708 M. Wilkinson and A. Morris Eur. J. Immunol. 1984.14: 70&713

43 Lazdins, J . K. , Stein, M. J., David, J. R. and Sher, A,, Exp.

44 Lawson, J. R. and Wilson, R. A., Parasitology 1983. 87: 481. 45 Stirewalt, M. A , , Cousin, C. E. and Dorsey, C. H., Exp. Parasitol.

46 Sher, A , , Hall, B. F. and Vadas, M. A., J . Exp. Med. 1978. 148:

47 Samuelson, J. C., Sher, A. and Caulfield, J. P., J . lmmunol. 1980.

48 Ross, G. D., Fed. Proc. 1982. 41: 3089.

Parasitol. 1982. 53: 39.

1983. 56: 358.

46.

124: 2055.

49 Dean, D. A., Exp. Parasitol. 1983. 55: 1. 50 Machado, A. J., Gazzinelli, G., Pellegrino, J. and Dias da Silva,

51 Rother, U . , Hansch, G., Menzel, J. and Rother, K., Z. 1mmun.-

52 Rumjanek, F. D. and McLaren, D. J . , Mol. Biochem. Parasitol.

53 Rumjanek, F. D., McLaren, D. J. and Smithers, S. R., Mol.

W., Exp. Parasitol. 1975. 38: 20.

Forsch. 1974. 148: 172.

1981. 3: 239.

Biochem. Parasitol. 1983. 9: 337.

Miles Wilkinson and Alan Morris

The E receptor regulates interferon-gamma production: four-receptor model for human lymphocyte activation

Department of Biological Sciences, University of Warwick, Coventry

The E receptor (binds sheep erythrocytes) is found on virtually all human T cells. Here we show that a monoclonal antibody 9.6, which recognizes and binds the E receptor, inhibited interferon-y production by human peripheral blood mononuclear leukocytes induced with the mitogens phytohemagglutinin, concanavalin A , Staphy- lococcal enterotoxin A and the monoclonal antibody OKT3. Metabolic activation (RNA and DNA synthesis) in human peripheral blood mononuclear leukocytes in response to mitogens was also sharply inhibited by 9.6. This inhibitory effect occurred early during the induction phase since 9.6 had much diminished inhibitory effects when added 15-24 h after induction; peak IFN-y production and DNA synthesis occurred 3-4 days post induction. An early event inhibited by 9.6 appeared to be interleukin 2 (IL 2) receptor formation since: (a) the ability of mitogen-stimulated peripheral blood mononuclear leukocytes to absorb IL2 was inhibited by 9.6, and (b) lines of T lymphocytes which already expressed IL2 receptors were largely resistant to the inhibitory effects of 9.6 on IFN-y production and DNA synthesis. The tumor promoters 12-0-tetradecanoyl phorbol-13-acetate and teleocidin largely reversed the inhibition by 9.6 of IFN-y production and metabolic activation induced by mitogens. A model for the control of IFN-y induction involving four receptors, those for mito- gens, tumor promoter, IL 2 and erythrocyte, is proposed.

1 Introduction

The interferons (IFN) are a class of proteins which have anti- viral activity and many other properties such as the ability to

specific receptors on the cell surface [2]. Identification of these regulatory receptors and their complementary ligands is important in developing an understanding of imrnunoregula- tion.

regulate t h i immune -response [i]. Different IFN types -are induced in different situations: IFN-a and -f~ are produced by a variety of cell types in response to virus infection while IFN-y appears to be produced exclusively by cells of the immune system in response to mitogens and antigens.

