soluble inhibitors of t lymphocyte proliferation: tools for dissecting pathways of t cell activation

Cellular Immunology

Immunol. Res. 7:93-112 (1988) �9 1988 S. Karger AG, Basel

0257-277X/8810072-009352.75/0

Soluble Inhibitors of T Lymphocyte Proliferation: Tools for Dissecting Pathways of T Cell Activation

John C. Reed, Peter C. Nowell

Department of Pathology and Laboratory Medicine, School of Medicine, University of Pennsylvania, Philadelphia, Pa., USA

Introduction

A number of the molecular events required for mitogen-stimulated T cells to enter and progress through the cell cycle have recently been elucidated. The data available indicate that following stimulation of resting (Go) human peripheral blood mononuclear cells (PBMC) with mitogens, T cells enter G l of the cell cycle, where they express receptors for interleukin-1 (IL2) and where some, but not all, T cells secrete this growth factor. The interaction of IL2 with its specific cellular receptor on activated T cells is then required for expression of transferrin receptors. The subsequent binding of transferrin to its receptor during late Gt of the cell cycle allows T cells to make the G~ --~ S phase transition, resulting in DNA synthesis and ultimately in cellular proliferation [reviewed in 1, 2]. Fig- ure 1 illustrates in oversimplified and sche- matic fashion this fundamental sequence of T cell activation events.

Though most investigations to date have focused on lymphokines and cytokines that amplify T lymphocyte functions, increas- ingly more attention is being given to immu- noregulatory molecules that negatively in-

fluence lymphocytes. Among these immunosuppressive agents are suppressive lymphokines, inhibitory monoclonal antibodies, prostaglandins, particular steroid hormones such as glucocorticosteroids and their synthetic derivatives, and various pharmacological agents including ciclosporin A (CsA). Here we summarize some of our recent findings regarding several inhibitors of T lymphocyte proliferation and illustrate their utility as tools for dissecting molecular mechanisms involved in the regulation of normal T cell activation and growth. All of these inhibitory molecules suppress the proliferation of normal T lymphocytes in an antigen-nonspecific fashion.

Relation of Inhibitors to Sequential Events in T Cell Activation

Though a myriad of natural and synthetic immunosuppressive compounds have been described, in many cases little is known about the mechanisms through which these agents down-regulate lymphocyte proliferation. Given the importance of IL2, transfer- fin, and their cellular receptors for the

94 Reed/Nowell

EVENTS OF TE.. CELL CYCLE IN HUMAN T-CELLS

, : . : " , O a . ~ 2

ZL2 ~ ~ 1 1

�9 G2/M 0111 " �9

S I~hose . late G~

Fig. 1. Events of the cell cycle in T lymphocytes. Schematically depicted are some of the events that occur in T lymphocytes following stimulation with antigen (presented by macrophages (ME}) or other accessory cells) or mitogenic lectins (PHA). The desig- nations 'early', 'middle', and 'late' Gt phase are arbi- trary. See text for details of IL2 (~), IL2 receptors (V), transferrin (e), and transferrin receptors (U).

growth of normal T cells, it seems likely that at least some of these inhibitory molecules might act by impairing events depicted in figure 1. For this reason, we selected several suppressive agents for detailed investigation and examined their effects on phytohemagglutinin (PHA)-induced production of IL2 and expression of receptors for IL2 and transferrin at both the protein and the mRNA levels.

In addition to inhibitors with well-de- fined mechanisms of action, such as monoclonal antibodies directed against the human IL2 receptor (anti-Tac) [3], the transferrin receptor (42/6) [4], we also investigated several suppressive agents with less evident mechanisms of action, including: (1) antibodies that recognize a 50-kd protein (p50) identical to or closely associated with the

sheep red blood cell receptor on human T cells (OKT11A, 9.6, 35.1) [5-7]; (2) CsA, a cyclic undecapeptide and fungal metabolite [8]; (3)dexamethasone (DEX), a synthetic glucocorticosteroid [9]; (4)wheat germ agglutinin, an inhibitory lectin [10], and (5) ' inhibi tor of DNA synthesis' (IDS), an immunosuppressive lympholine [11]. Each of these suppressive molecules was examined in relation to the sequence o f T cell activation events shown in figure 1. These investigations provided information about the inhibitory mechanisms of the above agents and allowed us to test certain aspects of the models shown in figure I.

Table I summarizes the results of many experiments. All inhibitors were used at concentrations that suppressed DNA synthesis by >__ 80% in cultures of human PBMC stimulated with the mitogenic lectin PHA. At these concentrations, none of these immunosuppressive molecules was cytotoxic. Consis- tent with previous reports [2], we determined that anti-Tac antibody (IL2 receptor) suppresses late events of T cell activation, including transferrin receptor expression, transferrin receptor mRNA accumulation, and DNA synthesis. Culturing T cells with anti-Tac antibody also partially down-modu- lates IL2 receptors and, as will be discussed in detail later, reduces levels of IL2 receptor mRNA by about 50% in PHA-activated PBMC [12], but has no effect on IL2 production. In contrast, 42/6 monoclonal antibody (transferrin receptor) inhibits DNA synthesis in cultures of PBMC but does not impair earlier events of T cell activation. Thus, these findings with anti-Tac and 42/6 antibodies are consistent with the sequence of events illustrated in figure 1.

To further confirm aspects of the model shown in figure 1, we also employed phar-

l n h i b i t o r s a s T o o l s f o r I n v e s t i g a t i n g T Ce l l A c t i v a t i o n 95

T a b l e I . S u m m a r y o f r e s u l t s fo r i n h i b i t o r s o f P H A - i n d u c e d p r o l i f e r a t i o n o f P B M C

I n h i b i t o r m R N A s I I L 2 2 I L 2 R 3 "I-FR a D N A 5

I L 2 I L 2 R T F R

IL2 restores 6

O K T 1 I A + + + + -~ + + y e s

D E X + + ~- + + + + y e s

C s A + + -~ + 4- 4- + r io

A n t i - T a c - + + - + + + y e s

C H X - - + N D N D N D + N D

W G A - - N D - + N D -v n o

4 2 / 6 . . . . . + + N D

H U . . . . . . + N D

I D S N D N D N D - - - + n o

+ = I n h i b i t s ; - = d o e s n o t i n h i b i t ; N D = n o t d o n e .

