TH2 paradigm in allergy

Immunotechnology 3 (1998) 233 – 244

Review article

The TH1/TH2 paradigm in allergy Enrico Maggi * Clinical Immunology Dept., Istituto di Medicina Interna e Immunoallergologia, Uni6ersity of Firenze, Firenze, Italy Received 8 September 1997; accepted 3 October 1997

Abstract Recent evidence has been accumulated to suggest that allergen-reactive type 2 helper T cells (Th2) play a triggering role in the activation and/or recruitment of IgE antibody-producing B cells, mast cells and eosinophils, i.e. the cellular triad involved in the allergic inflammation. Interleukin (IL)-4 production by a still unknown cell type (T cell subset, mast cell/basophil?) at the time of antigen presentation to the Th cell is critical for the development of Th2 cells. Other cytokines, such as IL-1 and IL-10, and hormones, such as calcitriol and progesterone, also play a favoring role. In contrast, cytokines such as interferon (IFN-a, IFN-g, IL-12 and transforming growth factor (TGF)-b, and hormones, play a negative regulatory role on the development of Th2 cells. However, the mechanisms underlying the preferential activation by environmental allergens of Th2 cells in atopic individuals still remain obscure. Some gene products selectively expressed in Th2 cells or selectively controlling the expression of IL-4 have recently been described. Moreover, cytokines and other gene products that dampen the production of IL-4, as well as the development and/or the function of Th2 cells, have been identified. These findings allow us to suggest that the up-regulation of genes controlling IL-4 expression and/or abnormalities of regulatory mechanisms of Th2 development and/or function may be responsible for Th2 responses against common environmental allergens in atopic people. The new insights in the pathophysiology of T cell responses in atopic diseases provide exciting opportunities for the development of novel immunotherapeutic strategies. They include the induction of nonresponsiveness in allergen-specific Th2 cells by allergen peptides or redirection of allergen-specific Th2 responses by Th1-inducing cytokines, altered peptide ligands, allergens incorporated into recombinant microorganisms or bound to appropriate adjuvants, and plasmid DNA vaccination. In severe atopic patients, the possibility of nonallergen-specific immunotherapeutic regimens designed to target Th2 cells or Th2-dependent effector molecules, such as specific IL-4 transcription factors, IL-4, IL-5 and IgE, may also be suggested. © 1998 Elsevier Science B.V. Keywords: Th1/Th2 responses; Cytokines; Atopy; Genetic and environmental factors; New therapies

* Tel.: +39 55 4221957; fax: + 39 55 412867. 1380-2933/98/$19.00 © 1998 Elsevier Science B.V. All rights reserved. PII S 1 3 8 0 - 2 9 3 3 ( 9 7 ) 1 0 0 0 5 - 7

234

E. Maggi / Immunotechnology 3 (1998) 233–244

1. Introduction Atopic diseases are genetically determined disorders characterized by an increased ability of B lymphocytes to synthesize IgE antibodies towards ubiquitous antigens (allergens) able to activate the immune system after inhalation or ingestion and after penetration through the skin. IgE antibodies are able to bind to high affinity Fco receptors (FcoRI) present on the surface of mast cells/ basophils. Allergen-induced FcoRI cross-linking triggers the release of vasoactive mediators, chemotactic factors and cytokines that are responsible for the allergic events. Eosinophils are also involved in the pathogenesis of allergic reactions, as these cells usually accumulate at the site of allergic inflammation and release toxic products contributing to tissue damage. The mechanisms linking IgE-producing B cells, mast cells/basophils and eosinophils in the pathogenesis of allergic reactions have remained unclear until distinct subsets of CD4 + helper T (Th) cells, based on their profile of cytokine secretion, were discovered. The cumulative study on the functional properties of helper Th cell subsets is known as the ‘Th2 hypothesis’ for the pathogenesis of allergic reactions [1]. This review summarizes the role of allergen-reactive Th2 cells in the pathogenesis of human allergic disorders and the possible mechanisms involved in the regulation of Th2 cell development. Finally, it will examine novel therapeutic strategies for atopic diseases, particularly those able to redirect the Th2 response.

