Definition of discrete signals involved in human T-cell activation

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Molecular Immunoiog!. Vol. 23. No. I I. pp. 1157-l 163. 1986 Printed in Great Britain.

DEFINITION OF DISCRETE IN HUMAN T-CELL S.

MEuER,t M.

HAUER, U.

MOEBIUS, E. SCHIEDHELM, K.

and K.-H. I. Med. Klinik

SIGNALS INVOLVED ACTIVATION* DEUSCH, M.

SCHYKOWSKI

MEYER ZUM B~SCHENFELDE

und Poliklinik, Johannes Gutenberg-Universitlt

Mainz, 6500 Mainz, F.R.G.

(Received 12 June 1986) Abstract-Mitogenic activities of monoclonal antibodies directed at defined receptor structures expressed on the surface of mature human T lymphocytes were employed to study. in detail, signals involved in primary T-cell activation. Based on differential requirements for stimulation, two discrete pathways of human T-cell activation can be defined: the antigen-induced mode of activation initiated through the Ti-T3 antigen-receptor complex and an alternative pathway which can be triggered by monoclonal antibodies directed at the Tl 1 glycoprotein. Perhaps more importantly, the approach taken here allows

the definition of stable intermediate cellular stages within the activation cascade and, thus, to analyze the signalling capabilities of individual receptor molecules.

INTRODUCTlON

A number of recently developed technologies, in combination, have allowed to collect new information on the biological significance of membrane molecules expressed by human T lymphocytes. These technologies include: (1) the generation of monoclonal antibodies directed against cell surface molecules (K6hler and Milstein, 1975; Reinherz et al., 1979); (2) the propagation of untransformed human T lymphocytes for prolonged periods of time in vitro with stable function and phenotype (Morgan et ai., 1976; Reinherz et af., 1983); (3) the production of pure sources of secreted cellular products (i.e. lymphokines) by recombinant technologies (Taniguchi et al., 1983; Rosenberg et al., 1984). In addition, functional in vitro systems exist which can be employed to dissect the complex cellular interactions underlying immunological responses in both physiologic and pathologic states (Reinherz and Schlossman, 1980). Thus, it is now accepted that specific-antigen recognition by T lymphocytes, an essential step for the initiation of specific immune responses, is mediated by a molecular membrane complex which is composed of glycoproteins bearing variable and constant components (Meuer et al., 1983~~; Acute et al., 1983). The former are expressed on both chains of Ti, a 90,000 disulfide-linked heterodimer. Moreover, Ti is non-covalently associated in the membrane with at least three additional non-polymorphic molecules, termed T3-complex (Meuer et al., 1983~; Borst et al.,

*Supported by a grant from the Deutsche For31 I-Immunpathoschungsgemeinschaft (A3/SFB genese). tAuthor to whom correspondence should be addressed.

1983). Whereas Ti mediates antigen contact, the T3 subcomponents are believed to be involved in signal transduction into the intracellular compartment. Finally, since T3 and Ti are generally co-expressed, one or the other T3-subunit may be responsible for expression of the regular T3-Ti antigen-receptor in the T-cell membrane (Royer et al., 1984; Weiss and Stobo, 1984). Monoclonal antibodies that were produced against T3 and Ti, respectively, are capable of competing with specific antigen for the binding site, i.e. to block antigen induced T-cell responses in vitro (Meuer et al., 1983~~). Perhaps more importantly, the same antibodies when coupled to the surface of a solid support, such as Sepharose beads, exert antigen-like effects in that they induce T-cell activation with regard to clonal proliferation and the production of immunoregulatory mediators (Meuer et al., 1983b,d). In addition to this antigen induced pathway of T-cell activation human T lymphocytes can be triggered in an antigen-independent fashion by simultaneous incubation with combinations of monoclonal antibodies reactive with individual epitopes of a T lineage restricted 50,000 glycoprotein, termed Tl 1 (Meuer et al., 1984b). The latter represents the earliest known T-cell specific surface molecule expressed during ontogeny and is maintained on thymocytes, resting, and activated T lymphocytes (Reinherz et al., 1980). Although a natural ligand that engages this “alternative pathway of T-cell activation” is not yet identified, it is believed that T-cell activation via Tl 1 is of considerable biological significance. The ligand-like agonistic effects of the above mentioned sets of monoclonal antibodies (anti-Ti, anti-T3 vs anti-T1 1) can now be used to study directly the signalling capabilities of the various molecules involved in T-cell activation.

