Springer Semin Immunopathol (1992) 13: 289 - 302

Springer Seminars in Immunopathology :~? Springer-Verlag 1992

Induction of T helper cell subsets Martin R6cken*, Kai M. Miiller, and Conrad Hauser Department of Dermatology, H6pital Cantonal Universitaire, 24, rue Micheli-du-Crest, CH-1211 Geneva 4, Switzerland

Introduction Antigen-specific immune responses are mediated by three types of cells: antigenpresenting cells (APC), T lymphocytes and B lymphocytes. CD4 § T helper/ inducer lymphocytes are considered to be the principal regulatory cells in this network. Activation of T cells in response to protein antigens is the consequence of a complex series of events. Antigen is taken up and modified (processed) by proteases within an acidic endosomal compartment of APC, such as macrophages, B cells and dendritic cells, including Langerhans cells. Antigen-derived peptides that fit into the peptide-binding groove of major histocompatibility complex (MHC) class II molecules are then expressed at the surface of APC. The complex of MHC class II molecules plus the antigen-derived peptide represents the natural ligand of the T cell receptor (TCR), the clonally distributed antigen-recognizing structure of T cells. Ligation of the TCR leads, together with other signals, to T cell activation. Other signals required for T cell activation include the co-ligation of the MHC class II molecules by the CD4 receptor, as well as the interaction of further surface molecules of both the APC and T cell. CD4 § T cells activated by APC, i. e., primed cells, are capable of antigen-specific interaction with either B lymphocytes or other antigen-loaded MHC class II § target cells. Thus, primed T lymphocytes may exert effector functions. The target cell-T cell interaction can consist of direct cell-cell contact and/or be mediated by cytokines secreted by T cells in response to activation. In contrast, nonactivated, i.e., resting T lymphocytes, are thought not to be capable of acting as effector cells. Cytokines are soluble polypeptides. They are potent hormone-like pleiotropic factors that control a wide range of biological functions in their target cells, such as cell growth and differentiation. They are usually active within a very short range and, thus, serve as cell-to-cell communication molecules. Because of their distinct effects and their high biological activity, T cell-derived cytokines, i . e . , *Present address: National Institutes of Health, National Institute of Allergy and InfectiousDiseases, Laboratory of Immunology, Building 10, Room llN311, Bethesda, MD 20894, USA Correspondence to: C. Hauser

290

M. R6cken et al.

lymphokines (LK), are considered to be important regulators of immune responses. Here we review some of the properties of LK with special reference to their function in effector T cell-target cell interaction. Since CD4 + T cells are the main source of T cell LK, we will focus on the LK produced by these cells. A brief account will be given on CD4 + effector T cell subsets that have been defined according to their LK pattern. Finally, we will review recently identified mechanisms that play a role in the differentiation of CD4 + effector T cell subsets.

Lymphokines and T helper cell function Nonprimed, resting CD4 + T lymphocytes produce IL-2 but no other LK when stimulated in vitro with T cell activators such as lectins, antigen-bearing APC, or ionomycin and phorbol esters. In contrast, previously activated CD4 + T lymphocytes may produce a large panel of LK, such as IL-2, IL-3, IL-4, IL-5, IL-6, IL-10 and IFN-3, when restimulated with the same T cell activators [5, 38, 48]. It has also become clear that, after primary activation, T cells have no effector functions other than the production of their autocrine growth factor IL-2. This is in contrast to previously activated CD4 + T cells that become capable of subserving various effector functions upon reactivation. From these observations it was inferred that the production of LK other than IL-2 may be associated with effector functions exerted on other cell types. This concept has recently been challenged [8]. Platelet-derived growth factor (PDGF) present in fetal calf serum (FCS), a routine constituent of media for lymphocyte culture, has been shown to inhibit IL-4, IL-5 and IFN--y production, and to amplify IL-2 production by freshly isolated T cells. PDGF, however, does not seem to affect the LK profile of cells previously cultured in FCS-containing media since exposure to PDGF desensitizes T cells to the effect of this growth factor. It remains to be shown whether this holds true for cells previously activated in vivo, for cells not activated in vivo, or for both.

Definition of T helper cell subsets according to their lymphokine profile In 1986 Mosmann and coworkers described that many long-term cultured CD4 + T cell clones did not produce a random pattern of LK [35]. They found that longterm cultured murine CD4 + T cell clones could be divided into two distinct subsets, according to the LK profile they secreted upon activation. One group of CD4 + T cell clones, named Thl, preferentially produced IL-2 and IFN-3, as distinct LK. The other group of CD4 + T cell clones produced IL-4 and IL-5 but neither IL-2 nor IFN-3,. This second group was defined as Th2 [35]. Extension of the analysis to further LK confirmed that the two subsets produced soluble polypeptides unique for each subset but shared the release of others (Table 1) [33, 34]. The biological significance was demonstrated in many experimental systems: the pattern of LK secreted by CD4 + T cells upon stimulation was

Induction of Thl and Th2

291

closely related to distinct functional activities both in vitro and in vivo. Later, it was reported that clones exist that produce LK typical for both Thl and Th2; they had an unrestricted LK pattern and were termed Th0 [12]. The unrestricted LK pattern of Th0 clones resembled polyclonal CD4 + T cell populations that were restimulated after primary activation. A Th0-1ike LK pattern has also recently been reported for single CD4 + thymocytes after in vitro activation [1].

