Serum factors influencing spontaneous rosette formation by lymphocytes of pregnant women

Serum. factors influencing spontaneous rosette formation by lymphocytes of pregnant women C. RUGARLI R. SCORZA SMERALDI M. G. SABBADINI G. FABIO P. BONARA Milan, Italy The effect of sera from pregnant women on the percentage of spontaneous rosette-forming peripheral lymphocytes was investigated. Pregnancy lymphocytes displayed a significantly lower capacity to bind SRBC than control male lymphocytes. However, after an exhaustive washing, it was possible to demonstrate a significant increase of spontaneous rosettes formed by pregnancy lymphocytes. It was found that the incubation of pregnancy-washed lymphocytes with pregnancy but not homologous male serum restored to depressed levels the values of rosette-forming peripheral lymphocytes. This blocking activity was significantly higher with autologous serum than homologous pregnancy serum. Control lymphocytes were unaffected both by washing and by incubation with pregnancy sera. The blocking activity was found in the same ion-exchange chromatography fraction of pregnancy serum where paternal HLA antigens could be demonstrated, and was reproduced by a soluble HLA preparation from the husband's lymphocytes. (AM. J. OasTET. GYNECOL. 129: 662, 1977.)

ful rejection because there are additional serum factors able to suppress, mostly aspecifically, the maternal immune responsiveness during pregnancy. 3 Some of these factors are hormonal in nature. In fact, cortisol and human chorionic gonadotropin can inhibit in vitro the lymphocyte response to aspecific mitogens as well as to allogenic stimulation. However. such hormonal effects are detectable only al hormone concentrations far higher than those physiologically reached in pregnancy. 21 Another nonhormonal aspecific immunodepressive effect has been ascribed to a-fetoprotein, which is found in maternal serum during pregnancy.ro. 2' Other ill-defined-but apparently more specificfactors also can weaken the maternal immune responsiveness. Maternal serum, immediately after delivery. can inhibit the mixed-lymphocyte reaction in vitro against lymphocytes from the uewborn infam as well as from the husband and other unrelated individuals.' Pregnancy serum is also active in specifically bloLking the MIF production by autologous lymphocytes stimulated in vitro by paternal cells. Such considerations suggest the existence in pregnant women of an immunologic status similar to the one demonstrated in cancer patients. In such patienL~, the cytotoxic immune reaction against autologous

PREGNANCY may be regarded as a successful natural allograft. Indeed, sensitization of the mother against fetal antigens of paternal origin is demonstrated by the existence of cytotoxic antibodies in maternal serum 17 · 2 " as well as by the elution from the placenta of antibodies directed against paternal histocompatibility antigens. 5 Moreover, maternal lymphocytes recognize fetal lymphocytes in a mixed lymphocyte culture in vitro/ produce soluble mediators, 28 and generate killer blast cells against target cells bearing paternal transplantation antigens. 4 • 11 However, the fetus is apparently spared by the maternal immune response, which, on the contrary, may afford the conceptus a small selective advantage. 1 It has been supposed that the allograft response of pregnant women to the fetus is too feeble to bring about a succes-

From the Laboratory for Cellular Immunology, JVIh Institute of Internal Medicine, University of Milano School of Medicine. Received for publication December 30, 1976. Accepted June 16, 1977.

Reprint requests: Dr. R. Scorza Smeraldi, Laboratory of Cellular Immunology, IV'h Institute of Internal Medicine, Universit_v of Milano, Via F. Sforza, 35- 20122 Milano, ltalv.

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Table I. Differences (means ± S.E.) from the basal per cent of E rosette-forming peripheral lymphocytes in pregnant women and male control subjects after some preliminary treatments of the cells (n = 9) Treatment of lymphocytes

Basal values (mean o/c ± S.E.) Differences after treatment: Six washings Six washings and incubation with "pregnancy" serum autologous or from wife homologous non-wife Six washings and incubation with male serum from husband or autologous non-husband homologous

Pregnant women

Male control subjects

43.55 ± 3.10

72.22 ± 3.06

+23.22 ± 3.63**

+ 1.24 ± 1.50

-5.77 ± 1.52** +6.33 ± 1.96*

-2.90 ± 1.75 -0.55 ± 0.78

+ 15.77 :t 4.05** :t 4.45**

+0.22 ± 0.89 + 1.45 0.87

+ 18.11

*P < 0.05. **P < 0.01.

