CELLULAR IMMUNOLOGY 141,409-42 1 (1992)

The Response of Human Decidual Leukocytes to IL-2

ASHLEY KING, RACHEL WHEELER, N. P. CARTER, D. P. FRANCIS, AND Y. W. LOKE

Department of‘ Pathology, University of Cambridge, Tennis Court Road, Cambridge CB2 lQP, United Kingdom

Received August 30, 1991; accepted January 13, 1992

The phenotype of human decidual leukocytes, composed predominantly of CD3-CD16-- CD56-’ cells, was examined after culture with IL-2 by immunofluorescence and flow cytometry. After IL-2 stimulation the phenotype became like that found on classical NK cells, with an increased proportion of cells expressing CD16. The IL-2Ra was absent before and after IL-2 stimulation. However, the intermediate affinity receptor, IL-2R/3, was expressed by CD56b”ph’ decidual cells, but this receptor was downregulated after IL2 stimulation. IL-2-induced proliferation of CD56+ decidual cells could be blocked using TU27, a monoclonal antibody to the IL-2RP. These findings indicate activation of decidual leukocytes by IL-2 occurs through the IL-2RP alone. 0 1992 Academic PKSS, h.

INTRODUCTION

The uterine mucosal lining, the endometrium, is transformed at the time of em- bryonic implantation into decidua which contains abundant CD3-CD 1 6pCD56b”gh’ large granular lymphocytes (LGL) (1). These cells exhibit NK activity against the K562 cell line, albeit at lower levels than peripheral blood classical CD 16b”b’ CD56dim NK cells, but they are not cytolytic to trophoblast (2, 3). However, they can be trans- formed by IL-2 to become potent lymphokine activated killer cells (LAK) which can then kill both normal and malignant trophoblast (4). Although it is not known if decidual LAK cells ever occur in viva, clearly these cells have the potential to induce pregnancy failure. The question, therefore, arises whether this important change in function after IL-2 activation is accompanied by an alteration in the phenotype of these LGL. To investigate this, we have compared the phenotype of decidual LGL before and after exposure to IL-2. We have also examined the expression of the ~55 and ~75 IL-2 receptors and the role of the ~75 receptors in the IL-2-induced prolif- eration of these cells.

MATERIALS AND METHODS

Extraction of Leukocytes from Decidua

Pieces of decidua were obtained from normal first trimester pregnancies from Ad- denbrooke’s Hospital and used immediately. The method for extraction of decidual leukocytes has been described elsewhere (2). Briefly, the fragments of decidua were

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410 KING ET AL.

identified macroscopically and washed in RPM1 1640 (Flow). The fragments were then finely minced between two scalpel blades and pushed through a 53-pm cell sieve (Gallenkamp), adding RPM1 1640 as required to aid filtration. The filtrate was then washed in RPMI, layered onto Lymphoprep, and spun at 400g for 20 min. The cells at the interface were removed, washed, and resuspended in RPM1 plus 10% fetal calf serum (FCS) (complete medium) at a concentration of 1-2 X IO6 cells/ml.

Peripheral blood leukocytes were obtained from normal adult donors. Heparinized blood was layered over Lymphoprep (Flow) and the mononuclear cells coilected from the interface and washed.

Culture of Decidual Leukocytes

Decidual leukocytes were cultured as described previously (4). Briefly, the cells were resuspended at a concentration of l-2 X lo6 cells/ml in complete medium, supple- mented with antibiotics, 2 mM L-glutamine and 100 U/ml of recombinant interleukin- 2 (rIL-2) (Sigma). Nonadherent leukocytes were removed after overnight culture leaving behind adherent stromal and glandular cells and macrophages. The cells were cultured in 25-ml flasks for l-4 days in 5% CO2 95% air at 37°C.