IFN-y production accompanies various other events of lym- phocyte activation: blastogenesis, lymphokine production, cell proliferation, and the generation of cytotoxic T lymphocytes. It is of interest to determine how these events are regulated and coordinated. The functions of cells of the immune system appear to be regulated by the binding of regulatory factors to

[I 43721

Correspondence: Alan Morris, Department of Biological Sciences, University of Warwick, Coventry, CV47AL, GB

Abbreviations: IFN: Interferon PHA: Phytohemagglutinin Con A: Concanavalin A SEA: Staphylococcal enterotoxin A TPA: 12-0- tetradecanoyl phorbol-13-acetate mAb: Monoclonal antibody(ies) IL 2: Interleukin 2 PBML: Peripheral blood mononuclear leuko- cytes PBS: Phosphate-buffered saline

00 14-298018410808-0708$02.50/0

One surface marker which has hardly been investigated is the E receptor; this receptor is defined by its ability to bind sheep erythrocytes. Since the E receptor is found on virtually all human T lymphocytes and most natural killer cells it may play an important role in the function of these cells. Palacios and Martinez-Maza reported that the E receptor-specific mono- clonal antibody (mAb), OKTlla, strongly inhibited mitogenesis in human T lymphocytes in response to both anti- genic and mitogenic stimulation [3]. They suggested that the OKTl la mAb mimics a natural suppressor factor and there- fore that the E receptor is an -‘inhibitory signal receptor” for T cell activation. Recently, Martin et al. demonstrated that two other mAb specific for the E receptor, 9.6 and 35.1, not only inhibited the T cell proliferative response, but also cytotoxic T cell function [4]. The antibodies were different, however, in that 9.6 inhibited natural killer cell cytotoxicity while 35.1 did not. The difference in their activities is probably due to the different epitopes recognized by these two E receptor mAb.

Since T cell activation and IFN-y production often occur together we investigated whether the E receptor also regulated

0 Verlag Chemie GmbH, D-6940 Weinheim, 1984

Eur. J. Immunol. 1984.14: 708-713 E receptor regulates interferon-y production 709

IFN-y induction. We found that both IFN-y production and metabolic activation in mitogen-stimulated human peripheral blood monoiluclear leukocytes (PBML) were strongly inhib- ited by the mAb 9.6 specific for the E receptor. We provide evidence that 9.6 coordinately regulates IFN-y production and metabolic activation by inhibiting early events in blas- togenesis, including interleukin 2 (IL 2) receptor formation. Tumor promoters, which also bind to a specific receptor on lymphoid cells [5], bypassed the inhibitory effects of 9.6. We therefore propose that lymphocyte activation and IFNy pro- duction are coordinately regulated by ligands binding to 4 cell surface receptors which modulate the function of each other: the mitogen, IL2, tumor promoter and E receptors.

2.5 Induction of IFN production and metabolic activation

Fresh PBML or growing lymphoblasts were washed and sus- pended in fresh medium. Added to these cultures were the mitogens SEA (20 ng/ml), PHA (10 Fgiml), Con A (20 ygiml), OKT3 (100 ngiml) and/or the tumor promoters teleocidin (3-10 ng/ml) or TPA (3-10 ngiml). Supernatants from lym- phoblast and PBML cultures were harvested 1 day and 3 days after incubation, respectively. Cultures treated with the E receptor mAb were given a pretreatment of the mAb 9.6 30 min before induction with mitogens and/or tumor promo- ters.

2.6 Assays 2 Materials and methods

2.1 Mitogens, tumor promoters and mAb

Staphylococcal enterotoxin A (SEA; supplied by the Food and Drug Administration of the USA; pure), phytohemag- glutinin (PHA; Sigma, St. Louis, MO; crude), concanavalin A (Con A; Sigma; crude) and the mAb OKT3 (Ortho Phar- maceuticals, Raritan, NJ) were used as T cell mitogens. Also used were the tumor promoters 12-0-tetradecanoyl phorbol- 13-acetate (TPA; Sigma), mezerein (L. C. Services corpora- tion, Woburn, MA) and teleocidin (the kind gift of Dr. H. Fujiki, National Cancer Research Institute, Tokyo). The E receptor mAb used was 9.6 (Lyt-3; New England Nuclear, Boston, MA): other mAb used were OKT4 and OKT8 (Ortho Pharmaceuticals).

2.2 Preparation of fresh PBML cultures

Buffy coat cells (provided by the Regional Blood Transfusion Centre, Birmingham, GB) were fractionated by centrifugation on Ficoll-Hypaque (Pharmacia, Uppsala, Sweden) as described in the Pharmacia handbook. PBML were resus- pended in RPMI 1640 medium, buffered with HEPES and supplemented with 10% fetal calf serum.