tumor cells is suppressed by serum factors 10 that are bound by lymphocytes. 8 • 9 In a previous article 18 we demonstrated that a prolonged washing restores to normal values the ability of peripheral lymphocytes from cancer patients to form spontaneous rosettes with sheep red blood cells (E rosettes). A similar result was observed by Holland, Browne, and Thormes 12 and by Whitehead and associates 25 after proteolytic enzyme treatment of lymphocytes. A sheep erythocyte-binding site is considered to be a marker of human T lymphocytes and a decrease in the percentage of these cells in peripheral blood is assumed to be a meaningful indication of depressed ceii-mediated immunity.26 It is apparent that a masking factor on the surface ofT lymphocytes depresses both their performance in the specific anti-tumor cytotoxic immune reaction~'~ and their ability to form E rosettes. Such a factor, in its soluble form, is apparently present in cancer serum since the incubation of autologous serum with peripheral lymphocytes from cancer patients, unmasked by prolonged washing or by proteolytic treatment, restored the depressed levels of E rosetteforming cells. 18 • 2 " The effect is apparently specific in that cancer serum did not affect the percentage of E rosette-forming peripheral lymphocytes in normal controls. 1" These findings in cancer patients have prompted a similar investigation in pregnant normal women in order to better define the nature and the degree of specificiiy of serum factors acting in the immune depression of pregnancy. Material and method

The subjects in our investigation were nine primigravid women, in their second trimester of pregnancy, between 21 and 30 years of age. Their husbands, ranging in age from 21 to 37 years, formed the control

group. All the subjects were clinically healthy and not receiving any drugs. They had no histories of blood transfusions. The presence in pregnancy sera of lymphocytotoxic antibodies against the husband's lymphocytes was excluded by testing the wife's serum against the husband's cells by means of the microlymphocytotoxicity technique. 14 Lymphocytes were purified by flotation on Lymphoprep.6 Cells were carefully collected and washed twice in Hank's solution (Eurobio) and finally resuspended in Hank's solution containing 20 per cent fetal calf serum (FCS, Eurobio) at a concentration of 4 x I 06 per miiiiiiter. Neutrophii and monocyte contamination were below 10 per cent. Spontaneous rosettes were determined by means of a standard technique. Briefly, sheep red blood cells (SR.BC, lstituto Sieroterapico !viilanese Serafino Belfanti) were stored in preservative solution until used. They were then washed three times in normal saline and suspended in Hank's solution containing 20 per cent fetal calf serum at a cell concentration of 160 x 106 per milliliter. A volume of 0.25 mi. of SRBC suspension was mixed with 0.25 mi. of the lymphocyte suspension (lymphocytes: SRBC ratio = I : 40). The mixture was incubated for 20 minutes at 37" C. and spun at 400 X g for 5 minutes. The tubes were then kept at 4° C. overnight, after which sediment was gently resuspended and rosettes read under a light microscope (x 400). About 200 lymphocytes were scanned for each sample, and all lymphocytes binding three or more SRBC were considered as rosette-forming cells. The test was performed on lymphocytes: (l) in basal condition, (2) after six additional washings with Hank's solution, (3) after incubation of washed lymphocytes with autologous serum, and (4) after incubation of washed lymphocytes with homologous serum. That is, pregnancy lymphocytes

664

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Rugarli et al.

pernatant fluids had been previously ultracentril'uged at 150,000 x g for 30 minutes to remove cell membrane fragments. Details of the preparation of such supernates and their contents of soluble HLA antigens are reported elsewhere.'"

Tabie II. Effect of ion-exchange chromatography fractions from pregnancy autologous serum on the percentage of E rosette-forming peripheral lymphocytes in two pregnant women and in their husbands E rosette-forming lymphocytes(%)

Results

After 6 washings arul incubation with fraction Basal values

After 6 washings

Pregnant R. S. R. S.'s husband

25 48

42 47

Pregnant G. C. G. C.'s husband

33 75

69 83

Lymphocyte donors

I

I

44 53 42 62

II

45 50 48 61

l

III

26 47 27 57

Table III. Effect of supernatant fluid from in vitro cultures of homologous male lymphocytes, pulse stimulated with PHA, on the percentage of E rosette-forming peripheral lymphocytes in two pregnant women t; rosette-forming lymphocytes (%) After incubation with supernate from Lymphocyte donors

R. S. G. C.

Basal values

After 6 washings

Husband

22 36

68 61

36 21

I

Not-husband

70 60

were incubated with homologous pregnancy serum. with the husband's serum, and with serum from another male control subject; control male lymphocytes were incubated with the wife's serum, with another pregnancy serum, and with homologous male control serum. Each test was performed in triplicate. Peripheral lymphocytes from two pregnant women and from their husbands were also assayed after washing and incubation with fractions of "pregnancy" serum submitted to ion-exchange chromato 0 • aphy, employing QAE Sephadex A-50 columns, according to the technique described by Bi!!ing and Terasaki. 2 The peak fractions were pooled and reconstituted into phosphate-buffered saline to a protein concentration of 1 per cent. Fraction I contained mainly IgG. fraction II contained mainly albumin, and fraction III contained, in soluble form, maternal HLA antigens and other HLA antigens coded by a paternal haplotype. 22 In another two instances, peripheral lymphocytes from two pregnant women were washed and incubated with the supernatant media harvested from short-term cultures of husband's and control lymphocytes, pulse stimulated with phytohemagglutinin (PHA). The su-