Proliferation Assays

The effect of rIL-2 on proliferation of decidual leukocytes was studied by culturing the leukocytes in the presence of various concentrations of rIL-2. The cells were first incubated overnight at 37°C 5% COZ in 48-well plates (Costar), and then from each well loo-p1 aliquots of nonadherent cells were transferred to round-bottomed 96-well plates (Coming), leaving behind adherent macrophages and stromal cells. After 3 days’ incubation, 1 &I [3H]thymidine (DuPont) was added to each well. Eighteen hours later, the cells were harvested onto filter papers using a Dynatech Automash 2000. Filters were placed in scintillation fluid (Optiphase Hisafe 11) and counted in a LKB 12 16 Rachbeta scintillation counter.

The ability of TU27 (a monoclonal antibody, MAb, against IL-2R/3) (5,6) to interfere with the effects of IL-2 was investigated by pretreating the cells with TU27 MAb (at various dilutions) for 30 min at 37°C and then adding the rIL-2 (100 U/ml). The cells were incubated overnight at 37°C 5% CO2 in 48-well plates, and then 100 ~1 of nonadherent cells was transferred to 96-well plates as before. On Day 3, 1 &i [3H]thymidine was added to each well. Eighteen hours later the cells were harvested as above.

Immunofluorescence and Flow Cytometry

All monoclonal antibodies (MAbs) were diluted in phosphate-buffered saline (PBS)/ 0.1% bovine serum albumin (BSA) and all incubations were conducted at 4°C. The MAbs that were used are shown in Table 1. The correct dilution of antibody was determined by titrating against a fixed number of cells. The point at which saturation occurs can be determined from the flow cytometer, since at saturation, increasing reagent concentration produces little increase in fluorescence intensity. The antibody was then used at twice the saturating concentration to compensate for differences in cell number and for differences in antigen expression.

RESPONSE OF DECIDUAL LEUKOCYTES TO IL-2 411

TABLE I

Monoclonal Antibodies Used in This Study

CD MAb Dilution Major cell reactivity Source

CD3 CD16

Leu-4 Leu-1 lb

l/20 l/IO

CD56 Leu-19 115

- Leukogate (LCA and l/10 Leu-M3)

CD25 - l/l0 - TU-27 l/100

CD45RA Leu- I8 l/l0 CD19 Leu- I2 l/IO

T cells NK cells FcR receptor NK cells NCAM Leukocytes and monocytes

BD” BD

BD

BD

IL-2Rcu (~55) IL-2R@ (~75) Isotype of CD45 B cells

BD Sugamura (5) BD BD

a BD. Becton-Dickinson

(a) Direct staining. Fluorescein (FITC)- or phycoerythrin (PE)-conjugated primary antibody at the correct concentration was incubated with 1.5 X 1 O6 cells for 30 min at 4°C in 100 ~1 PBS. The cells were then washed twice in cold PBS/O. 1% BSA and the pellets resuspended in 100 ~1 of PBS/O.l% BSA. The cells were fixed by adding 100 ~1 of 2% formaldehyde. The samples were stored in the dark at 4°C until analysis.

(b) Indirect staining. After incubation of 1.5 X lo6 cells with unconjugated primary antibody, 100 ~1 of FITC-conjugated goat anti-mouse IgG (GAM-FITC, Sigma), preincubated with heat-inactivated 10% human AB serum, was added to the washed pellet for 30 min at 4°C and then washed and fixed as before.

(c) Double staining. After indirect staining with primary antibody, 150 ~1 of mouse blocking IgG (Sigma) at a concentration of 200 pg/ml (i.e., a lo- to 15fold excess) was added and incubated for 10 min. After spinning down but without further washing, PE-conjugated Leu- 19 was added at appropriate concentration, incubated for 30 min at 4°C and then washed, fixed, and stored as before.

(d) Controls. Negative controls included (a) fluorochrome-conjugated isotype matched irrelevant MAb IgG, + IgG, (Simultest; Becton-Dickinson) and (b) an ir- relevant antibody, anti-CD19, (Leu-12) folIowed by GAM-FITC second antibody. Negligible numbers of B cells are present in decidua (7, 8).

