituted by optical density to 2 OD units measured at 280 A. To determine protein concentration, an E2O of 4 0 was used for AFP and 10 for MAF-AFP. The purity of AFP and MAF-AFP was examined by alkaline polyacrylamide gel electrophoresis (applying 100 Mg of protein), SDS-PAGE electrophoresis, analytical ultracentriguation and by Ouchterlony gel diffusion and immunoelectrophoresis using several anti-NMS and anti-MAF antisera. Rabbits were given multiple injections of the AFP preparations employed and the resulting antisera contained only antibodies to AFP when tested against MAF and newborn sera.

Lymph node cell proliferation inhibition

Radioimmunoassay (Gleich et al., 1971) was used to quantify the amount of AFP in the various preparations. Examples of the purity tested by gel electrophoresis of preparations obtained by this method have been previously reported (Labib & Tomasi, 1978). Immunization The following antigens were used for immunization: ovalbumin (OVA) (Sigma, St Louis, Mo); human gammaglobulin (HGG) (Miles Laboratory, Elkart, In); lysozyme (LYSO) (Sigma, St Louis, Mo); keyhole limpet haemocyanin (KLH) (Calbiochem, Ca); and purified protein derivative (PPD) of killed Mycobacterium tuberculosis H37Ra (Connaught Laboratory, Toronto, Canada). Immunization and the subsequent in vitro culture methods were essentially as described by Alkan (1978) with slight modifications. Antigens were emulsified in complete adjuvant (CA) containing killed Mycobacterium tuberculosis H37Ra organisms. CBA/J female mice were immunized subcutaneously at the base of the tail with 100 pg of antigen in a total of 50 p1 of emulsion.

Culture techniques Eight days after immunization the draining lymph nodes of the inguinal and para-aortic regions were removed from immunized mice and pooled in cold Hanks's balanced salt solution (Hanks's BSS, Flow Laboratory, Rockville, Md). Usually one to three mice were used in the assay for each antigen. Lymph nodes were teased apart, washed two times and the cell pellets resuspended in the culture media. The cell number was adjusted to 2 x 106/ml using a Coulter counter (Coulter Electronics, Hialeah, Fl). The culture media consisted of RPMI-1640 (GIBCO, Grand Island, N.Y.) supplemented with 5% heat-inactivated horse serum (GIBCO), 10 mm HEPES buffer, 2 x 10-5 M 2-mercaptoethanol (BME), 100 units/ml of penicillin, 100 pg/ml streptomycin, and 2 mm of glutamine. 0-2 ml of the cell preparation containing 4 x105 primed lymph node cells were pipetted into each well of a flat-bottomed microtitre plate (Falcon Plastics, Dividion of Bioquest, Oxnard, Ca). Twenty microlitres of antigen at the appropriate concentration in Hanks's BSS were distributed to each well. Twenty microlitres of samples to be tested for their immunosuppressive effect were added per well (in Hanks's BSS) and the plates incubated in a 5% CO2, 95% air, humidified atmosphere at 370 for 4 days. This period of culture was chosen on the basis of preliminary experi-

541

ments indicating that maximum or near maximum proliferative responses occurred at this time with each ofthe antigens employed. Sixteen hours before harvesting, the cultures were pulsed with 1 pCi of[3H]-methylthymidine (3H-TdR, New England Nuclear, Boston, Ma). Cultures were harvested on an automatic harvesting device (Skayton, Norway), and the radioactivity counted in a liquid scintillation counter (Beckman Instruments, Palo Alto, Ca). All determinants were done in triplicate and data expressed as a mean count per minute + standard error (SE).