The relation of lymphokine pattern and function in vitro The in vitro effector functions of CD4 + T cell clones and lines of the Thl and Th2 type have been extensively studied. Since the interaction of most CD4 + T cells is restricted to MHC class II + APC, these latter cells were chosen as targets. MHC class II § macrophages can serve as APC for CD4 + T cells. In turn, this interaction can modify effector functions including subsequent antigen presentation. Using parameters of macrophage function, it was found that Thl but not Th2 clones were capable of inducing macrophage activation (tumor cytotoxicity) [46]. This activity was inhibited by antibodies to IFN-3,, indicating that this LK was the responsible T cell-derived macrophage activating factor [46]. The capacity of macrophages to present antigen to Thl has been shown to be down-regulated as a consequence of previous interaction with Th2. IL-10, a recently cloned Th2 LK, was identified as the responsible factor [11, 34]. These findings explained how Th2 cells could indirectly influence the response of Thl cells. IL-10, however, may also directly affect Thl cells by inhibiting their LK production. IL-10 was also shown to de-activate macrophages by decreasing the production of reactive oxygen and nitrogen intermediates in activated cells [4]. When the two types of clones were tested for cytotoxic T cell activity, Th2 clones were weakly, if at all, cytolytic. In contrast, Thl clones showed both tumor necrosis factor (TNF)-dependent and TNF-independent cytotoxicity [23].

Table 1. Lymphokines produced by Thl and Th2 cells~ Lymphokine

Th1

IL-2 IFN-'y TNF-3 (LT) GM-CSF TNF-c~ IL-3 IL-4 IL-5 IL-6 IL-10 (CSIF)

++ ++ ++ ++ ++ ++

a Modified from [6] LT, lymphotoxin; CSIF, cytokine synthesis-inhibiting factor

Th2

+ + ++ ++ ++ ++ ++

292

M. R6cken et al.

The interaction of T helper cell subsets and B cells attracted a wide interest. The production of immunoglobulin isotypes by B cells was most frequently measured as a B cell response. While most Thl clones were unable to induce immunoglobulin production [26], some Thl clones with low-level IFN--y production stimulated IgG2A secretion by B cells [21, 45]. The inability of most Thl clones to stimulate immunoglobulin production was attributed to the high amounts of IFN- 7 produced by these cells, since IFN- T at high doses was previously shown to inhibit immunoglobulin production [44]. This view is supported by the observation that membrane preparations of activated Thl cells together with LK typical for Th2 were capable of inducing immunoglobulin production by B cells. In contrast to Thl clones, most Th2 clones were capable of inducing immunoglobulin synthesis in B cells [21, 26, 45]. However, the isotype pattern differed. High concentrations of IgG1 and IgE were observed. The addition of antibodies to IL-4 completely abrogated IgE but not IgG 1 production, suggesting that IL-4 was an absolute requirement for IgE production [21]. IL-4 was later shown to switch B cells from IgG1 to IgE production by inducing a rearrangement of the immunoglobulin constant region genes. IL-4 has remained until now the only known IgE-inducing factor. The requirement of IL-4 for IgE and IgG 1 production was recently shown in mice with a nonfunctional IL-4 gene. These mice showed greatly diminished serum IgE and IgG1 levels. They were also unable to mount an IgE response to parasitic infestation [27]. IL-5 and IL-6, two other Th2-characteristic LK, were shown to enhance immunoglobulin production including IgE but not to be essential for IgE synthesis [52]. Although both Thl and Th2 are very efficiently stimulated by antigen-bearing epidermal Langerhans cells to proliferate and to produce LK [20], it is at present unknown what functions they modify within this cell type that is closely related to other types of dendritic cells. It has been documented that Thl clones can be rendered nonresponsive to restimulation after interaction with keratinocytes that have been induced to express MHC class II molecules [16]. Reciprocally, it is unknown what functions are altered in nonprofessional MHC class II + APC after interaction with CD4 + T celt subsets. In conclusion, many in vitro studies demonstrated the close relation between the LK pattern of T helper cell subsets and the distinct functions they induce in target cells. In vivo occurrence and function of murine T helper cell subsets As expected from the in vitro experiments, the occurrence of Thl- and Th2-1ike cells has been found to be associated with various appropriate and inappropriate immune responses in vivo. Thl-like cells were demonstrated to be responsible for inflammatory reactions such as contact sensitivity and delayed-type hypersensitivity [6, 19, 54]. The latter was shown to be partially dependent on IFN- 7 [14]. Acute graft versus host reaction (GvH) was also shown to be mediated by CD4 + T cell clones with a Thl phenotype [29, 50]. It is conceivable that the high titers of virus-neutralizing isotype IgG2A observed in acute viral infections are induced by the activation of Thl-like cells [44]. Moreover, it has been demonstrated that the induction of Thl-like cells by infections and infestations is associated with protective