Positive controls included anti-CD3 (Leu4) and anti-CD56 (Leu-19) which were always used to confirm the effectiveness of the antibodies and to allow a scatter gating to be set. A positive dual control (Leukogate; Becton-Dickinson) was also used.

Just before analysis, the samples were whirlimixed for approximately 20 set to disperse any clumps of cells. The samples were analyzed on a FACStar Plus flow cytometer (Becton-Dickinson).

RESULTS

Identification of AutoJluorescent Cells in the Decidual Extract

On the negative controls of gated decidual lymphocytes, some fluorescent cells were identified in the upper right and/or lower right quadrants. The “tail” in the upper

412 KING ET AL.

PBL

*

I

*

-! LGL LEUCOGFtE

CD 14 t

CD45 -

CD56 t

9

CD3 -

CD16 -

* FIG. 1. Cytograms of peripheral blood lymphocytes (a-e) and a decidual cell preparation (f-k) for com-

parison. Data for PBL is shown gated around lymphocytes. (a and f) Simultest negative control to show positions of markers. (b and g) Leukogate (anti-CD45-FITC and anti-CDICPE). In this decidual sample

RESPONSE OF DECIDUAL LEUKOCYTES TO IL-2 413

FIG. 2. Coexpressian of CD16 and CD3 on freshly isolated CD56+ decidual lymphocytes. (a) Negative control (anti-CD 19 alone followed by GAM-FITC). B cells are present in negligible numbers in decidua. (b) Anti-CD45-FITC and anti-CD14 Leukogate indicating the number of bone marrow cells in this sample. (c) Anti-CD56 PE and anti-CD 16 followed by GAM-FITC. A small number of CD56+ CD 16+ cells are found. These express CD56 at lower density than the majority of CD56+ cells. (d) Anti-CD56 and anti-CD3 followed by GAM-FITC. The majority of CD56 ceils are CD3- with small numbers of CD3+ CD56+ cells. CD3+ T cells are shown in the lower right quadrant.

right quadrant was more pronounced upon exposure to IL-2. This autofluorescence was probably due in part to the abundant cytoplasm of certain cells in the decidual extract. Staining with propidium iodide (PI) indicated that dead cells were not re- sponsible for the fluorescence. This was confirmed by sorting the fluorescent cells into

83% lymphocytes and 7% macrophages are present. (c and h) Anti-CD56-PE: 12% of peripheral blood lymphocytes express this antigen at low density. in comparison, CD56 is “brightly” expressed by 76% of the decidual cell preparation. (d and j) Anti-CD3 followed by GAM-FITC. Very small numbers of CD3+ T cells are present in decidua compared to peripheral blood. (e and k) Anti-CD16 followed by GAM-F’ITC. The number of CD16+ cells is clearly far less than the number of CD56+ cells in this decidual sample, indicating most CD56+ decidual cells are CD16-. In addition, CD16 is expressed at a lower density on decidual lymphocytes compared to PBL.

414 KING ET AL.

Ne

Neg CD 25

FIG. 3. Contour plots of freshly isolated decidual leukocytes were stained with the following: (a) anti- CD19 followed by FITC-conjugated IgG. A few cells show nonspecific staining with the second antibody; (b) TU27 followed by GAM-FlTC and CD%-PE. IL2Rfl is clearly expressed on CD56+ decidual lymphocytes; (c) anti-CD25 followed by GAM-FITC and CD56-PE. The CD56+ cells are negative for CD25. No other cells in the decidual cell preparation express CD25.

cytospin buckets and staining the slides with Giemsa. The fluorescent cells were large viable cells with abundant cytoplasm, which could represent either glandular or stromal cells (not shown). Fluorescent cells seen with the irrelevant antibody (antXD19), followed by a secondary FITC conjugate probably reflect nonspecific binding by the