RESULTS The assay system employed is characterized by a proliferative response in the cultured cells of the draining lymph nodes from the inguinal and para-aortic regions and is highly specific for the immunogen. Dose responses and the kinetics of the response to each antigen were established in preliminary experiments. Usually 250-500 pg/ml of antigen were added in vitro (secondary stimulation) except with PPD in which 50 pg/ml gave a high stimulatory index (SI). As shown in Table 1, using five different antigens, we found that there were large differences in the magnitude of tritium incorporation at optimal concentrations. The maximum proliferative response in c.p.m. to a given antigen varied from experiment to experiment but the SI for a given antigen and the relative magnitude of the response compared to that given by PPD was quite constant. The response to PPD resulted from the use of complete adjuvant and, therefore, served as an 'internal' control with which to compare the response to the specific antigen. Both the specific antigen and the PPD responses were essentially abolished by pre-treatment of the sensitized LNC with anti-O plus complement (data not shown) indicating that the response was dependent on T cells. It was also noted that the magnitude of the response to the same antigens differed between mouse strains. For example, when C3H/Anf female mice were used in the assay, the responses were consistently about one-half to onethird that of CBA/J mice. The genetics of the strain differences in the responses in this assay will be the subject of a subsequent report (manuscript in preparation). CBA/J mice were used in all of the experiments reported in this paper. Figure 1 shows the dose-response profile of MAF, AFP, and MAF depleted of AFP (MAF-AFP) as well as normal mouse serum (NMS) on the proliferative

Koji Suzuki & T. B. Tomasi

542

Table 1. Antigen-specificity of lymph node cell proliferation -

Antigens used for initial in vivo priming (c.p.m. x 10-3 + SE)

added

Antigen dose

in vitro

(mg/ml)

OVA

KLH

HGG

LYSO

CA

Medium OVA KLH HGG LYSO PPD

250 250 250 250 50

5 5+0 3 123-6+5-1 6-7+0-2 5 6i+04 11 7+1-3 154 8+0-8

4-5+0-1 10-5+1*2 264-6+12-5 4-2+0-4 4-7+0 7 66-2+5-2

4-6+0-2 ND* 5-3+0 3 43-1+15 ND* 41-0+0-9

1-5+0-2 4-8 +0-2 4 4+0-1

2 0+0 2 3-9 +0-2 3-8+0-2 ND* ND* 38-8+2-2

Antigen

-

ND* 55-4+5 1 36-7+4-5

Animals were initially primed in vivo with the various antigens indicated and then secondarily stimulated in vitro by the same or different antigens (see Methods). The data illustrate that the proliferative response is specific for the antigen to which the animals were initially sensitized and that there are variations in the magnitude ofthe proliferative response induced by different antigens. OVA, ovalbumin; KLH, keyhole limpet haemocyanin; HGG, human gammaglobulin; LYSO, lysozyme; CA, complete adjuvant H37 Ra; ND* not determined.

response to PPD. All three MAF preparations show an inhibitory effect which is dose-dependent. Inhibition is not due to a cytotoxic effect since there was no significant difference in cell viabilities or recoveries between the experimental and control groups. In Fig. 1, AFP and MAF-AFP show similar inhibition curves. In other experiments using some eight different preparations, we observed that most frequently the order of inhibitory potency at the same concentration was MAF > MAF-AFP > AFP(data not shown). Data on the percentage stimulation (inhibition or enhancement of proliferation by MAF (c.p.m. with MAF added divided by c.p.m. without MAF x 100) for the various

antigens is illustrated in Fig. 2. This figure demonstrates that the order of inhibitory potency of MAF varies according to the antigen used in the assay. For example, KLH-induced proliferation is significantly more resistant to inhibition than that produced by PPD or HGG. Figure 2 also illustrates that at low concentration enhancement, rather than inhibition, may be seen. Enhancement was concentration-dependent and could be seen with many, but not all, preparations if careful dose-response curves were carried

c a

104

NMS ra *_,

,KLH

4-

'C

co

>e

.%I

U-

MAF -AFP AFP MAF 200

5 10 50 100 Sample concentration pg/mi

Figure 1. Dose-dependent inhibition of proliferation by MAF, AFP and AFP-depleted MAF. CBA/J female mice were primed with 50 1u of emulsioned CA with Hanks's BSS. Eight days later regional lymph node cells were tested with 50 ug/ml of PPD in the proliferation assay in the presence of various concentrations of NMS or the different inhibitors. Proliferation as measured by [3H]-thymidine incorporation (c.p.m.) is the mean of triplicate determinations.