1157

S. MEIJERet al.

1158 MATERIALS

AND METHODS

Derivation of lymphocyte populations

Peripheral blood mononuclear cells (PBMC) were obtained by Ficoll-Hypaque density centrifugation of blood from healthy donors (ages 20-40 yr). Subsequently, PBMC cells were plated onto glass petri dishes at 2.5 x IO6cells/ml and incubated for 2 hr at 37”C, 7% C02, humified atmosphere. Nonadherent cells were recovered and separated into E-rosette-positive (E +) and E-rosette-negative (E -) populations with 5% sheep erythrocytes. The rosette mixture was then layered over Ficoll-Hypaque and the recovered E+ pellet treated with ACK-buffer to lyse red blood cells. The T-cell population obtained was 90% reactive with anti-T3 and 95% reactive with anti-T1 1 monoclonal antibodies. To further enrich for T lymphocytes, the E+ population was treated with two cycles of the following protocol: a mixture of monoclonal antibodies anti-MO1 (Todd et al., 1981) and anti-12 (Nadler et al., 1981) (specific, respectively, for a monocyte lineage antigen and a constant portion of human Ia antigens) for 1 hr at 4°C. Subsequently, rabbit complement was added and the suspension incubated for 1 hr at 37°C in a shaking water bath. Cells were then washed twice, incubated overnight at 37°C 7% CO, humified atmosphere and utilized as purified resting T cells (Ta) in the various experiments. A human alloreactive IL-2 dependent T-cell line (Meuer et al., 1982), propagated in vitro for 2 months was employed as a source of “activated T lymphocytes”. Prior to use in the experiments these cells were propagated for 1 week in recombinant Interleukin-2 containing medium and, in order to avoid contamination with monocytes, in the absence of feeder cells.

Monoclonal antibodies anti-T3B, anti-T1 1, antiTll, (Meuer et al., 1984b) and anti-IL-2 receptor (lHT4-4H3) were kindly provided by Dr E. L. Reinherz (DFCI, Boston, MA). Antibodies anti MO-1 (Todd et al., 1981) and anti-12 (Nadler et al., 1981) were a generous gift of Drs R. F. Todd and L. M. Nadler, respectively (DFCI, Boston). In addition, a series of monoclonaf antibodies character&& by the “Second International Workshop on Human Leukocyte Differentiation Antigens” (Ab No, 135:TIA; Ab No. 141:Bl49,9; Ab No. 143:23A9.3; 145339C6.5; Ab No. 14633Bl.l; Ab No. 153:AA3; Ab No, 159: anti-Tat; Ab No. 140: I HT4-4H3) was employed to study the expression of IL-2 receptors on T lymphocytes by means of indirect immunofluorescence. The iatter anti,bodies were all grouped within the same cluster as anti-Tat. All antibodies were used in ascites form and employed in saturating concns.

Purification antibodies

and

surface

coupling

of monoclonal

Monoclonal antibodies anti-T3B and anti-T1 lZ were purified employing Sepharose-Protein A (Pharmacia, Uppsala, Sweden) according to the method of Ey et al. (1978). Following purification, antibodies were individually coupled to CnBr-activated Sepharose 4B (Pharmacia) at a concn of 3 mg of purified antibody per ml of swollen Sepharose beads. The amount of antibody coupled per bead was similar for all reagents produced. Lymphokines and mitogens

Affinity purified human Interleukin-1 was purchased from Genzyme Lab. (Haverkill, U.K.). Recombinant human Interleukin-2 was a generous gift of Sandoz Research Institute (Vienna, Austria). PHA-P was purchased from Wellcome Lab. (Burgwedel, F.R.G.) and phorbol-myristate acetate from Sigma (St. Louis, MO). Proit~erative assays

Responder cells (3 x 104) were incubated in the presence or absence of 5% adherent cells (600 rad irradiated) in round bottomed microtiter wells (Costar, Cambridge, MA) in 200 ~1 of final culture medium RPM1 1640, supplemented with 10% fetal bovine serum (Gibco, Paisley, U.K.) 1% ~nicillin-streptomycin and 2% glutamine (both purchased from G&co). To determine proliferative responses to Sepharose-linked monoclonal antibodies, beads were suspended at dilutions previously determined to yield optimal proliferative responses of Ficoll-Hypaque separated PBL. Following 3 days of in vitro culture, wells were individually pulsed with 1 HCi of ‘H-thymidine for 16 hr and mashed employing a Titertek cell harvester (Flow, M~kenheim, F.R.G.). 3H-thymidine uptake was measured in a Packard liquid scintillation spectrometer (Packard Instrument Co. Inc., Downers Grove, IL). RESULTS