Induction of Thl and Th2

293

immunity [43]. For example, in murine disease models with Leishmania major, TrichineUa spiralis and Schistosoma mansoni, the induction of Thl cells was followed by successful elimination of the parasites. Thl cells have even been implicated in immunity against cancer since tumor-infiltrating CD4 § T lymphocytes were reported to show a predominant Thl LK pattern [37]. Conversely, chronic GvH as well as some autoimmune diseases have been thought to be mediated by Th2-1ike cells [10, 17]. In the above-mentioned parasitic diseases, the induction of an immune response characterized by predominant Th2-1ike cells has been correlated with disease progression or persistence [43]. High IgE levels, commonly observed in parasitic diseases such as infestation with Nippostrongylus brasiliensis, were shown to be decreased by the administration of antibodies to IL-4 as well as by the injection of IFN-% the natural antagonist of IL-4 with regard to IgE synthesis. IL-5, another Tn2 LK, also plays an important biological role in parasitic diseases because in Nippostrongylus brasiliensis infestation blood eosinophilia was prevented by the administration of antibodies to IL-5 [7].

T helper cell subsets in the human Initial analysis of human CD4 + T cell clones has cast doubt on the existence of Thl and Th2 subsets in man since most of them produced a Th0-1ike LK pattern. Recent reports, however, have shown that Thl- and Th2-1ike cells can be cloned from peripheral blood as well as from tissue lesions with lymphocytic infiltrations [24]. Antigen-specific clones producing a predominant Thl profile were isolated from patients with allergic contact dermatitis [24] and also from patients with bacterial diseases such as Lyme Borreliosis [55]. Purified protein derivative (PPD)-specific T h cell clones from BCG-vaccinated volunteers also had a Thl phenotype [9]. Similar observations were made in leprosy [18]. The latter disease can manifest either as tuberculoid leprosy, a condition characterized by a low bacterial count and a relatively good immunity, or as lepromatous leprosy, a form with a high bacterial count and an inefficient immune response. Most interestingly, T cells derived from skin lesions of the tuberculoid variety had a predominant Thl phenotype, whereas T cells from lesions of the lepromatous type showed a Th2 LK pattern [42, 56]. These results suggest that the LK pattern may determine the form of clinical expression in leprosy. Th2 cells were obtained from patients with parasitic infestations such as toxocariasis [9]. Th2-1ike cells also seem to play a role in atopic diseases since allergen-specific clones secreting IL-4 were prepared from atopic donors. Such clones were obtained from vernal conjunctivitis [31] and atopic dermatitis lesions [24]. Although Th2 clones were also isolated from the blood, their frequency was higher from skin lesions, indicating that allergen-specific Th2 cells accumulated preferentially at the lesional site [24]. Most of these clones were capable of stimulating IgE synthesis that was critically dependent on IL-4 production. IL-5 and IL-6, two other Th2-typical LK, were shown to amplify IL-4-induced IgE release [51]. Further support for the involvement of IL-4 in de-regulated IgE

294

M. R6cken et al.

synthesis in atopics was provided by the observation that spontaneous IgE synthesis in vitro by lymphocytes from patients with atopic dermatitis was partially inhibited by anti-IL-4 antibodies and IFN-3~ [53]. In vitro analysis of human lymphocytes, however, bears the disadvantage of possible artefacts. To resolve this problem, in situ hybridization for LK messenger RNA was performed in skin biopsies from cutaneous late-phase reactions of patients with allergic asthma [25]. Positive hybridization signals for IL-4 and IL-5 but not IL-2 and IFN- 7 were identified, suggesting a Th2-1ike response in vivo.

Lymphokine induction and modulation in vitro

Factors influencing the lymphokine profile produced by T helper cells Since Thl- and Th2-1ike cells appear to be responsible for distinct biological effects, the origin and development of these T cell populations have become major fields of immunological research. Investigations were focused on two questions: 1. What are the signals required for the induction of T h1- or Th2-1ike cells? 2. Are Thl and Th2 subsets derived from two mutually exclusive, postthymic CD4 + T cell precursors that have a predetermined LK response pattern, or are they derived from a common precursor population that has the capacity to differentiate into either Thl or Th2? Experiments with long-term cultured T cell clones did not allow the identification of the signals required for the preferential induction of Thl or Th2. Therefore several short-term culture systems have been established to determine the factors that regulate the LK pattern in CD4 + T cells. Reactivation of primed T cells resulted in the secretion of a wide range of cytokines, such as IFN-T and IL-3 to -6. Subsequently, it was demonstrated that IL-2 and IL-4 were required for the induction of IL-4 secretion [2, 3, 28]. Furthermore, in vivo experiments have shown the induction of IL-4-producing cells after exposure to anti-CD3 monoclonal antibody (mAb) [13]. While the above studies suggested that several factors may influence the production of LK, the selective signals required for the development of either Thl-like or Th2-1ike subsets were not identified by these systems. Some reports documented that the development of IL-4-producing cells by antigen plus APC was inhibited by IFN--y and that the majority of outgrowing cells had a Thl phenotype [3, 15]. However, other investigators found that IL-4-producing cells could be induced even in the presence of high amounts of IFN--y [28, 40]. In vivo experiments suggested that the natural ligands of the T cells, i.e., antigen and MHC-encoded proteins, could determine whether an animal reacts with a Thl or a Th2 response [22, 30, 36]. This is in line with the report that the antigen used for priming in vivo determined the in vitro development of antigen-specific T helper cell subsets [47]. We observed that nonprimed CD4 + T cells activated in a 3-day culture developed into Thl-like cells when subsequently cultured in medium supplemented with IL-2. These results stand in apparent contrast to a recent report in which induction of Thl-like cells was observed after culturing activated cells