2.3 Preparation of conditioned medium containing IL 2

Cultures of fresh PBML (mixed donors) suspended at lo7 cells/ ml were pulsed for 8 h with 1% of a crude Staphylococcus aureus supernatant and 10 ngiml mezerein. The IL 2-contain- ing supernatants were harvested 15 h after completing the pulse. Such supernatants supported the proliferation of T lym- phoblasts at 1 : 10 to 1 : 20 dilution.

2.4 Growth of T lymphoblasts

Cultures of fresh PBML at lo6 cellsiml were stimulated with SEA (20 ngiml). After 2 days, the cells were thoroughly washed and then resuspended at 3 X lo5 cellsiml in medium supplemented with conditioned medium containing IL 2. Cul- tures were diluted with fresh IL 2-containing medium before they reached a cell density of 106 cellsiml. After 4 days in culture, >90% of the lymphoblasts formed rosettes with sheep erythrocytes (E), and no adherent cells were present. Thus, the cultures consisted mainly of T lymphoblasts.

IFN assay was done by inhibition of nucleic acid synthesis method [6] using WISH (human amnion) cells challenged with Semliki Forest virus. A laboratory IFN-y standard was included in all assays. IFN units quoted are in laboratory units (there is no international IFN-y standard). For assay of RNA and DNA synthesis cells were incubated with 5 yCi/ml [5-3H]uridine (29 Ciimmol or [Me-3H]thymidine (25 Ciimmol) for 24 h (supplied by Amersham International, Amersham, GB). Cells were than washed once with phosphate-buffered saline (PBS), twice with cold 5% trichloroacetic acid (TCA) and once with cold ethanol. The level of TCA-precipitable counts was assessed on a scintillation counter. For IL 2 assays lymphoblast cultures between the ages of 7 and 14 days were incubated with serial dilutions of IL 2-containing samples for 24 h. Five yCiiml [3H]uridine was then added for a further 24 h and the TCA-precipitable counts were assessed on a scintilla- tion counter

3 Results

3.1 mAb 9.6 inhibits IFN-y production by PBML

Table 1 depicts the effect of a mAb 9.6 to the E receptor, on IFN production in human PBML. 9.6 inhibited IFN-y produc- tion in response to the T cell mitogens PHA, Con A and SEA. The inhibitory effect of 9.6 at 1000 ngiml varied from donor to donor but was usually >90%. A pulse of 9.6 for only 1 h (2 washes in PBS and then resuspension in fresh medium with PHA) also substantially inhibited IFN-y production. 9.6 also inhibited IFN-y production in response to the mAb OKT3 which has been shown to induce IFN-y [7] (Table 1). IFN production in response to OKT3 was sharply reduced after 9.6 treatment in all the donors tested (usually > 99%); it was reduced to below detectable levels in many of the donors (Table 1). Concentrations of 9.6 as low as 30-100 ngiml had clearly inhibited IFN-y induction. Two other mAb binding to T cells (OKT4 and OKT8) did not inhibit IFN-y induction at similar concentrations, when added to PBML cultures either separately or together.

In addition to inhibiting IFN production in response to mito- gens, 9.6 inhibited the low levels of IFN which was constitu- tively produced by PBML from some donors (Table 1).

9.6 did not exert its inhibitory effect on IFN production by lowering cell viability since 9.6 did not decrease the number of viable cells as determined by trypan blue exclusion. Nor did 9.6 appear to induce the generation of suppressor cells SUP-

710 M. Wilkinson and A. Morris Eur. J. Immunol. 1984.14: 708-713

stimulus

none

OK13 9.6+OKT3

tel + OKT3 96btel+OKT3

Table 1. Effect of E receptor mAb") on IFN production

I A l

'3 86 * -I+ + 25

Donor Stimulus IFN (U 'ml ) Without 0.6 With 9.6'"

P6+PHA

tel+ PHA 46+tel+ PHA

1

2 78

2

none

OKT3 96+OKT3

3 -I

3. IC I *

3 94

Nonc 50 < I6 PHA 030 ' 5

OKT3 S(l0 < Ih

None < 16 < I6 SEA 50M) 320

Con A 250 < I6

0 KT3 3'0 < 16

OKT3 lo(1O < 3

5 Nonc 37 < 5 PHA 1 ( I00 < 5

PHA (pulsc 9h)"J I(HW1 13

a) Thousand ngiml 9.6. b) The cells were washed 3 times in PBS after a I-h treatment of 9.6,

and then induced with PHA.