z R e l a t i v e l e v e l s o f a c c u m u l a t e d m R N A m e a s u r e d b y R N A b l o t a n a l y s i s a s d e s c r i b e d p r e v i o u s l y [ 12, 13, 17,

271. 2 IL2 activity detected in culture supernatants by Ik2 bioassay [20]. 3.4 Relative levels of cell surface expression of receptors for IL2 (IL2R) and for transferrin (TFR) determined by immunofluorescence assays with anti-Tac and OKT9 antibodies, respectively [15]. 5 DNA synthesis measured by 3H-thymidine incorporation [20]. 6 Exogenous IL2 added to cultures (see text).

macological agents such as cycloheximide (CHX), an inhibitor of protein synthesis, and hydroxyurea (HU), a DNA synthesis inhibitor. The former of these pharmacological agents blocks elongation of peptides during translation, whereas the latter inhibits an enzyme necessary for DNA replication, di- nucteotide reductase. As shown in table I, experiments with CHX revealed that protein synthesis is not necessary for accumulation of mRNAs for IL2 and IL2 receptors, but is required for transcription of the transferrin receptor gene [ 13]. These findings with CHX support previous studies by Neckers and Cossmann [ 14] demonstrating that the interaction of IL2 with its cellular receptor is critical for the subsequent expression of receptors for transferrin. In contrast to CHX, and as expected from the model in figure 1, HU

impairs PHA-stimulated DNA synthesis but not other earlier events of T cell activation.

Having substantiated several aspects of our model for T cell activation and proliferation (shown in figure I) with the use of CHX, HU, and the monoclonal antibodies anti-Tac and 42/6, we next investigated the effects of other, less well characterized immunosuppressive molecules. As shown in table I, when used at concentrations that inhibit DNA synthesis by :> 80% in cultures of PHA-stimulated human PBMC, O K T I I A and similar anti-p50 monoclonal antibodies, CsA, and DEX appear to interfere with relatively early events of T cell activation, since they decrease expression of receptors for IL2 and transferrin and suppress production of IL2 (table I) [ 15-18]. These inhibitory, molecules profoundly inhibit the proliferation of

96 Reed/Nowell

PHA-stimulated PBMC when added at the initiation of cultures, but do not suppress T cell proliferation when added late into the culture period and do not diminish the IL2- induced growth of previously activated T cells [16, 18, 19]. OKTIIA, CsA, and DEX therefore interfere with the initiation of T cell proliferative responses but do not block ongoing responses.

In contrast to these early inhibitors, wheat germ agglutinin (WGA) acts later in the sequence ofT cell activation events. Pre- vious investigations in collaboration with Robb and Greene [20] demonstrated that this immunosuppressive lectin binds to high- affinity receptors for IL2 on activated T cells and prevents their maintenance on the cell surface. Accumulation of mRNAs for IL2 and IL2 receptors is not suppressed by WGA, indicating that the initial phases of T cell activation and cell cycle progression still occur in the presence of WGA. Furthermore, by binding to IL2 receptors on activated T cells, WGA diminishes proliferation even when added late into PHA-stimulated cultures and impairs the IL2-induced growth of T cell lines maintained in long-term cultures [20, 211.

Finally, in collaboration with Jegasothy [22], we demonstrated that the immunosuppressive lympholine IDS, similar to HU, acts late in the sequence of PHA-induced activation events, since IDS impairs DNA synthesis but does not interfere with earlier events ofT cell activation (table I). Thus, consistent with previous reports [23, 24], IDS arrests the growth of mitogen-stimulated T ceils, as well as a variety of other cell types, at a point late in Gl phase of the cell cycle.

Taken together, the data in table I demonstrate that T cell proliferation can be inhibited at multiple points in the normal se-

quence of activation events. The immunosuppressive agents discussed here thus pro- vide us with a panel of tools for investigating various phases of T cell activation and cell cycle progression.

Requirements for IL2 Receptor Expression and Function: Insights Provided by lnhibitors

Our preliminary studies of CsA, DEX, and anti-p50 antibodies such as OKTI IA suggested that these agents might be useful for investigating IL2 receptor expression, since they impair early events of T cell activation (table I). Accordingly, experiments by Reem and Yeh [25] using DEX and anti-Tac antibody and studies by us employing OKTI 1A and similar antibodies, in collaboration with Kamoun and Tadmori [15], demonstrated that IL2 can up-regulate expression of its cellular receptor on activated T lymphocytes. Table II shows typical data from experiments using DEX, CsA, and OKTI IA antibody to inhibit IL2 receptor expression (Tac) induced on PBMC by PHA. At concentrations that impaired DNA synthesis in these cultures by >__80%, DEX, CsA, and OKTI 1A antibody markedly reduced the levels of Tac antigen on PBMC stimulated with PHA. At least for CsA and DEX [17], further studies confirmed a pro- portional reduction in high-affinity IL2 bindings sites on inhibited cells, using radio- labeled purified IL2 in a binding assay as described previously [26]. Furthermore, the decreases in the levels of IL2 receptors on T cells exposed to DEX, CsA, or OKTI 1A- type antibodies were paralleled by similar reductions in the levels of IL2 receptor (Tac) mRNAs [ 12, 17].

Inhibitors as Tools for Investigating T Cell Activation 97

Attempts to restore proliferation in cultures of PBMC containing these immunosuppressive agents demonstrated that addition of IL2 brought the levels of IL2 receptor (Tac) toward normal for OKT11A and DEX. This IL2-mediated augmentation of IL2 receptor expression occurred at both the protein and mRNA levels as early as 24 h after the initiation of cultures [12, 17], i.e., before the first round of cellular division in PHA-stimulated PBMC cultures. Futhermore, pretreatment of PBMC with mitomycin C to prevent cellular proliferation did not abrogate the ability of IL2 to increase the levels of IL2 receptors on PHA-activated T cells [15]. Thus, the ability of exogenous IL2 to abrogate the inhibition of IL2 receptor expression by DEX and by OKTllA-type antibodies and to augment IL2 receptor levels on PHA-activated PBMC is not attributable to the ex- pansion of Tac § cells in these cultures.