..LYSO I< 0 5 10 50

100

OVA HGG

200

Sample concentration pig/ml

Figure 2. Inhibitory or enhancing effect ofvarious concentrations of MAF on the proliferation induced by different antigens. Percentage stimulation= c.p.m. with sample added x 100 c.p.m. without sample added _____

____

_____

____

Lymph node cell proliferation inhibition

543

Table 2.

Ovalbumin concentration

Sample added

(pg/ml)

Control (c.p.m. + SE)

MAF: 100 pg/ml (c.p.m. + SE)

10 50 100 250 500 1000

38,105 +2410 53,509+ 1296 58,826+ 5423 65,273 + 1068 70,899 + 7024 76,261 +6214

7,241 +384 (87) 12,141 +1102(79) 20,918 + 1826 (68)

28,862± 1283 (46) 32,678+2806(40) 40,523 +3256 (39)

34,963 + 2888 (51)

14,460+ 1520 (62) 28,719+2004 (54) 30,030+ 1856 (49) 40,901 +3246 (47)

50,578 + 2201 (29) 56,142 +4681 (22)

46,397 + 3307 (35) 58,242±4485 (24)

3,924 +104

MAF-AFP: 100 pg/mI (c.p.m. + SE)

AFP: 200 pg/ml (c.p.m. + SE)

(90%) 18,390 + 1142 (52)

46,218+3242 (40)

The effect of antigen (OVA) concentration on the suppression of lymph node cell proliferation by MAF (100 pg/ml), AFP (200 pg/ml), AFP depleted of MAF (100 pg/mI). Number in parentheses represents percentage suppression.

out although the absolute concentration at which enhancement was evident varies with different samples. Some preparations of AFP also showed low dose enhancement (data not shown). A similar dose-dependent stimulation of the growth of a fibroblast cell line (3T3-LI) has been found with MAF, but in this case increasing concentrations did not inhibit growth (R. Scott and T. Tomasi, unpublished observations). Since there is a significant variation in the degree of inhibition noted using the different antigens, we therefore examined whether this variation resulted from antigen specificity or was based on quantitative differences between the antigens in their capacity to produce proliferation, i.e. antigens such as KLH and OVA which produce the most vigorous proliferative responses were the least inhibited. In order to investigate this, the variations in the degree of inhibition with different antigen concentrations at a constant amount of MAF preparations were studied. Table 2 illustrates that the degree of inhibition of OVA-induced proliferation by three MAF preparations is dose-dependent. for example, when 10 ,ug of OVA was added to the culture, the proliferative response was over 90% inhibited by MAF whereas at 1000 pg only 40% inhibition was seen. Thus, the higher the proliferative response induced by the same antigen, the less it was inhibited by MAF. Similar data were obtained for inhibition by AFP and MAF-AFP, again indicating that the degree of inhibition is proportional to the magnitude of the proliferative response. For example, with OVA at 10 pg of antigen the response was significant (SI = 11-0) and inhibition by AFP was 52%. When the antigen dose was increased 100-fold and the proliferative response (c.p.m.) doubled, the degree of inhibition by

AFP was approximately halved (22%). Numerous experiments (data not shown) using different samples of inhibitors showed that in general the degree of inhibition by the three inhibitors (MAF, AFP and MAF-AFP) was greater for PPD- or HGG-induced proliferative responses than for OVA or KLH. This was essentially the same order as their ability to induce proliferation at optimal antigen concentrations.

DISCUSSION The present work confirms and extends the findings of Ettinger & Chiller (1977) that MAF suppresses the in vitro proliferative response of sensitized lymph node cells to HGG. In the human, Littman, Alkert & Rocklin (1977) have reported that low concentration of AFP inhibits the proliferation of peripheral blood mononuclear cells induced by SK-SD. In our study, inhibition of antigen-induced proliferation to five different antigens was found not only with AFP, but also MAF depleted of AFP by affinity chromatography. In this study, we have used a simple one-step affinity chromatography procedure for obtaining AFP and MAF-AFP. We have found most preparations of AFP isolated by this technique to be as homogeneous as those obtained using more complicated methods which we have previously employed (Murgita & Tomasi, 1975). Despite, however, the multiple tests for purity used, small amounts of MAF-specific or other serum components not detectable by our antisera may well be present. Therefore, we can conclude only that 'apparently homogeneous' preparations of AFP