I B I

TPA+ OK13 %+lPA+ OK13

lPA+OKT3 -+- 96+lPA+OKT3 + 47

Figure 1. The inhibition of macromolecular synthesis by mAb 9.6 is reversed by tumor promoters. Shown in (A) and (B) is ['Hlthymidine incorporation and in (C) ['Hluridine incorporation (between 72 and 96 h post induction) in PBML in response to the treatments indicated. The 9.6 concentration was 1000 ng/ml. The error bars indicate stan- dard deviation (SD) from the mean. Also shown is the % depression of macromolecular synthesis caused by 9.6 treatment. (A) is data from donor 7 and (B) and (C) are from donor 9. tel = Teleocidin. Abscissa = cpm x lo-'.

pressive for IFN production. PBML treated for 2 days with 9.6 or 9.6iPHA did not suppress IFN production in fresh PBML induced with PHA (data not shown).

In contrast to mitogens, IFN production in response to tumor promoters was not blocked by 9.6. Although the tumor pro- moters TPA and teleocidin induced low levels of IFN produc- tion in PBML, this production was barely inhibited by 9.6 (Table 2). Tumor promoters are known to act synergistically with mitogens in inducing IFN production. We found that

combined treatment of PBML with OKT3 and tumor promo- ters did induce the production of high titers of IFN-I/. This production was only slightly inhibited by doses of 9.6 which totally abrogated IFN production in response to OKT3 alone (Table 2 ) . Teleocidin also reversed the inhibitory effects of 9.6 IFN production in response to PHA (Table 2).

3.2 Effect of 9.6 on metabolic activation in PBML

The effects of 9.6 on DNA synthesis (as assessed by ["Hlthy- midine incorporation) in PBML induced with mitogens was also examined. Fig. 1 shows that 9.6 totally blocked DNA synthesis in response to OKT3. 9.6 also inhibited RNA synthe- sis ([3H]uridine incorporation). However, as with IFN produc- tion, the addition of the tumor promoters TPA or teleocidin substantially reversed the inhibitory effects of 9.6 on RNA and DNA synthesis. The inhibition of mitogenesis in response to PHA was similarly reversed by teleocidin.

3.3 Time dependence of 9.6 inhibition

mAb 9.6 was added to PBML cultures at various times after mitogen stimulation to examine at what phase of induction it acts. As before, IFN levels in cell supernatants were assessed 3 days after induction (the peak of IFN cumulative yields). If 9.6 was added 6 h after mitogen treatment, IFN production was still strongly inhibited (Table 3). However, by 15-24 h post induction, PBML were relatively refractory to the inhibi- tory effects of 9.6. The time dependence of 9.6 on mitogenesis (t3H]thymidine incorporation 72-96 h post induction) was also determined. 9.6 was less inhibitory if added 6 h post induction than if the cells were pretreated with 9.6 (Table 2 ) . As with IFN production, the inhibitory effect of 9.6 on mitogenesis was much reduced or absent if added 24 h after induction.

Table 2. mAb 9.6 inhibition of IFN production is reversed by tumor promoters

Donor Stimulus 9.6 concent rat iod' None I l l 0 n&! 1111 IIIOO 11: 1111

X

9 I

None TPA -I'el"

None 0 K.13

OKT3 Tcl PHA

PHA Tcl

None OK13 'I'PA

0 K13 3KT3,"FPA

N I> 10 I 0

< 3 1 3 500 < 3 630

ND ND N D

13 250

a) IFN titer in 3-day supernatants after pretreatment with these doses of 9.6.

b) Not determined. c) Teleocidin.