Experiments using OKTllA-type antibodies and DEX therefore demonstrated that IL2 can up-regulate expression of its cellular receptor on activated, but not resting, T ceils. These findings suggested that production of IL2 may be necessary to achieve optional levels of IL2 receptor expression in cultures of PHA-stimulated PBMC. To test this hypothesis, we used the anti-Tac antibody. In experiments wherein anti-Tac antibody was added at the initiation of PHA- stimulated cultures of PBMC, relative levels of IL2 receptor mRNAs were reduced signif- icantly [ 12]. The interaction of endogenously produced IL2 with IL2 receptors on PHA- activated cells thus up-regulates the expression of IL2 receptors (Tac antigens) at a pretranslational level and is necessary to achieve optimal levels of IL2 receptor expression.

Such experiments using OKTI I A antibody, DEX, and CsA have provided evidence that the expression of IL2 receptors is regulated by at least two pathways: one IL2- dependent, pertaining to the capacity of IL2 to up-regulate expression of its cellular receptor on activated T cells; the other IL2- independent, resulting directly from PHA- mediated events. Moreover, comparison of the effects of CsA with those of DEX and of OKTI 1A antibody have revealed important information not only about the requirements for optimal IL2 receptor expression, as discussed above, but also about the signals needed for IL2 receptor function. As shown in table II, though both CsA and DEX re- duce the levels of IL2 receptors on mitogen- stimulated PBMC (typically by about 50%), only CsA blocks the induction by PHA of responsivity to IL2. By 'responsivity' we re- fer to the ability of PHA-activated T cells to undergo increased proliferation when exogenous IL2 is added to cultures. Though we cannot exclude the possibility that some post-IL2 binding events still occur in PBMC treated with CsA, at least two IL2-mediated events, proliferation and IL2 receptor up- regulation, do not occur (table II) [ 17].

Previous studies by us [27] and by others [19, 28] have demonstrated that CsA (1-5 pg/ml) does not interfere directly with IL2 receptor function, since CsA fails to suppress IL2-induced proliferation in long-term cultures of activated T cells. What, then, is the basis for the observation that CsA inhibits the PHA-mediated induction of IL2 responsivity in primary cultures of PBMC but does not interfere with IL2 responsivity in long- term cultures of activated T cells? Clearly, the suppression of IL2 responsivity by CsA in primary, cultures is not attributable en- tirely to reduced IL2 receptor levels, since

98 Reed/Nowell

T a b l e II. Effects of CsA, DEX, and anti-p50 monoclonal antibodies on PHA-induced expression of IL2 receptors

Culture conditions CsA DEX Anti-p50 antibody

PHA inhi- IL2 % A FL 3H-TdR % A FL 3H-TdR % A FL 3H-TdR bitor Tac+ Tac+ Tac+

- - - 4 0.5 1.I 3 0.4 0.9 4 0.2 1.2 + - - 48 4.3 63.1 45 4, 1 62.7 42 2.8 54.4 + + - 26 2.5 8.2 22 2.3 1.2 12 0.5 7.0 § + + 29 2.8 11.4 48 4.4 87.3 40 3.6 80.1 + - + 61 5.3 101.9 60 5.1 93.2 62 5.5 132.5 - - + 5 1 . 0 1 1 . 0 6 1 .1 1 0 . 4 5 0.4 4.9

Indirect immunofluorescence analysis of Tac antigen (IL2 receptor) expression was performed as described previously [ 15]. Data (representative of several experiments) are given as the percentage of Tac-positive cells (% Tac+) and as the change in the relative mean fluorescence intensity (A FL) after correcting for nonspecific binding of a control antibody, R3-367. Incorporation of 3H-thymidine (K cpm) was measured in companion microcultures during the last 8 h of 3<lay cultures of PBMC containing 0.1% PHA-P, 1.0 ~g/ml CsA, 10 -4 M DEX, 0.5 I~g/ml anti-p50 monoclonal antibody (9.6), 100 U/ml rlL2, or various combinations of these reagents, and the data were expressed as mean cpm of triplicate cultures (SD = __< 15 %).

D E X also inhibi ted IL2 receptor expression (both low and high affinity [cf. 17]) to a similar extent as CsA yet did not block the acquisi t ion o f responsivi ty to IL2 in P H A -

s t imulated cultures. One possible interpreta- t ion consis tent with the findings reported

here and elsewhere [17, 19, 28] is that CsA (but not DEX) blocks the PHA-media t ed in- duct ion o f o ther signals (besides those sig-

nals leading to IL2 receptor expression) nec-

essary for resting T cells to become capable o f responding to IL2. Once T cells have received these signals and have expressed at least low levels o f IL2 receptors, they are

capable o f responding to IL2 and (as in the case o f long-term cultures o f IL2-responsive

T cells or preact ivated T cells) are, at least temporar i ly , no longer susceptible to inhibi- t ion by CsA.

The c o m b i n e d evidence from a variety o f

studies employing CsA therefore suggests

that the expression of high-affinity IL2 re-

ceptors is insufficient for IL2 responsivity, and that other signals are necessary. The sug- gestion that IL2 receptor-expressing cells

m a y require addi t ional signals to become responsive to IL2 is not unlike what is ob- served in other growth factor receptor sys-

tems. For example, fibroblasts possess high- affini ty receptors for epidermal growth fac-

tor (EGF) but cannot respond to E G F until rendered ' compe ten t ' by pr ior exposure to platelet-derived growth factor or o ther ap-

propr ia te stimuli [29]. Thus, CsA (but not D E X and O K T l l A ) appears to interfere

with the acquisi t ion o f the ' c o m p e t e n t ' state in T cells and dissociates IL2 receptor ex-

pression f rom IL2 receptor funct ion. The molecular nature o f the "signals' required to couple high-affinity IL2 receptors to prolif-

erat ion is unknown, but among the numerous possibilities are secretion o f soluble fac-


tors other than IL2, synthesis of regulatory proteins that associate with IL2 receptors in the membranes of T cells, expression of pro- to-oncogenes, and activation of intracellular protein kinases.