CD56 cm6

CD 16 TU 27 CD25

FIG. 4. Contour plots of decidual leukocytes cultured for 4 days with rIL-2. The cells were stained for the following: (a) anti-CD19 (negative control) followed by FITC-conjugated second antibody (GAM-RTC). Nonspecifically staining fluorescent cells are seen in the upper right quadrant; (b) anti-CD4WITC and anti- CDl4-PE (Leukogate). The vast majority are lymphocytes (lower right quadrant) with a few macrophages (upper right quadrant); (c) anti-CD3 followed by GAM-FITC. Small numbers of CD3+ T cells are present; (d) anti-CD16 followed by GAM-FITC and anti-CD56-PE. Coexpression of CD16 and CD56 is clearly demonstrated; (e) TLJ27 followed by GAM-FITC. A smaller percentage of cells expressing the IL-2R than freshly isolated decidual LGL, (f) anti-CD25 followed by GAM-FITC and CD56-PE. The CD56+ cells are mainly negative for CD25. Markers are set based on isotype controls (Simultest) and ceiIs stained with anti- CD19 + GAM-FITC.

RESPONSE OF DECIDUAL LEUKOCYTES TO IL-2 415

second antibody to Fc receptor as this is not seen in the Simultest negative control. The percentage of fluorescent cells in the negative control samples was noted and subsequently always subtracted from test values.

Phenotype of Freshly Isolated Decidual Leukocytes

Comparison of the phenotype of decidual leukocytes with PBL confirms our previous report (1) that the two populations are quite different (Fig. 1). The proportion of leukocytes in the sample was routinely assessed using Leukogate which consists of a FITC-conjugated MAb to the leukocyte common antigen (CD45) together with PE- conjugated MAb against the macrophage marker CD14. Average values of 84% (n = 10, range 70-90%) CD4Y cells, confirm the efficiency of the extraction method for decidual leukocytes. In addition, as CD14 macrophages only comprised 6% (n = 6, range 4-8%) of the cells analyzed, the vast majority are lymphocytes. A typical example is shown in Fig. lg.

CD.56. Average values from 10 samples show that 80% (n = 10, range 74-88%) of cells in the decidual extract are CD.56’. One sample is shown in Fig. 1 h. This confirms that the main population of decidual leukocytes have the CD56+ phenotype. As we have previously described (1) there are two subpopulations of CD56+ decidual LGL consisting of a small component of CD56di” cells and a large component of the CD56b”gh’ cells. Analysis of the scatter plots after gating around the dim and bright subpopulations showed that the CD56b”gh’ subpopulation was larger and more granular than the CD56dim cells (data not shown). Comparison with peripheral blood lympho- cytes confirms the increased density of CD56 on decidual leukocytes (Figs. lc and lh) (1).

CD1 6. Small numbers of CD 16+ cells were identified in decidual cell preparations. Comparison with PBL shows that the density of CD16 is greater on circulating lym- phocytes than on decidual LGL (Figs. le and lk). This may explain the difficulty in identifying this small population of CD16+ cells by immunohistology (7, 9).

Using two-color immunofluorescence, nonspecific fluorescence was a particular problem in analyzing the double positive CD56+ CD16+ decidual cells as these cell populations tend to overlap on the contour plot. The proportion of autofluorescent cells present in the negative control was always subtracted to account for this as de- scribed. Double staining with CD56 and CD16 revealed two main populations. The majority of CD56 cells were CD56+ CD 16-, most of these staining brightly for CD56. A smaller number of CD56+ CD 16+ ( 10% of CD56+ cells: (n = 10, range 2- 16%) cells were also present, most of these staining dimly for CD56. A typical example is shown in Fig. 2c.

CD3. CD3-CD56+ cells form the largest population. It can be seen that these are mainly CD56b”gh’ and hence probably correspond with the CD16- CD56b”gh’ group described previously. A smaller population of CD3+ CD56+ cells (-3%) was also distinguished as described before (1). A slightly larger population (7%: n = 7, range 5-l 1%) of CD56- CD3+ cells were also apparent. It is likely that these latter cells represent classical T lymphocytes (1). Representative samples are illustrated stained for CD3 alone (Fig. lj) and double stained for CD56 and CD3 (Fig. 2d).