Eur. J. Immunol. 1984.14: 708-713 E receptor regulates interferon-y production 71 1

3.4 Effect of mAb 9.6 on IFN production and metabolic activation in lymphoblasts

Lines of lymphoblasts were obtained by stimulating PBML with SEA and maintaining their growth in conditioned medium containing IL2. These lymphoblasts produced IFN in response to stimulation with mitogens. The effect of 9.6 on their IFN production was examined. Table 4 shows that, unlike fresh PBML, 9.6 failed to significantly inhibit IFN-y production in response to SEA or Con A. This was observed in lymphoblast cultures ranging in age from 4-11 days old. Even at high doses (3000 ngiml) 9.6 failed to significantly inhibit IFN-y production. This failure of lymphoblasts to respond is curious since these cells have E receptors on their cell surface as shown by their ability to rosette with sheep red blood cells. In contrast to SEA and Con A, the PHA response was somewhat inhibited by 9.6 treatment. However, 9.6 did not significantly inhibit PHA-induced IFN production by lym- phoblasts at doses which were sufficient to decrease IFN pro- duction in fresh PBML cultures (100 ng/ml); and at high doses of 9.6 (1000 ng/ml), the level of inhibition was only about 4-

Table 3. Time dependence of mAb 9.6 inhibition

Stimulus Time (h) 9.6 DNA synthesis (cpm)h' IFN (Uiinl)" :1dded"'

OKT3

OKT3 0 KT3

oKr3

o m PHA PHA PHA PHA

None 0 h 6 b, post

15 h. post 21 h , po\t

None 0 h 6 h. post

2 1 11. post

27871 (5 4909) 1749 (5 627)

14492 (2 2751) 21 724 (5 1615)

114 380 ( f 11 463) X235 (f 154)

32 166 (+ 4883) 45216( f X12h)

6502 (f 116.1)

a) mAb 9.6 (1000 ngiml) was added at the times indicated post induc- tion (post) to PBML (donor 16).

b) ["HIThymidine incorporation 72-96 h post induction. The error shown is the standard deviation from the mean.

c) IFN titers were assessed in 3-day supernatants.

Table 4. Effect of mAb 9.6 on IFN production of lymphoblasts

Donor Stimulus 9.6 concentration (ngiml)"' None 30 100 300 IOO() 3000

7 SEA 320 NDh' 320 ND 320 ND Con A 3200 ND 25oU ND 2000 ND PfIA 500 ND 630 ND 250 ND

I0 SEA IOU ND ND 80 I60 ND Con A 1000 ND ND 1000 800 8(K) 0 KT3 63 ND ND 50 130 XO PHA 16 13 0 3 3 ND

I I PHA 630 630 SO() 250 200 ND 12 PHA 200 ND 2OU 2OU 63 ND

a) IFN titer in 3-day supernatants after treatment with these doses of 9.6.

b) Not determined.

c.p.m. 0 5 x1n-4 10 15

b stimulus

none

donor PHA 13 [9.6+PHAI 4- 59

cl

donor

m I Figure2. mAb 9.6 does not block DNA synthesis in lymphoblasts induced with mitogens. Lymphoblasts proliferating in conditioned medium containing IL2 were washed thoroughly with PBS, and treated as indicated in the figure. The 9.6 concentration was 1000 ngi ml. ["Hjthymidine incorporation was assessed between 24 and 48 h post induction. The error bars indicate the SD from the mean. Also shown is the % depression caused by 9.6 treatment.

l6 I

12

c.p.m. x 10-4

8

4

2 4 8 16 32 dilution

Figure 3. mAb 9.6 inhibits IL2 receptor expression. PBML (donor 15) at 2.5 x lo6 cells/ml were treated with OKT3, 9.6, OKT3i 9.6 or left untreated for 3 days. The concentration of 9.6 was 1000 ngi ml. Then the cells were washed thoroughly in PBS and suspended at 2.5 X lo6 cellsiml in IL2-containing medium for 24 h. Shown is the IL2 activity (expressed as [3H]thymidine incorporation for dilutions of the samples) remaining after this incubation: untreated (7); 9.6 (+); OKT3 (a); OKT319.6 (W); and incubation of IL2 in the absence of cells (A).

fold, which was small in comparison with the degree of inhibi- tion for fresh PBML (10-30-fold; see Table 1).