Early Events of T Cell Activation: Studies with CsA and OKTI IA Antibody

Considerable progress has been made in our understanding of the intracellular events that result f rom the binding of mitogenic lectins to T lymphocytes. Early studies of lymphocyte activation had demonstrated that changes in cyclic nucleotide levels, phosphatidylinositol metabolism, cytosolic free Ca *~ concentrations, Na*-K + ATP p u m p activity, and intracellular pH occur in T cells within seconds to hours after exposure to PHA [for review, see 30]. Which, if any, of these early t ransmembrane events is important for signal transduction by mitogenic lectins re- mained obscure until recently. Based on the work of Berridge, Nishizuka, and others Ire- viewed in 31, 32], it now appears that in cases where receptor signaling does not occur via either adenylate or guanylate cyclase, with subsequent activation of cAMP- or cGMP-dependent kinase, respectively, that receptor signaling is often mediated through increases in phosphatidylinositol turnover and protein kinase C activation. Figure 2 schematizes the basic sequence of events. Though many of the details remain sketchy, binding o f ligand to its receptor (including binding of PHA to T cells) appears to initiate an increase in the rate of the phosphatidylinositol cycle. Two by-products of this futile cycle are inositol triphosphate, the purported 'second messenger' that regulates levels of cytosolic free Ca § and diacylglycerol,

Fig. 2. Early events ofT cell activation. T lymphocytes can be stimulated to increase their phosphoino- sitol turnover by mitogenic lectins such as PHA and ConA, by OKT3 monoclonal antibody (which recog- nizes a 20-kd molecule associated with the antigen receptor (Ti) on T ceils), or by antigen presented to T cells by accessory ceils bearing surface antigens en- coded in the major histocompatibility locus (MHC) [62, 63]. Increased phospboinositot metabolism produces inositol triphosphate, the purported regulator of cytosolic Ca ,~ concentrations [31 ], and diacylglycerol (DAG), an allosteric activator of protein kinase C [32], resulting in a cascade ofprotcin phosphorylation (serine and threonine residues) that ultimately leads to changes in gene expression. The calcium ionophores, A12387 and ionomycin, bypass some of these early events and directly induce increases in cytoso[ic free Ca **. Phorbol esters, such as TPA, are structur- ally similar to DAG: these agents bind directly to and activate protein kinase C [33],

100 Reed/Nowell

which binds directly to protein kinase C. The combination of increases in cytosolic free Ca ++ and in diacylglycerol produces protein kinase C activation. This protein kinase then presumably initiates a cascade of protein phosphorylation that results ultimately in changes in gene expression in ligand-stimulated target cells.

Clues that signal transduction mediated via receptors for antigen and for mitogenic lectins in T cells might occur by the phosphatidylinositol pathway come from investigations of the mitogenic effects of phorbol esters and calcium ionophores on T lymphocytes. Certain phorbol esters, such as tetra- decanoyl phorbol acetate (TPA), are struc- turally similar to diacylglycerol [33]. Like diacyiglycerol, TPA binds directly to and activates protein kinase C [34, 59]. Studies with human and murine T lymphocytes, as well as with a leukemic T cell line, have shown that calcium ionophores, mitogenic lectins, and OKT3 antibody (all of which increase cytosolic Ca ~ concentrations [35, 36]) can act synergistically with TPA to stim- ulate IL2 production and T cell proliferation [35, 37, 38]. A two-signal model has thus been proposed for T cell activation, wherein the combination of (1)increased cytosolic calcium ions and (2) diacylglycerol (or TPA) results in activation of protein kinase C. This model is doubtless grossly oversimplified, but it provides a useful framework for asking questions about mechanisms of lectin-me-

I

diated T cell activation. Because CsA and OKT 11A interfere with

early events of T cell activation, we won- dered whether these inhibitory agents might be useful for analyzing particular aspects of the model shown in figure 2. For this reason, CsA and the anti-p50 antibody OKTI1A were tested for their ability to inhibit the

proliferation of PBMC induced either by the phorbol ester TPA or by the calcium ionophores A23187 and ionomycin. As shown, OKTI1A antibody inhibited proliferation induced by PHA but had little effect in PBMC cultures stimulated with TPA or A23187. Experiments with a control antibody, 12.1, excluded the possibility of nonspecific inhibition by OKT 11A antibody (table III), and other experiments (not shown) demonstrated that OKT11A does not block binding of PHA to PBMC [16, 18]. In contrast to OKT 11A antibody, CsA did suppress proliferation of PBMC induced by TPA and ionomycin. Thus, phorbol esters and calcium ionophores can bypass the block in T cell activation events produced by anti-p50 antibodies but not by CsA.

Consideration o( these findings in the context of the model shown in figure 2 per- mits us to speculate on the mechanisms of action of inhibitory anti-p50 antibodies and CsA. For example, the data in table III are consistent with the possibility that antibodies such as OKT 1 IA inhibit events prox- imal to protein kinase C activation. If this speculation is correct, then a reasonable ex- planation for the inhibitory activity of OKTI 1A-like antibodies is that they impair phosphatidylinositol metabolism in PHA- stimulated T lymphocytes. Consistent with this hypothesis, OKTI IA antibody has been recently shown to impair at least one inositol pathway-dependent event, namely the increase in cytosolic Ca ++ concentrations induced in T lymphocytes by PHA [39]. In contrast to anti-p50 antibodies, CsA would be expected to interfere with T cell activation events at or distal to protein kinase C, since it suppresses proliferation induced by phorbol esters and calcium ionophores. Con- sistent with this supposition, CsA does not


Table III. Effects of CsA and anti-p50 antibodies (sheep erythrocyte receptor) on proliferation induced by phorbol esters and calcium ionophores

CsA Anti-p50 antibodies

culture 3H-thymidine, culture 3H-thymidine, conditions K cpm conditions K cpm

Unstimulated 2,001 _ 121 unstimulated 1,983 +_ 201 PHA 62,708_+3,014 PHA 70,110+_6,310 PHA + CsA (0.1 pg/ml) 30,081 _+2,515 PHA + OKTI 1A 34,474_+_2,758 PHA § CsA (1.0 ~tg/ml) 1,216_+63 PHA + 9.6 11,911 __.2,382 TPA 40,376_+ 1,778 PHA + 35.1 15,423_ 1,851 TPA + CsA (0.1 ~ tg /ml ) 21,680-+633 PHA + 1 2 . 1 84,755_+21,189 TPA + CsA ( 1.0 btg/ml) 1,200 4- 89 TPA 41,088 _+ 3,981 Ionomycin 22,610 4- 1,709 TPA + OKTI 1A 42,233 __+ 5,068 Ionomycin + CsA (0.1 lag/ml) 6,833_+271 TPA + 9.6 47,676-+2,384 Ionomycin + CsA (1.0 lag/rnl) 468 4- 195 TPA + 35. I 42,802 _+ 2,568