In the present study we have tried to dissect discrete signals involved in human T-cell activation by taking advantage of agonistic effects of monoclonal antibodies reactive with the T3-Ti T-cell antigen-receptor complex (Meuer et al., 1983d) or, alternatively, the Tll glycoprotein (Meuer et al., 1984b). The molecular characteristics of these surface molecules through which human T cells can be activated are described in Table 1. Employing, for example, monoclonal antibodies reactive with T3, a monomorphic subunit of the T-cell antigen receptor, for T-cell triggering avoids the ambiguities inherent in previous studies in which cell-cell interactions or lectins (PHA, ConA) were used.

Signals

involved

in T-cell

activation

1159

Table I. Surfwe structures involved in human T-cell activation T-cell surface Mol. wt molecule ~._____ T3 20,000/ All mature T 20.000~ thymocytes 25.000

Ti

Tl

I

55.oiw

Distribution -._ lymphocytes and a majority

Functional effects of monoclonal antibodies IO the structure of

(I) Inhibits antigen-specific T-ceh proliferation and cytotoxic effector function of CTL. (2) Enhances IL-2 responsiveness. (3) Modulates by external shedding. (4) Triggers polyclonnl T-cell activation when surface linked in an antigen-like fashion.

Specific for an individual T-cell clone (clonotypic). Similar disulfide-linked heterodimers are expressed on all peripheral T lymphocytes and T3+ thymocytes

(1) Identical to anti-T3 effects but inhibits response only of the individual clone with which it reacts.

All cells of the 7 lineage (i.e. thymocytes. resting and activated T lymphocytes). Three individual epitopes (Ti I,. Tl lr. 71 I,) can be defined by monoclonal antibodies

(1) The combination of anti-Ttl,+ anti-Tii> is mitogenic for T lymphocytes m the absence of monocytes and/or IL- I. (2) Anti-T1 I, inhibits E-rosette formation with SRBC.

“Non-reduced state; reduces to mol. WI 41.000-43.000

(2) Co-modulates with T3. (3) Triggers individual T-cell clone in an antigenlike fashion.

(bela chain) and 49.000-53.000

Since monocytes are known to contribute at one or the other level of the activation process, it was first necessary to obtain a preparation of monocyte depleted pure resting T lymphocytes from peripheral blood of healthy individuals. This was achieved by Ficoll-Hapaque sedimentation, adherence on glass Petri dishes, E-rosetting and complement lysis employing monoclonal antibodies directed at human monocytes and Ia antigens. The latter antibody was utilized to allow a more vigorous depletion of monocytes and at the same time eliminate Ia positive “preactivated” circulating T lymphocytes. Table 2 demonstrates that in the course of the purification procedure monocytes are efficiently removed from the T-cell preparation as judged by the loss of responsiveness of the latter to PHA (27.845 vs 1.054 cpm 31-I-thymidine uptake). Perhaps more importantly, there was also a complete loss of proliferative T-cell responses to anti-T3Sepharose (16.31I vs 686cpm). Proliferation to both stimuli could be fully restored by addition of 5% autologous monocytes (adherent cells; 5000 rad irradiated). Note that in the control experiment, anti-T1 1, covalently linked to Sepharose had no effect on either monocytedepleted or monocyte-reconstituted TR indicating that proliferation to Sepharose-anti-T3 was de-

(alpha-chain).