Induction of Thl and Th2

295

with a combination o f l L - 2 and transforming growth factor-/3 (TGF-/3) [49]. According to these studies, however, TGF-/3 levels present in FCS-containing culture media may suffice to induce Thl-like cells in the presence of exogenous IL-2 alone. Thus, our results are compatible with the latter report. In contrast, resting CD4 + T cells differentiated into Th2-1ike cells when activated and then cultured for 2 weeks in the permanent presence of a T cell activator in addition to IL-2 [39]. Based on these findings, we have been investigating the signals that regulate the differentiation of CD4 § T cells towards a Thl or Th2 phenotype, and identified a postthymic precursor cell for T~I and Th2.

Prolonged exposure to T cell mitogens induces the development of Th2-1ike cells In vitro activated, polyclonal CD4 § T cells produced large amounts of IL-2 but little or no IL-4 when restimulated after a culture period of 2 weeks in medium supplemented only with IL-2 as a T cell growth factor (Fig. 1, Table 2). These cells therefore resembled Thl cells. This observation was largely independent of the way the T cells were primed in vitro [39]. However, repetitive in vitro stimulation of these IL-2 producing, Thl-like CD4 + T cells with antigen or polyclonal T cell activators regularly induced CD4 § T cells that produced IL-4 but no IL-2

9 I

Activation ]

\ IL-2

IL-2 + agent ]

lRestimulati~ ] Lymphoklne Production

Fig. 1. An in vitro culture system that allows the development of Thl and Th2 cells. CD4 + T cells prepared from nonimmunized mice were activated in co-culture with antigen-presentingcells in the presence ofa T cell mitogen. Subsequent culture for 10-12 days with IL-2 induced cells presenting with a Thl phenotype upon reactivation, whereas cultures with IL-2 plus a T cell mitogen present during the whole culture period yielded Th2-1ike cells

296

M . R 6 c k e n et al.

T a b l e 2. I n f l u e n c e o f the c u l t u r e c o n d i t i o n s o n the l y m p h o k i n e ( L K ) p a t t e r n o f T h cells Cell t y p e

Culture condition

LK production IFN-3~

CD4 CD4 CD4 CD4 CD4 CD4

+ + + + + §

TCR TCR TCR TCR TCR TCR

~3 a3 c~3 c~3 V38 + V38-

IL-2 IL-2 IL-2 IL-2 IL-2 IL-2

+ + + + +

lectin anti-CD3 a n t i - T C R a3 anti-TCR V38 anti-TCR V38

+ + + . . . . . . nd nd nd

IL-2

IL-3

IL-4

+ + +

+ + + + + + + + + nd nd nd

+ + + +

. . - - + + +

+ + + +

IL-5

+ + + +

+ + + + + + nd nd nd

nd, Not done

[21]. These data suggested that signals delivered during the stimulation of polyclonal CD4 § T cells favor the development of Th2-1ike cells. Thus, the outgrowth of IL-4-producing T cells was most likely due to mitogenic signals and/or to cytokines released during the repetitive stimulation of T cells. Since expansion of activated T cells in the presence of IL-2 alone induces a Thl-like phenotype within 2 weeks of culture (Table 2), this system allowed the investigation of signals that are capable of modulating the LK phenotype of CD4 + T cells. To this end, activated CD4 + T cells were expanded with IL-2 or with IL-2 plus single defined agents that could be responsible for the modulation of the LK pattern towards a Th2-phenotype (Fig. 1). Potential agents that regulate the induction of the Th2 phenotype are cytokines. However, CD4 § T cell blasts that were expanded for 2 weeks with IL-2, plus single defined cytokines (IL-1, IL-3, IL-4, IL-5, IL-6, IFN-3/, M-CSF, GM-CSF), as well as T cells that were expanded with supernatants of activated T cells, produced the same LK pattern as T cells cultured with IL-2 alone [39]. These results suggested that mitogenic signals may be required for the induction of IL-4 and the down-regulation of IL-2. To study the influence of mitogenic signals on the induction of IL-4, freshly isolated and activated CD4 § T cells were expanded in the presence of polyclonal T cell activators such as the plant lectins concanavalin A (Con A) or phytohemagglutinin (PHA). In contrast to cytokines, both mitogens strongly influenced the LK response pattern of CD4 + T cell blasts when added during the culture period with IL-2:CD4 + T cells that were expanded for 2 weeks in the presence of IL-2 and Con A produced up to 100 times more IL-4 and up to 100 times less IL-2 than cells of the same origin that were expanded with IL-2 alone (Table 2). Similar results were observed with PHA. The induction of IL-4 and the down-regulation of IL-2 production required prolonged binding of the lectin to the T cell surface, since o~-methyl-mannoside, which competes for the binding sites of Con A, completely abrogated its effect on the IL-2/IL-4 pattern [39]. Since Con A binds to the TCR and since T cell activation by Con A requires cross-linking of the TCR, these data suggested that the LK pattern of CD4 + T cells can be modulated via the TCR. However, lectins bind to a large number of other surface glycoproteins, which raised the possibility that surface molecules other than the TCR could be responsible for the modulation of the LK pattern.