TPA+ 1 2 . 1 48,411-+11,619 A23187 23,217_+ 3,883 A23187 + OKT11A 21,804~3,217

PBMC (106/ml) in complete medium were cultured in flat-bottom microtiter wells with PHA-P (0.5/ag/ml), PHA-M (25 lag/ml), TPA (100 ng/ml), A23187 (1 lag,/ml), ionomycin (2.5 lag/ml), CsA (0.1 or t.0 pg/ml), OKTI IA (150 ng/ml), 9.6 (500 ng/ml), 35.1 (1:250 ascites), 12.1 (1:100 ascites), or various combinations of these reagents as shown. Eight hours before termination of culture, wells were pulsed with 0.5 ~tCi 3H-thymidine and harvested, usually at 72 h. Filters were counted in an automated scintillation counter and K cpm (mean -+ SD for triplicate cultures) was determined.

d imin ish increases in cytosolic Ca ++ induced in hepatocytes by specific agonists [40].

Though the simple two-signal model in figure 3 has p rov ided a useful f r amework for ou r discussions o f the potential mechan i sms o f inhibi tory ant i -p50 ant ibodies and CsA, recent studies o f CsA have compl ica ted the

picture. Based on the recent work o f Hess et al. [41], it now appears that the act ion o f CsA m a y be at tr ibutable, at least in part, to inhi- b i t ion o f ca tmodul in . These invest igators

showed that CsA binds to and inhibits the in vi tro activi ty o f ca lmodul in - a Ca *+ b inding

pept ide required for the act ivat ion o f several enzymes including some kinases, phospha- tases, and phosphodiesterases [reviewed in

42]. The observat ion that CsA impairs cal-

modu l in ' s action, together with the data in table III , raises several interesting questions, including: (1) Is act ivat ion o f bo th protein kinase C and ca lmodul in -dependen t kinases necessary for T cell act ivat ion and prolifera- t ion or, instead, do these kinases represent al ternat ive act ivat ion pa thways? (2 )Does

CsA have addi t ional inhibi tory mechan isms that explain its ability to suppress TPA- induced prol i ferat ion? (3 )Are there regula-

tory interact ions between those kinases activa ted by kinase C and those turned on by ca lmodul in?

Given the above comments , it is likely that our model o f v e ~ early T cell act ivat ion

102 Reed/NoweU

Fig. 3. Inhibition of c-myc mRNA accumulation by anti-p50 antibody OKTI I A and by CsA. PBMC (2 X 106/ml) were cultured in complete medium with PHA-P ( I: 1,000 vol:vol), OKTI I A (0.5 ~edml), CsA ( 1 lag/ml), or various combinations of these reagents. After various times, as shown, cells were harvested, total cellular RNA was isolated, and 10 ~tg/lane analyzed for relative levels of c-myc mRNA by RNA blot assay. Measuring relative levels of 13-actin mRNA accumulation in these experiments demonstrated that OKTI IA and CsA do not nonspeeifically impair gene expression in PBMC (not shown). Data are representative of several experiments.

events (fig. 2) will require modification. Un-

doubtedly, anti-p50 antibodies and CsA will prove useful tools for addressing questions about signal transduction mechanisms in-

volved in mitogen- and antigen-induced T cell activation. The ultimate goal of such

investigations will be to determine com- pletely the sequence of events beginning with the binding of antigen or lectins to their spe-

cific surface receptors on T cells and ending with changes in gene expression in the nu- cleus.

As a first step towards this goal, we have

investigated the effects o f O K T 1 1A antibody

and CsA on the expression of the proto-

oncogenes c -myc and c-fos. Accumulation of mRNAs for c-fox and c-myc begins within minutes of exposure of T lymphocytes to PHA or ConA and represents one of the ear-

liest detectable changes in gene expression following stimulation of T cells [13]. As

such, the increase in these mRNAs induced by mitogens serves as a convenient marker of early T cell activation events. In figure 3

are typical RNA blot data wherein PBMC were cultured with PHA in the presence or

absence of CsA or OKTI1A, and relative levels of c-myc mRNA were measured after

lnhibitors as Tools for Investigating T Cell Activation t03

various times. As shown, OKTI 1A and CsA reduced the accumulation of c-myc mRNA even at the earliest times assayed. Similar data were obtained for c-fos mRNA accumulation (not shown). Thus, these findings (fig. 3) corroborate other evidence that OKTI 1A and CsA impair very early events o fT cell activation and begin to establish the p50 molecule and calmodulin as interme- diates involved in a sequence of molecular events that ultimately connects events at the cell surface initiated by the binding of PHA with changes in gene expression in the nu- cleus of the T lymphocyte.

IL2 and Mitogenic Lectins Induce Expression of Overlapping Sets of Genes in T Lymphocytes: Studies with Anti-Tac Antibody and CsA

Figure 2 depicts some of the early trans- membrane events thought to be important for lectin-stimulated T cell activation. Re- cent investigations by Farrar et al. [43, 44] have suggested that at least some of the early events induced in T cells by mitogenic lectins, such as increased phosphatidylinositol metabolism and activation of protein kinase C, are also induced in activated T cells by IL2. These apparent similarities in signal transduction pathways used by receptors for mitogenic lectins and receptors for IL2 suggested to us that PHA and IL2 might induce expression of some of the same genes in T lymphocytes. The data in table II support this hypothesis, since both PHA and IL2 increased IL2 receptor levels. In previous investigations of IL2 receptor gene expression, we employed the anti-Tac antibody to block the effects of endogenously produced

IL2 in cultures of PHA-stimulated PBMC. These experiments using anti-Tac demonstrated that IL2 up-regulates expression of the IL2 receptor gene [12].