pendent on functional anti-T3 antibody present on the bead surface. Thus, in striking contrast to activated T l~phocytes (TA) (Table 2) antigen-receptor oligomerization, referred to as signal 1, is in itself not sufficient to trigger proliferation of T,. In a next set of experiments the nature of signal 1 was further investigated. We analyzed whether, in addition to surface binding of anti-T3 monoclonal antibody, T-cell receptor oligomerization is indeed critical for T-cell activation. To this end, functional effects of soluble vs multime~c anti-T3 were compared on monocyte reconstituted T,. As demonstrated in Fig. 1, both reagents were capable of producing a proliferative in vitro response when the standard culture system was employed [Fig. 1, upper panel (B) and (C)l. However, in the presence of high serum concns (20% human AB serum) and a pool of unrelated monoclonal antibodies (the combined panel of anti-T6 monoclonals submitted to the Second International Workshop on Human Leucocyte Differentiation Antigens; 1: 100 final concn) the mitogenie activity of soluble anti-T3 was almost completely inhibited (4.879 vs 973 cpm) [Fig. 1, lower panel (B)]. In contrast, this treatment did not influence PHA responses (not shown) and, perhaps more importantly, did not reduce the capacity of

Table 2. Activation of resting T lymphocytes by monoclonal antibodies via T3-Tj requires an accessory cell dependent signal Responder population PBL EC E+ (Ab+C)=T, E+ (Ab+C)+MO Blasts = T,

Medium 0.483” 0.684 0.892 0.732 0.513

a-T3-Sepharose

Stimulus a-T1 I-Sepharose

16.31 I 2.688 0,686 23.835 8.816

0.389 0.572 0.486 0.601 0.321

PHA 27.845 24.381

I .054 24.935 1.973

‘3 x 10’ responder cells/well were incubated with various stimuli (anti-T3Sepharose. antiTl I-Sepharose. PHA 0.25 pgjmt) or medium m microtiter plates for 3 days and then pulsed for 18 hr with I rCi ‘H-TdRfwell. Results are given as cpm of ‘H-TdR uptake. SD. was ~15%.

1160

0 A

Ei

C

D

Fig. 1. T-cell receptor triggering requires T3-Ti oligomerization. Monocyte reconstituted T, were incubated with medium (A), soluble anti-T3, (ascites, final concn I:300 in medium) (B). anti-T3sSepharose (C). Sepharoseanti-TlIr,(D), in the standard 3 day culture system and proliferatton determined by means of ‘H-TdR uptake. Upper panel: medium supplemented with 10% human serum. Lower panel: medium supplemented with 20% human serum plus a pool of unrelated monoclonal antibodies (ascites, final concn I : 100).

As shown in Fig. 2, triggering of monocytedepleted Ta by Sepharose-anti-T3 induced receptiveness to Interleukin-1 as indicated by a dosedependent proliferative in vitro response. Although not shown, proliferation to IL-1 was only observed when T cells were first treated with antiT3-Sepharose but not vice versa indicating that responsiveness to signal 2 (IL-l) occurs as a consequence of the initial signal I, i.e. antigen-MHC or anti-T-cell receptor-antibody binding (in multimeric form) by the T3-Ti antigen-receptor complex. Since previous studies firmly established the concept that T-cell proliferation is mediated through a hormonal system based on the activity of Interleukin-2 and the presence of Interleukin-2 receptors (Morgan et al., 1976; Smith et al., 19806; Cantrell and Smith, 1983; Leonard et al., 1982) it was necessary to investigate the effects of the above defined signals 1 and 2 on the activation of the IL-2 hormonal system. It was reasoned that if antigen-receptor triggering (signal 1) which in itself did not lead to proliferation of T, was at all related to this system, then signal 1 could only induce its partial activation, e.g. IL-2 receptor expression without IL-2 secretion or vice versa. If the former were the case then incubation of ‘Ta with Sepharose-anti-T3 should induce responsiveness to Interleukin-2. To test this notion, a source of recombinant human Interkeukin-2 was employed and incubated with untreated or anti-T3-Sepharosetreated T,. Again, anti-Tll,-Sepharose served as a control. As shown in Table 3, untreated Ta do not proliferate to Interleuk~n-2. However, following incubation with anti-T3-Sepharose but not anti-

Sepharose-bound anti-T3 to induce T-cell proliferation [Fig. 1, lower panel (C)l. Thus, this experi-

ment in which Fc-receptor mediated multimeric surof the anti-T3 monoclonal is face binding competitively inhibited provides further support for the view that multimeric ligation of antigen-receptors is a critical requirement for efficient antigen recognition by T cells. In addition, this experiment excludes the possibility that the mitogenic effects observed with Sepharose-anti-T3 were due to antibody leaking from the bead surface. A number of previous studies had suggested that Interleukin-I, a soluble monocyte product, plays a role in primary T-cell activation (Larsson et al., 1980; Smith et al., 1980a; Farrar et al., 1980; Wakasugi et at., 1984; Chu et al.. 1985; Oppenheim et al., 1982). To analyze this point, a source of affinity purified human Interleukin- I was utilized to investigate whether this lymphokine instead of adherent cells would be capable of serving as signal 2 in the response of resting 7, lymphocytes to Sepharoseanti-T3. Thus. 7, in the presence of Sepharoseanti-T3 were incubated with varying concns of Interlcukin- I and proliferation determined in the standard 3 day culture system.