In addition, stimulation lymphoblast DNA synthesis by mito- gens was hardly inhibited by 9.6 (Fig. 2 ) . In response to SEA, no effect of 9.6 was observed. In response to PHA, a drop of DNA synthesis was observed in lymphoblasts, but this decrease was much smaller than observed for PBML treated with PHA.

3.5 mAb 9.6 inhibits IL 2 receptor expression

Since 9.6 inhibited lymphocyte activation at an early stage, its effect on the expression of IL2 receptors was tested. PBML

712 M. Wilkinson and A. Morris Eur. J. Immunol. 1984.14: 708-713

were treated with PHA or OKT3 in the presence or absence of 9.6 for 3 days and then the ability of these cells to absorb IL2 was examined. IL 2 absorption is a standard method for deter- mining relative levels of IL2 receptor expression [8]. Fig. 3 shows that PBML induced with OKT3 very efficiently removed IL2 activity from conditioned medium. In contrast, PBML treated with OKT3 and 9.6 removed very little IL2 activity; IL 2 levels were similar to those from incubation with uninduced PBML which lack IL2 receptors. This implies that 9.6 inhibited the expression of IL2 receptors. Similar results were obtained for PBML induced with PHA (data not shown).

We considered the possibility that the E receptor and the IL2 receptor are associated or identical molecule(s), and that 9.6 inhibits IL2 receptor formation by capping it from the cell surface. Evidence that the IL2 receptor is an "unmasked" E receptor includes the finding that mAb to these two receptors are known to immunoprecipitate receptors of similar molecu- lar weight and cause similar inhibitory effects on T lymphocyte proliferation (see Sect. 4). However, we found the I T 2 and E receptors to be different since capping of the E receptor from lymphoblasts by 9.6 treatment (as judged by loss of ability to form E rosettes) did not inhibit the ability of lymphoblasts to absorb IL2 (data not shown).

3.6 mAb 9.6 does not act by inhibiting the calcium flux required for lymphocyte activation

Taken together, these results show that the coordinate regula- tion of IFN-y production and metabolic stimulation by anti- bodies binding to the E receptor occurs only under certain circumstances. The anti-E receptor mAb 9.6 was not inhibi- tory in lymphocytes which had already undergone blas- togenesis (PBML 24 h after induction, or in IL2-dependent T cell lines) or in fresh PBML in response to tumor promoters with or without mitogens. Thus, 9.6 must act on induction processes occurring early during blastogenesis which are bypassed by tumor promoters. 9.6 may act on the initial cal-

Table 5. IFN production and DNA synthesis in both PBML and lym- phoblasts is dependent on calcium

Cells Donor Stimulus EGTA concentration (inv)." None 1 3 I0

IFN production PBML I6 PHA 310 I ? 5 < j Blasts 17 PHA mI 13 < 5 < s

PHAll'cl 800 700 < .5 < 5 Con A 630 ND"' < 5 < 5

DNA synthesis" PBML 16 PHA I14380 1838 2170 NI) Blasts 17 PHA 137215 14052 1714 .i30

PHA 1264ll 287h 3253 ND PHA'Tcl I05803 10687 4513 ND

Cells were pretreated with these concentrations of EGTA 15 min prior to induction and the IFN in cell supernatants was assessed (1 and 3 days after induction for blasts and PBML, respectively). Not determined. DNA synthesis was determined by ["Hlthymidine incorporation 24-28 h or 72-96 h for blasts and PBML, respectively. The values given are cpm.

cium flux stage necessary for lymphocyte activation since tumor promoters are known to induce events independently of calcium in some cell types. We further postulated that 9.6 may not be inhibitory in already activated lymphoblasts because these cells may not require a calcium flux for re-induction in response to mitogens. We tested the calcium dependence of IFN production and DNA synthesis under these circumstances using the specific chelater of calcium, EGTA. We found that EGTA totally abrogated IFN production and DNA synthesis induced with PHA in both PBML and lymphoblasts [Table 5 ) . The tumor promoter teleocidin did not reverse this depend- ence on calcium. Thus, it appears that 9.6 does not act by inhibiting the calcium flux required for IFN-y production and lymphocyte activation. Formally, it is not clear if IFNy pro- duction and DNA synthesis are inhibited by EGTA because of an inhibition of a calcium flux necessary for these events or because calcium is continuously required for these events to occur. However, the inhibitory effects of high concentrations of EGTA (10 mM) on IFN-y production in lymphoblasts are no longer observed if added 3 h after induction (data not shown).