Therefore, in collaboration with Hoover and Prystowsky, we took a similar approach for studies of the c-myc proto-oncogene in T cells. As shown in table IV, anti-Tac antibody had little effect on c-myc mRNA levels at 3 h, before IL2 receptors are expressed in cultures of PHA-stimulated PBMC, but did produce reductions in c-myc mRNA levels at 24 h. Conversely, purified IL2 augmented levels of c-myc mRNA on PHA-activated PBMC at 24 h, after IL2 receptors are expressed in these cultures, but not at 3 h. These findings suggest that expression of the c-myc proto-oncogene, like that of the IL2 receptor gene, is induced by both PHA and IL2 in T cells.

To confirm these findings further, we and our colleagues undertook studies with a variety of cloned T lymphocytes. Table IV shows typical data from experiments using one of these cloned noncytolytic routine T cells. Cloned T cells are maintained in culture by periodic stimulation with antigen and IL2. When rendered quiescent, these recently 'activated' T cells, which express receptors for both antigen and IL2, can be stimulated either with ConA or with IL2. As shown, both ConA and IL2 induced c-myc mRNA accumulation in cloned T cells. Addition of CHX to these cultures stimulated by ConA inhibited [L2 production by these cells but did not impair c.rayc m R N A accumulation, thus indicating that ConA directly induces c-rnyc gene expression in cloned T cells. In contrast, inclusion of CsA in cultures of cloned T cells suppressed ConA-induced c- myc mRNA accumulation but had little effect on IL2-stimulated cells.

104 Reed/Nowell

Table IV. Evidence that mitogenic lectins and IL2 induce c-myc proto-oncogene expression in T lymphocytes

Peripheral blood mononuctear cells ~ Cloned T lymphocytes 2

culture condition time, h Relative level culture condition time, h relative level mRNAs ~ mRNAs

c-rnyc 13-actin c-myc [3-actin

Unstimulated 24 0 +++ unstimulated 8 0 +++ PHA 24 +++ ++§ ConA 8 +++§ +++++ PHA + anti-Tac 24 + +++++ ConA + CsA 8 + ++++ PHA § IL2 24 +++++ +++++ ConA + CHX 8 +++++ ++++ IL2 24 0/+ +++ CHX 8 + +++ Unstimulated 3 0 +++ excipient control 1 0 +++ PHA 3 +++++ ++++ IL2 1 ++++ +++++ PHA + anti-Tac 3 +-~+ ++++ IL2 + CsA 1 ++++ +++++ PHA + IL2 3 +++++ ++++ IL2 + CHX 1 ~ i i ~ ~ +++++ IL2 3 0/4 +++ CHX l + +++*

n PBMC were cultured in complete medium with 0.01% PHA-P, 50 U/ml ptarified recombinant IL2, 0.2 btg/ml anti-Tac antibody, or various combination of these reagents as shown. Total cellular RNA was isolated after either 3 or 24 h. -" Cloned T cells (L2 cells [cf. 27]) were cultured with 10 Ixg/ml ConA, 1 lag/ml CsA, 15 pg/ml CHX, 100- 1,000 U/ml rlL2, excipient control for IL2, or various combinations of these reagents as shown. Total cellular RNA was isolated after I or 8 h. 3 Relative levels of c-rnyc and ~act in mRNAs were determined by scanning densitometry of RNA blots as described previously [64]: ~ ~ ~ ~ + = highest mRNA level within an experiment (arbitrarily set at 100%); a ) ) ~ = 75-99% of maximum, +++ = 50-74%; ++ = 25-49%, + = 1-24%, 0 = undetectable.

T h e c o m b i n e d d a t a in t ab le IV d e m o n -

s t r a t e t h a t e x p r e s s i o n o f the c-myc p r o t o -

o n c o g e n e in T cel ls is r e g u l a t e d by t w o

p a t h w a y s : o n e I L 2 - i n d e p e n d e n t a n d C s A -

s ens i t i ve , t h e o t h e r I L 2 - d e p e n d e n t a n d

C s A - i n s e n s i t i v e . U s i n g s i m i l a r e x p e r i m e n t a l

a p p r o a c h e s , we a n d o u r co l l e agues h a v e de-

t e r m i n e d that : (1) c-myc, p53 ( a n o t h e r p r o t o -

o n c o g e n e ) , IL2 r e c e p t o r , a n d 15-actin exp re s -

s ion a r e s t i m u l a t e d by b o t h m i t o g e n i c l ec t in s

a n d I L 2 in a c t i v a t e d T cells; (2) IL2 a n d c-los

e x p r e s s i o n a r e i n d u c e d p r i m a r i l y by m i t o -

gen i e lee§ a n d (3) t r a n s f e r r i n r e c e p t o r , c-

myb, a n d h i s t o n e gene e x p r e s s i o n a re s t i m u -

l a t e d p r e d o m i n a n t l y by IL2 [13, 45]. Thus ,

m i t o g e n i c l ec t ins a n d IL2 i n d u c e e x p r e s s i o n

o f o v e r l a p p i n g b u t n o n i d e n t i c a l sets o f genes

in a c t i v a t e d T l y m p h o c y t e s .

T h a t m i t o g e n i c lee§ a n d IL2 i n d u c e

e x p r e s s i o n o f s o m e o f the s a m e genes is c o n -

s i s t en t w i t h e v i d e n c e t h a t c o r n m o n a l i t i e s ex-

ist in s ignal t r a n s d u c t i o n p a t h w a y s u s e d by

m i t o g e n i c l ec t ins a n d IL2. D e s p i t e these

s i m i l a r i t i e s in l ec t in - a n d I L 2 - i n d u c e d

e v e n t s , d i f f e r e n c e s in s ignal t r a n s d u c t i o n

m e d i a t e d by r e c e p t o r s fo r m i t o g e n i c l ee / in

lnhibitors as Tools for Investigating T Cell Activation 105

Table V. lmmunosuppressive lymphokines and cytokines that inhibit T lymphocyte proliferation

Factor Producing cells Mr pl Cytotoxic Ref.