The over-all results are reported in Table I. The basal values of E rosette-forming peripheral lymphocytes are significantly depressed in pregnant women as compared with the corresponding values in their husbands. However. after six washings. the perf(nmance of lymphocytes in the E rosette test was markedly improved, reaching levels in the normal range. When washed lymphocytes were incubated with autologous serum, this improvement was suppressed. The same thing happened. but to a definitely lesser degree, upon incubation with homologous serum from other pregnant women. On the contrary, no clear-cut suppressive effect was observed upon incubation of washed lymphocytes from pregnant \vomen \vith serum from the husband or a male control subject. No significant variation of the percentage of E rosette-forming peripheral lymphocytes was observed in male control subjects after simple washing or washing and incubation of the cells with blood serum from pregnant or control sources. Table II depicts the effect on washed lymphocytes from two pregnant women of ion-exchange chromatography fractions of autologous serum. It is apparent that only fraction III, containing soluble HLA antigens, was effective in resuppressing the levels of E rosette-forming lymphocytes. This effect was not seen on the husband's lymphocytes. Table III reports a similar experiment performed with supernates from PHA pulse-stimulated lyphocyte cultures containing homologous soluble HLA antigens. It is apparent that only the supernates harvested from the husband's lymphocytes are effective in resuppresin pregnant women. Comment

The percentage of E rosette-forming peripheral lymphocytes is definitely lower in pregnant women than in control healthy men. Since no difference between the sexes has been previously reported with reference to this test, nor found by us in our experience, we conclude that pregnancy causes a depression of the percentage of E rosette-forming lymphocytes. This finding is in keeping with the depressed levels of

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peripheral T lymphocytes demonstrated by Strelkanskas and associates23 between 7 and 20 weeks of gestation. However, in our system, such a depression cannot be attributed to a true decrease in the percentage of T lymphocytes, since it is possible to shift the results into the range of normal values by prolonged cell washing. The most likely explanation is that some of the peripheral lymphocytes of pregnant women bear on their surfaces a factor masking the sheep erythrocytebinding sites. It is apparent that blood serum is the source of this masking factor, since unmasked lymphocytes from pregnant women are resuppressed upon incubation with serum from a pregnant donor, whereas they are unaffected by normal maie serum. So far, the agreement of our present results with previous observations in cancer patients12 • 18 • 23 lends support to the hypothesis of the role of serum blocking factors both in malignancy and in pregnancy. However, some additional postulates may be advanced, arising from the patterns of sensitivity of lymphocytes from different donors to the assumed serum blocking activity. Indeed, serum from pregnant women was suppressive only on lymphocytes from the same donor (and, to a lessor degree, from other pregnant donors), whereas it was ineffective on control lymphocytes. Since, according to our previous data, the same lack of effect was manifested also on lymphocytes from nonpregnant women, it is possible to conclude that aspecific effects are of a lesser relevance in our system and that pregnancy entails both the emergence of serum factors suppressing E rosettes and a peculiar sensitivity of blood T lymphocytes to their suppressive effect. This kind of specificity suggests that such an interplay is immunologic and possibly related to the maternal immunization by fetal antigens. First, it is worth considering the possible role of maternal alloantibodies against those fetal histocompatibility antigens coded by the paternal haplotype. It is not unlikely that surface-bound alloantibodies might exert a sort of hindrance of the sheep erythrocytebinding sites of T lymphocytes. However, maternal alloantibodies have their effect on paternal and not on autologous lymphocytes; this suppression can be discarded on the basis of our present evidence. An alternative explanation is that the blocking activity might be mediated by autoantibodies. Indeed, autolymphocytotoxins were found in sera from pregnant women. 16 However, such autoantibodies are directed

Rosette formation by lymphocytes

665

against a non-HLA "public" antigenic structure, richly represented on human lymphocytes, and should be active both on autologous and on homologous lymphocytes from nonpregnant donors. We think that the suppressive effect of ion-exchange chromatography fractions of "pregnancy" serum is not consistent with the assumption that such an activity is due to antibodies, but lends support to the hypothesis of a role of soluble fetal antigens released into maternal blood. A recruitment of maternal lymphocytes, specifically committed to bind fetal antigens, may be induced through sensitization, thus explaining why only lymphocytes from pregnant women are affected by their sera in suppressing the E rosette test. It is possible to suppose that soluble fetal antigens in maternal sera interact with the recruited sensitive lymphocytes. Such a mechanism would interfere also with the aspecific function of T lymphocytes of binding sheep erythrocytes. Suitable candidates for such an interaction may be fetal or developmental antigens as \
666

Rugarli et al.

Am.

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November 15, 1977 J. Obstet. Gynecol.

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