IL-2 Receptor Expression

Double staining with MAb TU27 to the ~75 chain of the IL-2 receptor (IL-2RP) and CD56 shows the presence of CD56+ cells which are also positive for IL-2RP (Fig.

416 KING ET AL.

FIG. 5. Overlay histograms of decidual leukocytes before (a) and after (b) IL-2 stimulation stained with an irrelevant antibody anti-CD19, anti-CD25 (~55) and anti-IL-2R@ (~75) (TU-27) followed by GAM- FITC. Decidual lymphocytes express IL-2RP but not CD25. After IL-2 stimulation, IL-2RP expression is downregulated with maintenance of CD25-negative phenotype.

3b). Only a very few CD25+ cells were identified on freshly isolated decidual CD56+ lymphocytes (Fig. 3~).

Changes in Phenotype of Decidual Leukocytes after Culture with rIL-2

Our previous findings indicate that after short-term culture with rIL-2, decidual leukocytes acquire the functional characteristics of lymphokine-activated killer (LAK) cells capable of killing both normal and malignant trophoblast (4). We now show that

\lY ted

Nw CD45 CD 56

FIG. 6. The expression of CD45 (b and e) and CD56 (c and f) on decidual lymphocytes before and after culture with IL-2. The majority of CD56 cells express the antigen brightly when freshly isolated (c). After IL2 stimulation there is an increase in the CD56*” cells resulting in a broadened peak. The Simultest negative control is shown in (a) and (d).

RESPONSE OF DECIDUAL LEUKOCYTES TO IL-2

FIG. 7. Contour plots showing expression of CD4RA on decidual lymphocytes before (a) and after(b) IL- 2 stimulation. CD45RA is present on CD56’ and CD?& cells. Expression on all cells is downregulated after IL-2.

the phenotype of these decidual LAK cells differs from freshly isolated decidual leu- kocytes. The following surface markers were examined.

(i) CD45. After 4 days exposure to IL-2, 79% (n = 8, range 66-91%) of CD45+ cells were obtained as illustrated in a typical sample in Fig. 4b.

(ii) CD3. The fraction of CD3+CD56+ cells remained approximately the same as that prior to IL-2 culture (data not shown). There was also no appreciable change in the CD3+CD56- constituent as shown in Fig. 4c.

(iii) CD16. There was a significant increase in the double positive CD56+ CD16+ population, with 48% of CD56+ cells coexpressing CD16 (n = 6, range 35-58%). In addition, the density of CD16 appeared increased after exposure to IL-2. These findings are shown in a representative sample in Fig. 4d.

(iv) IL-2 receptor. The expression of the IL-~RQ! and IL-2RP was examined after the cells had been cultured for 4 days with rIL-2. There was a reduction in IL-2Rfl+ CD56+ cells with a downward shift of the contour plot, indicating there was a greater proportion of CD56di” IL-2Rfi cells compared with CD56b”ph’ IL-2R/3+ cells (Fig. 4e). After exposure to IL-2 for 4 days CD25 was still only expressed by a few CD56+ cells (Fig. 4f). These findings are summarized in overlay histograms which clearly show the downregulation of the intermediate affinity receptor, IL-2RP, after IL-2 activation (Fig. 5).

(v) CD56. Although the percentage of total CD56+ cells remained the same there was an alteration in the proportion of CD56dim and CD56b”gh’ cells. The two distinct dim and bright peaks seen on the CD56 histogram of freshly isolated cells were no longer apparent due to an increase in the CD56dim population (Fig. 6). Measurement of the mean fluorescence intensity confirmed that the density of CD56 was 2.2X greater before culture with IL-2.

(vi) CD45RA. A variety of phenotypes with respect to CD45RA and CD56 antigens were distinguishable: (a) CD56’CD45RA+, (b) CD56-CD45RA+, and (c) CD56+- CD45RA. The majority of cells lay in the CD56+CD45RA+ and the CD56CD45RA quadrants. After culture with IL-2 the phenotype changed to almost exclusively CD56+CD45RAP (Fig. 7).