Inhibitor of DNA synthesis T cells 80 kd 3.4 - 11 (20-kd subunit)

Lymphotoxin T ceils and MO 60-70 kd + 65 (25-kd subunit)

Tumor necrosis factor M~ 30-40 kd 5.0 + 66 (17-kd subunit)

Transforming growth factor-13 T ceils and most 25 kd 10.0 - 67 other cells

T leukemia-derived suppressor leukemic T cell 90 kd 6.4 - 68 lymphokine lines (45-kd subunit)

Soluble immune suppressor T cells or Mr 30-45 kd ND - 50 supernatant of T cell proliferation

Histamine-induced suppressor T cells 25-40 kd ND - 69 factor

Nonspecific inhibitor of DNA T cells 40-60 kd 6.4-6.9 - 70 synthesis

ND - Not determined; () = noncovalently associated subunits; MO = monocyte/macrophage cells.

and for IL2, whether quantitative or qualita- tive, also exist, since: (1)CsA markedly inhibits c-rnyc mRNA accumulation induced by mitogenic lectins in activated T cells but

has far less effect on IL2-induced gene expression; (2) IL2 induces little or no increase

in the levels of c-fos and IL2 mRNA in activated T cells, and (3) mitogenic lectins fail to induce detectable expression of c-my& transferrin receptor, and histones in cloned cyto- lytic T cells that to not produce IL2 [13, 27,

45, and unpubl, data]. Though many questions remain unanswered, the use of inhibitors of T cell proliferation has begun to pro- vide clues about the mechanisms of signal

transduction utilized by receptors for mitogenic lectins and IL2.

Inhibitory Lymphokines

Besides pharmacological or synthetic inhibitors of T cell proliferation, a number of

soluble peptide and protein factors have been described that possess immunosuppressive activity. Many of these soluble factors

are secreted in cultures of lymphocytes stimulated with mitogenic lectins, and are hy- pothesized to play a role in down-regulating

normal immune responses both in vitro and in vivo. Table V gives a partial listing of lymphokines and cytokines known to inhibit T cell proliferation in an antigen-nonspecific

fashion. Each of these molecules appears to be distinct, based on biochemical and functional criteria, though formal proof awaits

106 Reed/Nowell

cloning and sequencing of their genes. Un- like the synthetic inhibitors described previously, relatively little is known about the mechanisms by which these soluble factors suppress the growth of T cells.

Together with Jegasothy and coworkers, we have begun to characterize the inhibitory effects of one of these immunosuppressive factors, inhibitor of DNA synthesis (IDS). IDS is produced by ConA-stimulated T lymphocytes and has been purified to apparent molecular homogeneity [ 11]. Previous investigations of IDS by Jegasothy et al. [46] had revealed that this immunosuppressive lymphokine activates adenylate cyclase in the membranes of PHA-stimulated PBMC. How levels of cyclic adenosine monophosphate (cAMP) relate to the interleukin pathway of T cell activation, however, is unknown. For this reason, we investigated the effect of IDS on IL2 production and on IL2 receptor expression by human lymphocytes stimulated by the mitogenic lectin PHA.

As reviewed in table I, experiments with IDS determined that this immunosuppressive lymphokine has no inhibitory effect on the production of IL2 or on the expression of receptors for IL2 and for transferrin in cultures of PBMC stimulated with PHA. These findings (table I) [22] suggest that human IDS has its inhibitory action late in G~ phase of the cell cycle, after the IL2-dependent stages of T cell activation [47]. This conclu- sion is consistent with data from a number of previous investigations, including: (l) cell cycle studies using synchronized murine L cells, which demonstrated that rat IDS blocks growth of L cells 4 h before entry into S phase [24]; (2) related kinetic studies measuring increases in adenylate cyclase activity induced by IDS in lymphocyte membrane preparations that revealed that T lympho-

cytes stimulated by PHA become responsive to IDS just 8 h before the onset of DNA synthesis [46]; (3)experiments demonstrating that IDS inhibits proliferation even when added 36 h after the initiation of mitogen- stimulated lymphocyte cultures [46], and (4) observations that IDS inhibits DNA synthesis induced by PHA, yet has negligible effects on the increase in protein and RNA synthesis that occurs during G~ phase of the cell cycle following exposure of lymphocyte to this mitogenic lectin [48]. The combined data therefore suggest a role for cAMP in regulating critical events of T cell activation during late G~ phase of the cell cycle. These findings also demonstrate that, unlike many of the immunosuppressive agents studied to date, IDS inhibits human T lymphocyte proliferation through an IL2-independent mechanism.

In addition to IDS, we were also inter- ested in another of the immunosuppressive factors in table V, "soluble immune suppressor supernatant of T lymphocyte proliferation' (SISS-T). Interestingly, this factor shares many properties with the inhibitory lectin WGA, including ability to bind molecules containing N-acetyl-D-glucosamine and competition for binding sites on human T lymphocytes [49]. For this reason, we initiated studies with WGA to delineate its inhibitory mechanism as a model for how SISS-T might act. These investigations, in collaboration with Robb and Greene, demonstrated that WGA binds to the IL2 receptor. Through an undetermined mechanism, this inhibitory lectin prevents the maintenance of high-affinity IL2 binding sites on the surface of activated T cells, thereby impairing proliferation in WGA-containing cultures without inhibiting IL2 production (table I) [20]. Additional studies in collabo-


ration with Taylor showed that inhibition by WGA was direct and did not involve the induction of suppressor T cells or the elaboration of endogenous immunosuppressive soluble factors [50]. Whether these findings with WGA bear any semblance to the mechanism of inhibition by SISS-T remains to be determined.

Summary and Future Directions

In this chapter we have described our findings regarding a variety of immunosuppressive molecules that inhibit the proliferation of normal T lymphocytes. Investigation of the mechanisms of action of these agents has revealed several phenomena of importance for the regulation of T cell proliferation. For example, studies of OKT1 IA and other anti-p50 antibodies [12, 15], as well as experiments using DEX and anti-Tac antibody [17, 25], helped demonstrate that IL2 can up-regulate expression of its cellular receptor on activated T lymphocytes. Similar- ly, contrasting the inhibitory properties of CsA with DEX demonstrated that IL2 receptor expression can be insufficient for responsivity to IL2 and suggested that other unknown molecular events are necessary for inducing 'competence' for IL2 in T lymphocytes [ l 7]. As another example, adding CsA to cultures of cloned T cells hinted that c- myc gene expression is regulated by two pathways [27]. In the case of IDS, our work provided further evidence for an IL2-independent phase of T cell activation occurring late in G~ phase of the cell cycle [47], estab- lished that not all down-regulators of T cell proliferation act by interfering with IL2- independent events, and finally suggested a possible role for cAMP in regulating very late events of T cell activation [22].