IO

IA

~ 6-T

b

T/I

PPTPPP 015

03

06

I2

25

5

10

i?fl

Fig. 2. In vitro responses of human T lymphocytes (Ts) to Interleukin-I. Purified resting T cells were incubated with anti-T3-Sepharose (0). anti-T1 I:-Sepharose (0) or medium (m) in the presence of varying concns of affinity purified human fnterleukin-1. Following 3 days, cultures were individually pulsed with ‘H-thymidine and harvested 18 hr later. Results are expressed as means of triplicate cultures with ranges of maximal and minimal levels of ‘H-TdR incorporation.

Signals involved in T-ceil activation Table

3. Antrpn-receptor

Responder pop&lion TX T, + IL-2 (20 ng/ml) T, + IL-2 (4 ng:ml)

1161

triggering of resling T lymphocytes induces responsiveness to Interleukin-2 Slimulus Anti-T1 I Sepharose

Anti-T1 1: + anri-TI I,

539

491

18.647

457

12,803

600

n.t.

553

6982

565

rl.L.

Medium

Anti-T3Sepharose

430”

“‘H-thymidine incorporation. S.D. < 15%. Purified resting T lymphocytes (3 x 104/well) were incubated with the various stimuli or medium in the presence or absence of recombinant human Interleukin-2. Cultures were treated as described in the footnote to Table 2.

Tl 1,Sepharose.

a dose-dependent proliferation to IL-2 occurred. Although not shown, IL-2 responsiveness was only observed when anti-T3 was added in Sepharose-linked form but not to soluble anti-T3 antibody, provided the experiment was carried out under conditions where Fc receptor activity could be excluded (compare Fig. 1). Moreover, no IL-2 activity could be detected in supernatants collected from Sepharose-anti-T3 treated TR at various points of time (up to 72 hr following initiation of culture). We therefore conclude that signal 1 (T-cell receptor oligomerization?) serves to induce receptiveness to Interleukin-1 and Interleukin-2 whereas signal 2, i.e. most likely IL-l, is required to trigger IL-2 production and/or secretion. As demonstrated above (Table 2), and by earlier studies, the latter does only apply to Ta and clearly not to presensitized T lymphocytes (T,) as investigated here. Table 3 also demonstrates that in the absence of monocytes T, can be triggered to proliferate when incubated with two monoclonal antibodies directed at distinct epitopes of the 50,000 Tll sheep erythrocyte receptor glycoprotein (termed Tl 1z and Tl 1J, respectively (Meuer et al., 1984b). This mode of human T-cell activation was recently discovered and suggested to represent a previously unknown “alternative pathway” of human T-cell activation (Meuer er af., 19846) which in marked contrast to activation via the T3-Ti antigen-receptor complex circumvents the requirement of accessory cells and/or IL-l in a primary immune response.

either invariant components (i.e. T3) or variable determinants (i.e. Ti) of the human Ti-T3 antigenreceptor complex mimic effects that are indistinguishable from those mediated by the natural ligand of Ti-T3, namely antigen itself (Meuer et ul., 1983b.d. 1984~; Reinherz et al., 1982). In addition. functional activities of, respectively, anti-T3 and anti-Ti were found to be identical, thus, supporting the notion that T3 and Ti are intimately linked to each other in the membrane of all mature human T Iymphocytes. The view which emerges from the present analysis is that activation of resting human T lymphocytes occurs as a series of precisely orchestrated events which are regulated by discrete signals mediated via surface receptors. Based on this information it is possible to construct a unifying model of the activation cascade (Fig. 3) in which a physical interaction between T cells and antigen presenting celts (signal 1) initiates all subsequent steps. This interaction may have to result in T-cell receptor oligomerization. since soluble ligands of the T3-Ti molecular complex can not efficiently mediate signal 1 (Fig. I). T-cell receptor triggering of resting lymphocytes induces responsiveness to both IL-l and IL-2 but does not lead to T-cell proliferation. An additional signal provided by monocytes is required for the initiation of DNA synthesis and mitosis. The present report suggests that one contribution of monocytes in