418 KING ET AL.

0 100 l/l00 11200 l/500

Concentration of IL2 (U/ml) Concentration of TU27 with 100 U/ml IL2

-7 E d 2. 2 ‘Z [ 10000 -

n 0 12 25 50 100 l/l 00 11200 11500 1 I1 000 115000

Concentration of IL2 (U/ml) Concentration of TU27 with 100 U/ml IL2

? 4000 - E 4 0 s ‘S

B

g .-

A

0 20 50 100 l/100 l/200 11500 l/1000 l/5000

Concentration of IL2 (U/ml) Concentration of TU27 with 100 U/ml IL2

FIG. 8. Bar charts with standard deviations showing the effect of IL-2 on decidual leukocyte proliferation and the ability of TU27 to inhibit this effect.

RESPONSE OF DECIDUAL LEUKOCYTES TO IL-2 419

Proliferation Assays

The effect of increasing concentrations of rIL-2 on decidual leukocytes was to increase proliferation (Fig. 8) confirming previous studies (2, 3). The effect of TU27 on this rIL2-induced proliferation was investigated in four experiments. Representative results are shown in Fig. 8. There is a clear blockade of rIL2-induced proliferation by a monoclonal antibody to IL-2RP, TU27.

DISCUSSION

Analysis of freshly isolated leukocytes from early decidua has shown that the major population (-90%) of LGL is CD3-CD16-CD56b”gh’ with a minor subpopulation (- 10%) which is CD3-CD16+CD56dim (1). We have now shown that after IL-2 stim- ulation in vitro the latter population is expanded. Christmas et al. ( 10) generated CD3- decidual lymphocyte clones in the presence of IL-2 from decidual CD3’-depleted leukocytes and also observed that half of these clones were CD16+. Thus, it appears that IL-2 drives decidual LGL from CD 16-, CD56b”ph’ cells into a more classical NK phenotype (CD 16+, CD56d’m) ( 11). These CD 16+ cells might be the cells transformed by IL-2 into decidual LAK cells which are responsible for the observed cytolysis of trophoblast in vitro (4). The expansion of CD 16+ cells could be due either to a selective proliferative response to IL-2 by the minor CD16iCD56dim subset or to stimulation of the main population of decidual CD1 6-CD56b”ph’ cells into classical NK cells. CD1 6+ cells are rarely observed in vivo (7,9) indicating that the decidual environment, while capable of maintaining proliferation of LGL, does not normally activate them into LAK cells. Proliferation of CD56+ LGL in vivo appears to be hormonally regulated as it is seen in the luteal but not the proliferative phase of the nonpregnant endometrium (1, 12). Although proliferation continues in early decidua it is less pronounced. This may reflect pregnancy-associated differentiation of the decidual CD56+ LGL with a concomitant low proliferative frequency which has been observed by us and others ( 10).

In this study we have observed that most of the CD56b”ph’ decidual LGL lose the CD45RA antigen after IL-2 stimulation. There are three isotypes in the CD45 family: RA, RB, and RO with variations in the length of the extracellular domains defining the isotypes (RA being the longest and RO the shortest). These different isotypes emerge at different stages of T cell maturation with the CD45 RA molecule being expressed by naive virgin T cells (13, 14). Therefore, our finding that decidual LGL have lost this antigen after exposure to IL-2 may be indicative of an increased mat- uration.