Induction of Thl and Th2

297

T cell receptor mediated differentiation towards a Thl or Th2 phenotype

To work with T cell populations that have a defined TCR, and to assess effector functions other than LK production under cognate conditions, subsequent studies were performed with staphylococcal enterotoxin B (SEB). This toxin is a so-called superantigen that allows activation of T cells with the TCR V~3, 7, 8, and 17 chain families in the presence of APC [32]. CD4 + T cells that had been activated with SEB plus APC released large amounts of IL-2 when restimulated after culture with IL-2. These cells also produced IL-3 and IFN-y but neither IL-4 nor IL-5 and, thus, expressed a cytokine pattern that defines Thl cells (Table 2) [40]. In contrast, when cultured with recombinant (r)IL-2 plus a mitogenic mAb to the TCR-associated CD3 complex, the cells released IL-3, IL-4 and IL-5, but neither IL-2 nor IFN-3, upon restimulation (Table 2) [40]. This cytokine profile is characteristic for Th2 cells. Similarly, a mitogenic mAb to a common epitope of the o~/3 TCR also modified the LK pattern towards a Th2 phenotype [40]. The ability to induce Th2-1ike cells appeared to be restricted to mitogenic mAb, since nonmitogenic antibodies to T cells were not capable of modulating the LK pattern. These data demonstrate that TCR-derived signals are critically involved in the generation of a patterned LK response.

The induction o f the Th2-pheno~.pe depends on T cell receptor occupancy and on IL-4

We observed that activated CD4 + T cells produced IFN-y, IL-3, IL-4, and IL-5 during culture with IL-2 and antibodies to the TCR or to the CD3 complex. Thus, cytokines produced in an autocrine fashion could alone be responsible for the induction of Th2. To address this point, TCR V~8 + and Vr SEB-reactive T cell blasts were co-cultured in the presence of an mAb to V~8 (Fig. 2). After restimulation, V~8 + cells had a Th2-1ike phenotype and Vr cells had a Thl-like LK profile similar to ceils cultured with IL-2 alone (Table 2) [40]. This demonstrates that TCR ligation, but not LK alone, is capable of inducing Th2-1ike cells. TCR-mediated induction of Th2, however, was also dependent on IL-4 since the addition of an antibody to IL-4 prevented the development of Th2 cells (unpublished data). The resulting phenotype was Thl-like. Thus, we propose a model for the induction of T~2 cells that comprises at least two signals: one is provided by IL-4, either exogenously added or produced in an autocrine fashion, and the other consists of TCR ligation (Fig. 3).

Thl- and Th2-like cells can be derived from a common CD4 + precursor cell

To analyze the precursor cell for Thl or Th2 cells, pairs of subclones were derived from single CD4 + T cells by the split-clone technique. With this approach, it could be demonstrated that subclones expanded with IL-2 alone regularly give rise to a predominantly IL-2-producing phenotype. In contrast, sister clones derived from the same precursor but expanded with IL-2 and a

298

M. R6cken et al.

IL-2

< IL-2 + (xV[58

0

Fig. 2. Prolonged T cell receptor (TCR) occupancy is required for T cell differentiation toward a Th2 phenotype. Activated polyclonal CD4 ÷ T cells, comprising both, V~8 ÷ and V~8- cells, were cultured either in the presence of IL-2 alone or with IL-2 plus a mAb specific for Va8. After 2 weeks, V;~8÷ and V~8- cells were separated by cell sorting and restimulated. Only V~8 + cells from cultures with anti-V~8 released IL-4

II

Fig. 3. A two-signal model for T h cell differentiation toward a Th2 phenotype. Prolonged TCR occupancy may exert a dual mode of action on activated T h cells: (1) it induces the auto- or paracrine secretion of IL-4 in these cells, and (2) it enables the cells to develop a Th2-1ike phenotype, at least partially in response to shed IL-4

Induction of Thl and Th2'

299

mitogenic mAb to CD3 consistently gave rise to predominantly IL-4-producing subclones [41]. Analysis of 66 pairs of clones revealed that the large majority of them was not predetermined for a distinct LK response pattern. Sixty-eight percent of the clones gave rise to both one Thl-like and one Th2-1ike subclone. The latter released up to 50 times more IL-4 but 90 times less IL-2 than the corresponding Thl-like subclones. Less than 2 % of the split clones gave a mixed LK pattern (Th0 phenotype) under both culture conditions. These data demonstrate that one single, freshly isolated, postthymic CD4 § T cell can give rise to both Thl and Th2. Since none of the investigated clones was predetermined for the production of either IL-2 or IL-4, the Thl or Th2 phenotype of CD4 § T cells represents a functional differentiation state that is acquired during culture. Several of the described short-term IL-2-producing T cell clones could be switched to IL-4 producers by prolonged stimulation with anti-CD3 plus IL-2 [41]. This demonstrates that Thl-like cells can function as precursors for Th2. Together, these results show that freshly isolated CD4 + T cells have a LK pattern that can be modulated. This stands in sharp contrast to long-term cultured T cell clones, in which the LK pattern is fixed.