Antigen mduced pathway DISCUSSION

in the present paper we have investigated signals involved in T-cell activation in primary immune responses. So far, such studies have been largely hampered by the requirement of Ia bearing antigen presenting cells that, in addition to their physical interaction with T lymphocytes, release soluble mediators. Thus, it was virtually impossible to accurately determine the prerequisites for resting T-cell prohferation even if homogenous populations of responding T cells were utilized. The approach taken here was based on recent findings which demonstrated that monoclonal antibodies reactive with

1Alternatwe pathwayj

Fig. 3. A model of human T-cell activation.

1162

S. MEUER er al.

this process is to elaborate a soluble product, possibly IL-l (i.e. signal 2 in Fig. 3). Moreover, receptiveness

to IL-l is a consequence of signal 1. The function of IL-l in primary T-cell activation appears to be the engagement of IL-2 production and/or secretion by T lymphocytes since functional IL-2 receptor expression is achieved in the absence of IL-l. Given that T-cell proliferation only occurs following binding of IL-2 to its surface receptors, IL-2 has to be considered the third signal necessary for expression of the functional T-cell repertoire. Note that human T cells can be experimentally arrested at each of the various activation steps (Fig. 3). For example, in the absence of monocytes, antigen-r~eptor t~ggering {signal I) transfers the resting T lymphocyte (T,) to stage TA,. A next stable intermediate activation stage can be reached by addition of IL-l (or in the presence of monocytes) provided, monoclonal antibodies to the IL-2 receptor are employed to prevent specific binding of IL-2. This step T,* could be defined as the IL-Zproducing non-proliferating T Iymphocyte. Finally, following IL-2-IL-2-receptor binding, DNA synthesis and mitosis are initiated and T cells begin to express, e.g. Ia antigens as a sign of full activation (stage TA3). As previously shown (Meuer et al., 1984~) in the absence of further antigenic restimulation, T cells in culture lose responsiveness to IL-2. This does, however, not mean that these cells now revert to stage Ta for a number of reasons: firstly, Ia antigens are still detectable on the surface; and secondly such cells differ markedly from stage T, lymphocytes with regard to their restimulation requirements. Thus, as shown in Table 2 and in previous studies (Meuer et al., 1984a), triggering of the T3-Ti antigen-receptor in the case of Ia + T cells induces both IL-2 receptor expression and IL-2 secretion, even in the absence of monocytes and/or IL- 1. The mode of the T-cell activation via the T3-Ti antigen-receptor complex can be precisely distinguished from T-cell activation mediated through the 50,000 Tl 1 glycoprotein f”alternative pathway of T-cell activation”) (Meuer et al., 19846). Whereas the former is monocyte/IL-I dependent (Table 2 and Fig. 2), T-cell growth in response to anti-T1 12/T11, clearly occurs in the absence of accessory cell activities (Table 3). Moreover, under the experimental conditions employed here, PHA seems to act similarly to anti-T3 (Table 2), i.e. to preferentially trigger the antigen-receptor dependent pathway but not-as suggested very recently-the alternative Tl l-pathway (O’Flynn et al., 1985). Whether the latter discrepancy is due to different lectin concns employed remains to be determined but is a likely possibility. Supernatants obtained from separated adherent cells after varying periods of time did not contain IL-l activity. Therefore, our present data can only be explained by the existence of an as yet unidentified additional signal (in Fig. 3) involved in T-MO interaction which, following multimeric antigen-receptor

ligation, is directed from the responding T cell towards the accessory cell in order to engage IL-l production, Preliminary evidence exists in our laboratory that this signal requires physical T-MO interaction. In conclusion, the present experimental system provides excellent tools to precisely dissect discrete steps involved in primary T-cell activation, to study the role of Interleukin-I in T-cell differentiation and, perhaps, help to identify physiologic receptor structures for this lymphokine. Moreover, it may serve as an approach to analyze signals necessary for the generation of functional immunoregulatory and effector cells such as helper, suppressor and cytotoxic T lymphocytes. Finally, given that surface-linked anti-T3 represents a defined T-cell specific mitogen, this system should allow to gain further insight into failures underlying certain stages or primary and acquired immunodeficiency. REFERENCES

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