544

Koji Suzuki & T. B. Tomasi

(within the limits of our detection methods) are inhibitory. Since the MAF-AFP samples were analysed by a radioimmunoassay and were found to contain less than 200 ng/ml of AFP (the approximate concentration of AFP in most adult NMS), it is unlikely that residual AFP in these preparations was responsible for the suppressive activity. If a highly potent species of AFP were selected, it would have to be poorly reactive with the anti-AFP affinity column since recycling does not further deplete the suppressive potency. The nature of the inhibitory factor(s) in MAF-AFP preparations is currently under investigation. The assay system employed was found to be highly reproducible; it could be performed on a single mouse and was applicable to the study of a number of different antigens. Work from other laboratories (Alkan, 1978; Corradin, Ettinger & Chiller, 1977; Rosenwasser & Rosenthal, 1978), using very similar assays, strongly suggests that the proliferating cell is predominantly the T cell. Since this has not been formally proven with all the antigens used, however, we prefer to refer to the assay as T-cell dependent. It is to be noted that different antigens induced different degrees of the proliferative response. The most plausible explanation for the different degree of inhibition by the three inhibitors (MAF, AFP, MAF-AFP) for various antigens is that the degree of proliferation in the presence of the inhibitor depends not only on the concentration and potency of the inhibitor but on the magnitude of the proliferative response induced by each antigen. The data on the percentage inhibition by OVA, as a function of antigen dose, suggests that at suboptimal doses where the degree of proliferation is less, the inhibition becomes larger. Another explanation is that since there still remains a possibility of some B-cell proliferation in the responses to certain antigens such as KLH, this may cause the different degree of inhibition by the three MAF preparations, depending on the antigen employed. The data in Fig. 2 show that an enhancing activity is evident at low concentrations over a narrow dose range with MAF. The dose concentration at which enhancement occurs varies from preparation to preparation. The mechanism for the enhancing effect is unknown. There is some evidence (Yachnin, 1976) that only certain species of AFP are immunosuppressive in both the human and mouse and it is conceivable that both suppressive and enhancing forms of AFP exist, or there may be a single species of AFP which both enhances and inhibits depending upon its concentration. Alternatively, completely different factors

may be responsible for enhancement and suppression. We have noted in studying the mitogenic response to PHA that changing the culture conditions (cell numbers and supporting serum) determines the degree of suppression and whether enhancement is observed. Enhancement was observed when the cultures were of lower cell density, and was more evident when the culture media contained 5% human serum instead of 5% FCS (data not shown). Although we have shown the presence of suppressive factor(s) in MAF, the key question still remains regarding the biological relevance of these findings. Can the results of the in vitro studies be translated into the in vivo situation? We have previously suggested (Tomasi, 1977, 1978) that suppressive factors in MAF and neonate serum could conceivably play a role in the immaturity of the foetus, in the development of neonatal and self tolerance and in the failure of the foetal allograft to be rejected. The earlier in vivo work of Ogra, Murgita & Tomasi (1974), demonstrating that the administration of MAF to newbom mice prolongs the period of immunological immaturity, supports this view. More recent work in adult mice demonstrates that MAF administered in vivo suppresses the primary antibody response to SRBC (T. Tomasi & K. Suzuki, in preparation). A report by Gershwin, Castles, Ahmed & Makishima (1978) showed that AFP administered in vivo accelerates tumour growth, increases mortality and lowers the threshold dose necessary to induce tumours by the Maloney sarcoma virus. Also, work by Murgita, Goidal, Kontainen, Beverley & Wigzell (1978) has shown that AFPinduced inhibitory T cells from adults have the same functional properties and Ly phenotype as splenic T cells from newborn mice and these workers suggest that AFP may function as an immunoregulatory agent in vivo during ontogenetic development. On the other side of the ledger are observations that in some clinical conditions serum AFP levels may be quite elevated in the absence of any demonstrable immune suppression. For example, in experimental animals with hepatomas, very high levels of AFP are noted without significant immune suppression (Sell, Sheppard & Poler, 1977). Thus, the whole question of the biological relevance of AFP and/or other inhibitors in neonatal fluids remains to be answered.