Th2-, but not Thl-like cells provide B cell help Short-term cultured Thl- or Th2-1ike polyclonal T cell blasts share not only the LK pattern of established long-term clones, but also other biological properties of Thl or Th2. Like Th2 clones, the IL-4-producing T cell blasts were capable of providing B cell help for IgG l, IgG2A and IgE synthesis under cognate conditions [40]. In contrast, the IL-2-producing cells were not capable of providing B cell help. They did, however, interact with B cells since they produced LK in cognate interaction.

Conclusions Long-term murine CD4 + T cell clones can be classified into two distinct subsets which produce either IL-2 and IFN-3, (Thl) or IL-4 and IL-5 (Th2). This functional separation of CD4 + T cells has subsequently been shown to be of great biological relevance, since these T helper cell subsets can be associated with protective or pathogenic immune responses. While a large number of in vitro and in vivo experiments underlined the biological relevance of Thl and Th2 phenotypes, the ontogeny of Thl or Th2 cells remained unknown until recently. Experiments published during the last 3 years have shown that cytokines such as IL-2, IL-4 and IFN-3, can influence the LK response pattern of CD4 + T cells. However, further investigations demonstrated that these cytokines alone had no effect on the modulation of the LK response pattern. Prolonged TCR binding by mitogenic mAb delivered a signal required for the induction of the Th2 phenotype. Whereas this signal cannot be replaced by soluble factors, ligation of the TCR together with LK leads to the development of T~2. The fact that TCR-mediated induction of Th2 in vitro takes the same time

300

M. R6cken et al.

as the a n t i g e n - m e d i a t e d c o m m i t m e n t of Thl- or Th2-1ike cells in vivo underlines the role o f the T C R in T helper cell subset differentiation. Since the two subsets can be derived from a c o m m o n postthymic C D 4 § precursor, the Thl or Th2 p h e n o t y p e o f a T cell is acquired d u r i n g T cell differentiation and is not secondary to the e x p a n s i o n of distinct subpopulations which are predetermined for the production o f a specific L K pattern. The structure o f the natural ligands of the T C R , i. e., antigens and M H C class I I - e n c o d e d molecules, have been shown to influence the d e v e l o p m e n t of T helper cells with distinct L K profiles in vivo. This suggests that the T C R - l i g a n d interaction is decisive for the c o m m i t m e n t of a peripheral C D 4 + T cell towards a T e l or a Th2-1ike response pattern. Acknowledgement. This work was supported in part by grants from the Deutsche Forschungsgemeinschaft to MR (Ro 764/1-1) and from the Swiss National Foundation for Scientific Research to CH (3.115-0.88 and 32-27/59.89).

References 1. Bendelac A, Schwartz RH (1991) CD4 + and CD8 + T cells acquire specific lymphokine secretion potentials during thymic maturation. Nature 353:68 2. Ben-Sasson SZ, Le Gros G, Conrad DH, Finkelman FD, Paul WE (1990) IL-4 production by T cells from naive donors: IL-2 is required for IL-4 production. J Immunol 145:1127 3. Betz M, Fox BS (1990) Regulation and development of cytochrome c-specific IL-4-produeing T cells. J Irmnunol 145:1046 4. Bogdan C, Vodovotz Y, Nathan C (1991) Macrophage deactivation by interleukin 10. J Exp Med 174:1549 5. Budd RC, Cerottini JC, MacDonald HR (1987) Selectively increased production of interferon-3, by subsets of Lyt-2 + and L3T4 + T cells identified by expression of Pgp-1. J Immunol 138:3583 6. Cher DJ, Mosmann TR (1987) Two types of murine helper T cell clone. II. Delayed-type hypersensitivity is mediated by Thl clones. J Immunol 138:3688 7. Coffman RL, Seymour BWP, Hudak S, Jackson J, Rennick D. Antibody to interleukin-5 inhibits helminth-induced eosinophilia in mice. Science 245:308 8. Daynes RA, Dowell T, Araneo BA (1991) Platelet-derived growth factor is a potent biologic response modifier of T cells. J Exp Med 174:1323 9. Del Prete GF, De Carli M, Mastromauro C, Biagiotti R, Macchia D, Falagiani P, Ricci M, Romagnani S (1991) Purified protein derivative of Mycobacterium tuberculosis and excretorysecretory antigen(s) of Toxocara canis expand in vitro human T cells with stable and opposite (type 1 T helper or type 2 T helper) profile of cytokine production. J Clin Invest 88:346 10. Doutrelepont JM, Moser M, Leo O, Abramowicz D, Vanderhaegen ML, Urbain J, Goldman M (1991) Hyper IgE in stimulatory graft-versus-host disease: role of interleukin-4. Clin Exp Immunol 83:133 11. Fiorentino DF, Zlotnik A, Vieira P, Mosmann TR, Howard M, Moore KW, O'Garra A (1991) IL-10 acts on the antigen-presenting cell to inhibit cytokine production by Thl cells. J Immunol 146:3444 12. Firestein GS, Roeder WD, Laxer JA, Townsend KS, Weaver CT, Horn JT, Linton J, Torbett BE, Glasebrook AL (1989) A new murine CD4 + T cell subset with an unrestricted cytokine profile. J Immunol 143:518 13. Flamand V, Abramowicz D, Goldman M, Biernaux C, Huez G, Urbain J, Moser M, Leo O (1990) Anti-CD3 antibodies induce T cells from unprimed animals to secrete IL-4 both in vitro and in vivo. J Immunol 144:2875 14. Fong TAT, Mosmann TR (1989) The role of IFN-3, in delayed-type hypersensitivity mediated by Thl clones. J Immunol 143:2887