4 Discussion

In this report we show that the E receptor plays a role in regulating lymphocyte activation and IFN-y production. By using the mAb 9.6 as a probe we demonstrate that the E receptor modulates both IFN-y production and metabolic activity (RNA and DNA synthesis) stimulated by mitogens in human PBML. The inhibition of these responses by 9.6 was observed in response to a variety of mitogens including the mAb OKT3. The E receptor regulates lymphocyte activation early in the induction phase since: (a) addition of 9.6 12-24 h after induction of PBML had much less of an inhibitory effect on both IFNy production and DNA synthesis than if added simultaneously with the mitogen stimulus, and (b) lympho- blasts already activated and proliferating in IL2 were hardly affected by 9.6. Similar observations were made by Palacios and Martinez-Maza for the inhibition of DNA synthesis by OKTl la [3]. Our finding that IFN-y production is inhibited with similar kinetics and dose response as mitogenesis suggests that the E receptor regulates both of these processes by the same or similar mechanisms perhaps by regulating the IL2 system. Palacios and Martinez-Maza showed that OKTlla inhibits IL2 production as well as the ability to respond to IL2 [3]. We found that the loss of the ability to respond to IL2 is due to the inhibition of the formation of IL 2 receptors. This is an early step in activation since IL2 receptor expression is first detectable 3 h after induction [9]. IL2 interaction with cells is important in proliferation; IFN-y production may also require IL2 [1&13].

The mechanism by which the E receptor regulates lymphocyte activation was further examined by studying the effects of tumor promoters. Tumor promoters bind to a specific, satur- able receptor found on lymphocytes [5] and a variety of other cell types [14], and have many activities in addition to the ability to promote the growth of tumors [15]. In cells of the hemopoetic system, tumor promoters induce the differentia- tion of immature macrophages and B and T lymphocytes [16-181 and induce cell proliferation in lymphoid cells from many species, including human PBML [19], and act synergisti- cally with mitogens [ 5 , 20, 211.

Eur. J . Immunol. 1984.14: 708-713 E receptor regulates interferon-y production 713

Palacios and Martinez-Maza showed that, unlike classical mitogens, the induction of DNA synthesis by the tumor pro- moter TPA in human T cells was not inhibited by the E recep- tor mAb OKTlla [3]. In this report we show that a distinct mAb to the E receptor, 9.6, failed to inhibit IFN production in response to TPA, and that TPA largely reversed the inhibitory effects of 9.6 on both mitogen-induced metabolic activation and IFN-y production. Another tumor promoter, teleocidin, chemically unrelated to TPA, also partially bypassed the inhibitory effects of 9.6 on IFN-y production and DNA syn- thesis.

These data suggest that the E receptor modulates metabolic activation and IFN-y by a similar mechanism which tumor promoters bypass. One possible candidate step is the calcium flux known to be required to initiate IFNy production and DNA synthesis in response to mitogens in fresh PBML. How- ever, this is probably not the case since neither tumor promo- ter induction nor lymphoblast induction was independent of calcium in the medium (as shown using the specific calcium chelater, EGTA).

The results from this study provide evidence for a 4-receptor model for lymphocyte activation. A basic activation stimulus (mitogen) binds to an activation receptor. This leads to the generation of IL2 receptors which are necessary for T cell proliferation. Binding of 9.6 to the E receptor blocks the expression of IL 2 receptors and hence also blocks T cell prolif- eration. Since IFN-y and IL2 production are also blocked by 9.6 this suggests that IL2 receptors are also necessary for these events (though it is also possible that IL 2 receptor expression is irrelevant; 9.6 may inhibit an earlier step which is necessary for both proliferative events and lymphokine production). In contrast to the inhibitory effects of 9.6 on T cell function, tumor promoters stimulate mitogen-induced responses; this implies that the tumor promoter receptor is a stimulatory receptor. Binding of ligands to this receptor bypass the inhibi- tory effects of 9.6 binding to the E receptor.