ACKNOWLEDGMENTS We acknowledge the excellent technical assistance of Gary Bren and Geoffrey Curran.

Lymph node cell proliferation inhibition This work was supported by grants CA-09127 and CA-1 8204, awarded by the National Cancer Institute, DHEW and by National Institutes of Health Grant HD-09720. REFERENCES ABELEV G.I. (1971) Alpha-fetoprotein in oncogenesis and its association with malignant tumors. Adv. Cancer Res. 14, 295. ALKAN S.S. (1978) Antigen-induced proliferation assay for mouse T lymphocytes. Response to a monovalent antigen. Europ. J. Immunol. 8,112. CHARPENTIER B., GUTTMAN R.D., SHUSTER J. & GOLD, P. (1977) Augmentation of proliferation of human mixed lymphocyte culture by human a-fetoprotein. J. Immunol. 119,897. CORRADIN G., ETTINGER H.M. & CHILLER, J.H. (1977) Lymphocyte specificity to protein antigen. I. Characterization of the antigen-induced in vitro T-cell dependent proliferative response with lymph node cells from primed mice. J. Immunol. 119,1048. ETTINGER H.M. & CHILLER J.M. (1977) Suppression of immunological activities by mouse amniotic fluid. Scand. J. Immunol. 6, 1241. GERSHWIN M.E., CASTLES J.J., AHMED A. & MAKISHIMA R. (1978) The influence of a-fetoprotein on Maloney sarcoma virus oncogenesis: evidence for generation of antigen nonspecific suppressor T cells. J. Immunol. 121, 2292. GLEICH G., AVERBACK A.K. & SWEDLUND H.A. (1971) Measurement of IgE in normal and allergic serum by radioimmunoassay. J. Lab. clin. Med. 77, 690. LABIB R.S. & TOMAsI T.B. (1978) Immunosuppressive factors

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in mouse amniotic fluid and neonate serum. Immunol. Commun. 7, 223. LIrTMAN B.H., ALKERT E. & ROCKLIN R.E. (1977) The effect of purified x-fetoprotein on in vitro assay of cell-mediated immunity. Cell. Immunol. 30, 35. MURGITA R.A., GOIDAL E.A., KONTAINEN S., BEVERLEY C.L. & WIGZELL H. (1978) Adult murine T cells activated in vitro by a-fetoprotein and naturally occurring T cells in newborn mice: identity in function and cell surface differentiation antigens. Proc. natn. Acad. Sci. (U.S.A.), 75, 2897. MURGITA R.A. & TOMAsI T.B. (1975) Suppression of the immune response by a-foetoprotein. I. The effect of mouse a-fetoprotein on the primary and secondary antibody response. J. exp. Med. 141, 269. OGRA S.S., MURGITA R.A. & TOMASI T.B. JR (1974) Immunosuppressive activity of mouse amniotic fluid. Immunol. Commun. 3,497. ROSENWASSER L.J. & ROSENTHAL A.S. (1978) Adherent cell function in murine T lymphocyte antigen recognition. 1. A macrophage-dependent T cell proliferation in the mouse. J. Immunol. 120, 1991. SELL S., SHEPPARD H.W., JR & POLER M. (1977) Effects of n-fetoprotein in murine immune response. Studies on rats. J. Immunol. 119,98. SHEPPARD H.W., SELL S., TREFUS P. & BAKU R. (1977) Effects of a-fetoprotein on murine immune response. I. Studies on mice. J. Immunol. 119,91. TOMAsI T.B., JR (1977) Structure and function of alpha-fetoprotein. Ann. Rev. Med. 28,453. ToMAsi T.B., JR (1978) Suppressive factors in amniotic fluid and newborn serum: is a-fetoprotein involved? Cell. Immunol. 37,459. YACHNIN S. (1976) Demonstration of the inhibitory effect of human alpha-fetoprotein on in vitro transformation of human lymphocyte. Proc. natn. Acad. Sci. (U.S.A.), 73, 2857.