Induction of Thl and Th2

301

15. Gajewski TF, Joyce J, Fitch FW (1989) Antiproliferative effect of IFN-3, in immune regulation. III. Differential selection of Thl and Th2 murine helper T lymphocyte clones using recombinant IL-2 and recombinant IFN-3,. J Immunol 143:15 16. Gaspari AA, Jenkins MK, Katz SI (1988) Class II MHC-bearing keratinocytes induce antigenspecific unresponsiveness in hapten-specific Thl clones. J Immunol 141:2216 17. Goldman M, Druet D, Gleichmann E (1991) Th2 cells in systemic autoimmunity: insights from allogeneic diseases and chemically-induced autoimmunity. Immunol Today 12:223 18. Haanen JBAG, De Waal Malefijt R, Res PCM, Kraakman EM, Ottenhoff THM, De Vries RRP, Spits H (1991) Selection of a human T helper type l-like T cell subset by mycobacteria. J Exp Med 174:583 19. Hauser C (1990) Cultured epidermal Langerhans cells activate T effector cells for contact sensitivity. J Invest Dermatol 95:436 20. Hauser C, Katz SI (1990) Generation and characterization of T-helper cells by primary in vitro sensitization using Langerhans cells. Immunol Rev 117:67 21. Hauser C, Snapper CM, Ohara J, Paul WE, Katz SI (1989) T helper cells grown with haptenmodified cultured Langerhans cells produce interleukin 4 and stimulate IgE production by B cells. Eur J Immunol 19:245 22. Jardim A, Alexander J, Teh HS, Ou D, Olafson RW (1990) Immunoprotective Leishmania major synthetic T cell epitopes. J Exp Med 172:645 23. Ju ST, Ruddle NH, Strack P, Dorf ME, DeKruyff RH (1990) Expression of two distinct cytolytic mechanisms among murine CD4 subsets. J Immunol 144:23 24. Kapsenberg ML, Wierenga EA, Bos JD, Jansen HM (1991) Functional subsets of allergen-reactive human CD4 + T cells. Immunol Today 12:392 25. Kay AB, Ying S, Varney V, Gaga M, Durham SR, Moqbel R, Wardlaw AJ, Hamid Q (1991) Messenger RNA expression of the cytokine gene cluster, interleukin-3 (IL-3), IL-4, IL-5, and granulocyte/macrophage colony-stimulating factor, in allergen-induced late-phase cutaneous reactions in atopic subjects. J Exp Med 173:775 26. Killlar L, Mac Donald G, West J, Woods A, Bottomly K (1987) Cloned, Ia-restricted T cells that do not produce interleukin-4 (IL-4)/B cell stimulatory factor 1 (BSF-1) fail to help antigenspecific B cells. J Immunol 138:1674 27. Kiihn R, Rajewsky K, Mfiller W (1991) Generation and analysis of interleukin-4 deficient mice. Science 254:707 28. Le Gros G, Ben-Sasson SZ, Seder R, Finkelman FD, Paul WE (1990) Generation of interleukin-4 (IL-4)-producing cells in vivo and in vitro: IL-2 and IL-4 are required for in vitro generation of IL-4-producing cells. J Exp Med 172:921 29. Lehmann PV, Schumm G, Moon D, Hurtenbach U, Falcioni F, Muller S, Nagy ZA (1990) Acute lethal graft-versus-host reaction induced by major histocompatibility complex class II-reactive T helper cell clones. J Exp Med 171:1485 30. Liew FY, Millott S M, Schmidt JA (1990) A repetitive peptide of leishmania can activate T helper type 2 cells and enhance disease progression. J Exp Med 172:1359 31. Maggi E, Biswas P, Del Prete G, Parronchi P, Macchia D, Simonelli C, Emmi L, De Carli M, Tiri A, Ricci M, Romagnani S (1991) Accumulation of Th-2-1ike helper T cells in the conjunctiva of patients with vernal conjunctivitis. J Irnmunol 146:1169 32. Marrack P, Kappler J (1990) The staphylococcal enterotoxins and their relatives. Science 248: 705 33. Mosmann TR, Coffman RL (1989) Thl and Th2 cells: different patterns of lymphokine secretion lead to different functional properties. Annu Rev Immunol 7:145 34. Mosmann TR, Moore KW (1991) The role of IL-10 in crossregulation Of Thl and Th2 responses. lmmunol Today 12:A49 35. Mosmann TR, Cherwinski H, Bond MW, Giedlin MA, Coffman RL (1986) Two types of murine helper T cell clone. I. Definition according to profiles of lymphokine activities and secreted proteins. J Immunol 136:2348 36. Murray JS, Madri J, Tite J, Carding SR, Bottomly K (1989) MHC control of CD4 + T cell subset activation. J Exp Meal 170:2135 37. Nagarkatti M, Clary SR, Nagarkatti PS (1990) Characterization of tumor-infiltrating CD4 + T cells as Thl cells based on lymphokine secretion and functional properties. J Immunol 144:4898 38. Powers GD, Abbas AK, Miller RA (1988) Frequencies of IL-2 and IL-4 secreting T cells in naive and antigen-stimulated lymphocyte populations. J Immunol 140:3352