Natural ligands have been tentatively identified for all of these receptors except for the E receptor. The mitogen receptor is linked with the antigen receptor [22-231 and therefore anti- gens are probably the natural activator of this receptor. Clearly, IL2 binds to the IL2 receptor. The natural ligand for the tumor promoter receptor may be diacylglycerol [12]. In contrast, there is no clear candidate for the natural ligand of the E receptor. If 9.6 and other E receptor mAb mimic the action of natural ligands for the E receptor then the normal role of the E receptor may be to bind suppressor factor(s) as suggested by Palacios and Martinez-Maza [3]. On the other hand, 9.6 may act by preventing the binding of ubiquitous helper factor(s) to the E receptor.

In conclusion, the E receptor may have a central role in reg- ulating the primary immune response initiated and controlled by other receptors. Ligands binding to the E receptor may modulate lymphocyte function at many levels, including con- trol of the expression of other receptors and the production of immunoactive lymphokines such as IFN-y.

We are grateful to the Cancer Research Campaign for financial support and the Birmingham Blood Transfusion Service for human buffy coat preparations.

Received December 19, 1983; in revised form April 21, 1984.

5 References

1 Stewart, W. II., The interferon System, Springer-Verlag, Wien and

2 Bluestone, J. and Hodes, R., immunol. Today 1983. 4: 256. 3 Palacios, R. and Martinez-Maza, O., J . Immunol. 1982.129: 2479. 4 Martin, P., Longton, G., Ledbetter, J., Newman, W., Braun, M.,

5 Sando, J . , Hilfiker, M., Salomon, D. and Farrar, J., Proc. Nail.

6 Atkins, G., Johnston, M., Westmacott, L. and Burke, D. C., J .

7 Pang, R., Yip, Y. and Vilcek, J . , Cell. Immunol. 1981. 64: 304. 8 Bonnard, G., Yasaka, K. and Jacobson, D., J . immunol. 1979.

9 Larsson, E. and Coutinho, A., Nature 1979. 280: 239.

New York 1979.

Beatty, P. and Hansen, J. , J . Immunol. 1983. 131: 180.

Acad. Sci. USA 1981. 78: 1189.

Gen. Virol. 1974. 25: 381.

123: 2704.

10 Farrar, W., Johnson, H . and Farrar, J . , J . immunol. 1981. 126:

11

12

13

14 15

16 17

18

19

20

21

22

23

1120. Kasahara, T., Hooks, J., Dogherty, S. and Oppenheim, J., J . Immunol. 1983. 130: 1784. Yamamoto, J., Farrar, W. and Johnson, H., Cell. immunol. 1982. 66: 333. Handa, K., Suzuki, R., Matsui, H., Shimizu, Y. and Kumagai, K., J . Zrnmunol. 1983. 130: 988. Weinstein, I., Nature 1983. 302: 750. Weinstein, I., Lee, L., Fisher, P., Mufson, A. and Yamasaki, H . , J . Supramol. Struct. 1979. 12: 195. Weinberg, J., Science 1981. 213: 655. Okamura, J., Letarte, M., Stein, L., Sigal, N. and Gelfand, E., J . Immunol. 1982. 128: 2276. Ryffel, B., Henning, C. and Huberman, E., Proc. Natl. Acad. Sci. USA 1982. 79: 7336. Abb, J., Baybliss, G. and Deinhardt, F., J . Imrnunol. 1979. 122: 1639. Wang, J., McClain, D . and Edelman, G., Proc. Natl. Acad. Sci. USA 1975. 72: 1917. Yip, Y., Pang, R., Oppenheim, J., Nachbar, M., Henriksen, D., Zerebeckyj-Eckhardt, I. and Vilcek, J., infec. Zmmun. 1981. 34: 131. Reinherz, E., Meuer, S. , Fitzgerald, K., Hussey, R., Levine, H. and Schlossman, S. , Cell 1982. 30: 735. Munro, A., Nature 1983. 303: 570.