The response to IL-2 depends on the presence and density of the IL-2 receptor which is composed of at least two distinct subunits: the low affinity IL-2Ra (~55) chain and the intermediate affinity IL-2RP(p75) chain ( 15). When expressed together, the high affinity CD25 IL-2R is formed. We observed that freshly isolated CD16- CD56b”gh’ LGL expressed only the IL-2RP chain and not the IL-2Ror chain. The CD25 receptor could not be demonstrated in decidua by immunohistology ( 16). After IL-2 stimulation the IL-2RP chain was downregulated but the IL-2Ra chain remained absent. Similar results have recently been reported by others (17) but there was one conflicting report which described induction of IL-2Ra on decidual CD2+ lymphocytes after IL-2 stimulation (18). We have also found that the proliferative response of LGL to IL-2 could be totally inhibited by TU27, a MAb to the ~75 IL-2Rfl chain. These findings are similar to that reported for the major population of NK cells in peripheral

420 KING ET AL.

blood which also constitutively express only the IL-2RP chain. IL-2 interaction with this receptor alone on CD56+ PBL is sufficient for induction of proliferation and increased cytolytic ability (I 9-2 1). In addition the p55-negative phenotype is main- tained (22, 23). However, the phenotype of this major population of NK cells is CD 16b”*‘CD56dim. In contrast, the small subset of CD16-CD56b”gh’ NK cells in peripheral blood (which phenotypically correspond to decidual LGL) are found to constitutively express the high-affinity IL-2 receptor (CD25) and preferentially respond to low physiological levels of IL-2 (24, 25). There is, thus, a dichotomy in CD1 61 CD56 and IL-2R expression between decidual LGL and peripheral blood NK cells. We have previously suggested that circulating agranular CD56b”ph’CD25+ cells might migrate to the uterine lining where they proliferate and differentiate acquiring cyto- plasmic granules and increased levels of CD56 (1). It seems that IL-2Ra expression is also downregulated as they mature in the uterus. This difference in IL-2R expression between decidual and circulating NK subsets could also explain why CD56b”ph’ cells are preferentially expanded on exposure of PBL to IL-2 in vivo or in vitro (of which 50% are CD167 (26-28), whereas, in contrast, as we have shown in this paper, cells with a classical NK phenotype (CD 16+CD56+) are generated when decidual lympho- cytes are cultured with IL-2.

The lack of the high affinity CD25 heterodimer on decidual LGL, even after IL-2 stimulation, could be a mechanism for controlling the generation of LAK cells in the decidual environment. The downregulation of the IL-2RP chain after IL-2 stimulation could be an additional negative feedback mechanism to ensure that decidual LGL are not activated too readily to kill trophoblast. Decidual LAK cells are unlikely to be present in vivo in normal pregnancy but it is not known if they occur in, or are responsible for, failing pregnancies. As we have clearly demonstrated that these cells have the potential to kill first trimester extravillous trophoblast, mechanisms must exist to prevent IL-Zinduced activation of LGL. One possibility is regulation by cy- tokines present in the decidual environment. TGF-P, which is present in the uterine lining (29), inhibits several IL-2R&mediated events including LAK induction and IL- 2Ra expression (30).

Other biological responses may occur after IL-2 induction such as cytokine pro- duction. Different cytokines are produced by T cells depending on whether activation is via the IL2Ra or the IL2RP, with GM-CSF produced after IL-2R/? activation and IFN-y after IL-2Rol activation (3 1). IL-2R/3 also mediates induction of NK cell GM- CSF production (32). Therefore, activation of decidual LGL may not necessarily result in proliferation and augmented cytotoxic activity but, instead, could lead to the pro- duction of cytokines important in the control of trophoblast proliferation and differ- entiation. We have recently shown that extravillous trophoblast expresses GM-CSF receptors and can be induced to proliferate by rGM-CSF (33). Investigations into whether decidual LGL could be the source of GM-CSF are now underway in our laboratory.

ACKNOWLEDGMENTS We gratefully acknowledge grants from East Anglian Regional Health Authority, Sir Halley Stewart Trust,

Isaac Newton Trust, Medical Research Council, and World Health Organization Special Programme of Research, Development and Research Training in Human Reproduction. We thank Dr. Sugamura for the generous gift of the TU27 monoclonal antibody.

RESPONSE OF DECIDUAL LEUKOCYTES TO IL-2 421

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