302

M. R6cken et al.

39. R6cken M, Mfiller KM, Saurat JH, Hauser C (1991) Lectin-mediated induction of IL-4-producing CD4 + T cells. J Immunol 146:577 40. R6cken M, Miiller KM, Saurat JH, M011er I, Louis JA, Cerottini JC, Hauser C (1992) Central role for TCR/CD3 ligation in the differentiation of CD4 + T cells toward a Thl or Th2 functional phenotype. J Immunol 148:47 41. R6cken M, Saurat JH, Hauser C (1992) A common precursor for CD4 § T cells producing IL-2 or IL-4. J Immunol 148:1031 42. Salgame S, Abrams JS, Clayberger C, Goldstein H, Convit J, Modlin RL, Bloom BR (1991) Differing lymphokine profiles of functional subsets of human CD4 and CD8 T cell clones. Science 254:279 43. Scott P, Kaufmann SHE (1991) The role of T-cell subsets and cytokines in the regulation of infection. Immunol Today 12:346 44. Snapper CM, Paul WE (1987) Interferon-7 and B cell stimulatory factor-1 reciprocally regulate Ig isotype production. Science 2 3 6 : 9 4 4 45. Stevens TL, Bossie A, Sanders VM, Fernandez-Botran R, Coffman RL, Mosmann TR, Vitetta ES (1988) Regulation of antibody isotype secretion by subsets of antigen-specific helper T cells. Nature 334:255 46. Stout RD, Bottomly K (1989) Antigen-specific activation of effector macrophages by IFN-y producing (Thl) T cell clones: failure of IL-4 producing (Th2) T cell clones to activate effector function in macrophages. J Immunol 142:760 47. Street NE, Schumacher JH, Fong TAT, Bass H, Fiorentino DF, Leverah JA, Mosmann TR (1990) Heterogeneity of mouse helper T cells: evidence from bulk cultures and limiting dilution cloning for precursors of Thl and Th2 cells. J Immunol 144:1629 48. Swain SL, McKenzie DT, Weinberg AD, Hancock W (1988) Characterization of T helper 1 and 2 cell subsets in normal mice: helper T cells responsible for IL-4 and IL-5 production are present as precursors that require priming before they develop into lymphokine-secreting cells. J Immunol 141:3445 49. Swain SL, Huston G, Tonkonogy S, Weinberg A (1991) Transforming growth factor-/3 and IL-4 cause helper T cell precursors to develop into distinct effector helper cells that differ in lymphokine secretion pattern and cell surface phenotype. J Immunol 147:2991 50. Tary-Lehmann M, Rolink AG, Lehmann PV, Nagy ZA, Hurtenbach U (1990) Induction of graft versus host-associated immunodeficiency by CD4 § T cell clones. J Immunol 145:2092 51. Vercelli D, Jabara HH, Arai KI, Geha RS (1989) Induction of human IgE synthesis requires interleukin 4 and T/B cell interactions involving the T cell receptor/CD3 complex and MHC class II antigens. J Exp Med 169:1295 52. Vitetta ES, Fernandez-Botran R, Myers CD, Sanders VM (1989) Cellular interactions in the humoral immune response. Adv Immunol 4 5 : 1 53. Vollenweider S, Saurat JH, R6cken M, Hauser C (1991) Evidence suggesting involvement of interleukin-4 (IL-4) production in spontaneous in vitro IgE synthesis in patients with atopic dermatitis. J Allergy Clin Immunol 87:1088 54. Wong RL, Ruddle NH, Padula SJ, Lingenheld EG, Bergman CM, Rugen RV, Epstein DI, Clark RB (1988)Subtypes of helper cells: non-inflammatory type 1 helper T cells. J Immunol 141:3329 55. Yssel H, Shanafelt MC, Soderberg C, Schneider PV, Anzola J, Peltz G (1991). Borrelia burgdorferi activates a T helper type l-like T cell subset in lyme arthritis. J Exp Med 174:593 56. Yamamura M, Uyemura K, Deans RJ, Weinberg K, Rea TH, Bloom BR, Modlin RL (1991) Defining protective responses to pathogens: cytokine profiles in leprosy lesions. Science 254:277