Taken together, the data presented here illustrate the usefulness of these inhibitor)' agents for investigating basic questions of T lymphocyte activation and proliferation. Clearly, the list of inhibitors described here is not exhaustive. Several other immunosuppressive molecules, including prostaglandins, histamine, ct-l,25-dihydroxyvitamin D, cyclophosphamide, somatostatin, sero- tonin, carcinoembryonic antigen, and acidic isoferritins, have inhibitory effects on T cells and could potentially serve as tools for investigating various molecular and cellular events involved in T cell activation and proliferation.

Though the immunosuppressive agents discussed here have been useful in dissecting the pathways ofT cell activation and growth, in some cases their use may be hampered by their capacity to interfere with more than one event in T cell activation. Because WGA binds to numerous glycoproteins on the surface of T lymphocytes, for example, we cannot exclude the possibility that it has other effects besides the inhibition of high-affinity IL2 receptor expression. Similarly, comparison of the ability of IL2 to restore proliferation in PBMC cultures inhibited by either CsA or by DEX reveals that CsA has an additional inhibitory effect absent in DEX [17].

At present, monoclonal antibodies represent the most specific reagents for selective inhibition of various molecular events involved in T cell activation and proliferation. Monoclonal antibodies, however, have limitations. The anti-Tac antibody, for instance, competes poorly with IL2 for high-affinity IL2 binding sites on activated T ceils and, particularly in circumstances where IL2 is produced locally, as in PHA-stimutated cultures, it inhibits proliferation by only about

108 Reed/Nowell

50% [12, 51]. A similar problem has been reported for most anti-IL2 antibodies because they only partially neutralize the biological activity oflL2 [52]. Thus, more effec- tive monoclonal antibodies are needed for use as specific inhibitors of IL2 and IL2 receptors. In this regard, the use of synthetic peptides as immunogens may allow develop- ment of better monoclonal antibodies that recognize the active sites of molecules such as IL2 and IL2 receptors.

Besides difficulties in generating neutral- izing monoclonal antibodies, these reagents have other limitations. In particular, they are not useful as tools to inhibit the functions of intracellular proteins (e.g., the proto- oncogene products), except in the case of microinjection which is at present impossi- ble in lymphocytes [53]. Techniques of DNA transfection or use of retroviral expression vectors may be of use in this latter regard. By introducing into T cells DNA sequences en- coding anti-sense copies of genes, one could selectively ablate particular molecular events of T cell activation and assess their impact [54]. Future efforts should therefore be directed toward solving technical problems of introducing macromolecules into normal lymphocytes and toward perfecting newer technologies such as anti-sense oligonucleo- tides [55].

Though many questions remain to be an- swered about the inhibitory agents discussed here, the combined data (table I) indicate that the proliferation of T lymphocytes can be down-regulated at numerous points in the sequence of events that follows stimulation with antigen or mitogens. The capacity to inhibit T cell growth through several mechanisms could be of physiological importance in regulating in vivo immune responses. For example, suppressing the initiation of T cell

activation (as by OKT1 IA, CsA, and DEX) could be important for maintaining the antigen specificity of immune reactions and could help to ensure tolerance to self antigens. In contrast, inhibiting later events of T cell activation and proliferation (as by WGA and IDS) potentially could play a role in down-regulating ongoing immune responses, thereby ensuring that T cell proliferation does not continue uncontrollably. Defects in these control mechanisms could play a role in the pathological conditions of immunode- ficiency, autoimmune disease, and neopla- sia.

What is the evidence that our studies of these inhibitory reagents may be applicable to in vivo situations? The sheep erythrocyte receptor (E-receptor), for example, is one of the earliest surface antigens acquired in the thymus and has been postulated to play a role in cell-cell interactions between T cells and non-T ceils [56]. In fact, recently, it has been shown that this surface antigen can spe- cifically bind to a membrane glycoprotein termed LFA-3, that is ubiquitously distrib- uted throughout tissues [57]. In addition to cell surface ligands for the E-receptor, soluble factors obtained from the serum of pa- tients with various disease conditions have been described that block E-rosette formation and impair T cell proliferation. Studies by Hann et al. [58], for example, demonstrate that some tumors secrete acidic isoferritins with these inhibitory properties. Thus, competition between stimulatory and suppressive soluble factors that act through the E-receptors may play a role in modulating in vivo immune responses.

As concerns CsA and DEX, at concentrations attainable iatrogenically, these agents impair several T cell activation events induced by PHA in vitro [9, 60]. With regard


to WGA, this lect in shares m a n y characteris-

tics with SISS-T, a factor secreted in cul tures

of PBMC s t imula ted with ConA [49]. Thus ,

W G A may m i m i c the effects o f an endoge-

nous inhibi tor . Final ly, concern ing IDS, pro-

duc t ion of this l ymphok ine by rat spleno-

cytes has been demons t r a t ed in an in v ivo

mode l of an t igenic compe t i t ion [61].

The above facts argue that the inh ib i to ry

m e c h a n i s m e lucidated for O K T I 1 A ant i -

body, CsA, DEX, WGA, and IDS are of

physiological relevance. Fu tu re invest iga-

t ions of these and other inhibi tors , and their

use as tools for address ing ques t ions abou t

i m m u n e responses, should con t inue to shed

light on the mechan i sms that regulate the

ac t iva t ion and prol i fera t ion of T lympho-

cytes, and perhaps suggest add i t iona l ap-

proaches to cl inical i n t e rven t ion in disorders

of i m m u n e regulat ion and lymphocyte

growth.

Acknowledgements

For their important roles in these investigations, we thank our many collaborators including Drs. W.C. Greene, R.G. Hoover, B.V. Jegasothy, M. Kamoun, M.B. Prystowsky, and R.J. Robb. We also thank the numerous investigators who supplied various reagents, as well as M.J. Larsen for artwork, W. Fore for photography, and L. Delpino for manuscript prepara- tion.

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Dr. John C. Reed Department of Pathology and Laboratory Medicine School of Medicine University of Pennsylvania Philadelphia, PA 19104-6082 (USA)

soluble inhibitors of t lymphocyte proliferation: tools for dissecting pathways of t cell activation

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