2. The Th1/Th2 paradigm Murine CD4 + Th1 cells secrete interferongamma (IFN-g), interleukin (IL) 2 and tumor necrosis factor (TNF)-b, which promote macrophage activation, production of opsonizing and complement-fixing antibodies, antibody-dependent cell cytotoxicity, and DTH [2]. For these reasons, it is possible to refer to Th1 cells as cells responsible for phagocyte-dependent host responses [3]. On the other side, Th2 cells, which produce IL-4, IL-5, IL-6, IL-9, IL-10 and IL-13,

provide optimal help for antibody responses, including IgE and IgG1 isotype switching facilitation to IgA synthesis, and promote mast cell and eosinophil growth, differentiation and activation. In addition, some Th2-derived cytokines, such as IL-4, IL-10 and IL-13 inhibit several macrophage functions [2]. Therefore, it is appropriate to refer to Th2 cells as cells responsible for phagocyte-independent host responses [3]. In the absence of clear polarizing signals, CD4 + T cell subsets with a less restricted lymphokine profile than Th1 or Th2 cells, designated Th0, usually arise, that mediate intermediate effects depending upon the ratio of lymphokines produced and the nature of the responding cells [2,4]. The cytokine response of the single Th0 cell can remain unrestricted or further differentiate into the polarized Th1 or Th2 pathway under the influence of strong (and/or chronic) microenvironmental signals or because of a particular genetic background [5]. It is customary to define CD4 + T cells that have been differentiated to produce IL-4, but not IFN-g as Th2 (or Th2-like) cells and those that produce IFN-g but not IL-4 as Th1 (or Th1-like) cells, without considering the other set of Th1 or Th2 cytokines. With these reservations in mind, the Th1/Th2 model provides a useful paradigm for the understanding of several pathophysiologic processes, including allergic disorders and possibly for the development of novel immunotherapeutic strategies. In the last few years, strong evidence for the existence of human CD4 + Th cells with cytokine patterns and functions that are comparable to murine Th1 and Th2 cells has been provided [6–9], although in humans the expression of some cytokines, such as IL-2, IL-6, IL-10 and IL-13 may be less restricted [3,4]. Human Th1-like and Th2-like cells also differ for their cytolytic potential, the majority of Th1, but only a minority of Th2, clones being cytolytic. Moreover, Th1 and Th2 cells differ in their mode of help for B-cell antibody synthesis. Only Th2, but not Th1, clones provide B-cell helper activity for IgE synthesis. At low T:B cell ratios, both Th1 and Th2 clones can provide help for IgM, IgG, and IgA synthesis. At high T:B cell ratios, the B-cell helper activity of Th1 cells strongly declines, whereas there is a

E. Maggi / Immunotechnology 3 (1998) 233–244

dose-dependent increase of the B-cell helper activity of Th2 cells [10]. Interestingly, the decline in helper activity of Th1 cells appears to be related to their ability to kill the B cells used as antigenpresenting cells (APC). Finally, human Th1 and Th2 clones exhibit different abilities to activate monocytic cells: Th1 clones induce procoagulant activity (PCA) and tissue factor (TF) production by human monocytes, whereas Th2 clones do not [11]. Moreover, supernatants of Th2 clones, as well as IL-4 and IL-10, inhibit both PCA and TF production by Th1 clones. More recently, it has been shown that Th1 and Th2 cells can exhibit the preferential expression of some surface molecules. For example, CD30, a member of the TNF receptor superfamily [12], as well as CCR3 antigen, a member of chemokyne receptors, are preferentially expressed by activated Th2 cells ([13]; Lanzavecchia, personal communication). In contrast, LAG-3, a member of the immunoglobulin superfamily [14], surface IFN-g and the b chain of IL-12R preferentially associate with the production of Th1 cytokines [15 – 17]. Although the great majority of CD8 + T cells produce IFN-g, but no IL-4, CD8 + T cell clones producing IL-4 can be obtained by stimulation of murine CD8 + T cells with anti-CD3 antibody [18], mitogen or allostimulation [19,20] and antigen [21,22] in the presence of IL-4. Based on these findings, the names Tc1 and Tc2 for cytotoxic CD8 + T cells secreting Th1-like and Th2-like cytokines were proposed [20]. CD8 + T cell clones that produce IL-4 have also been generated from the skin of immunologically unresponsive individuals with leprosy [23], the peripheral blood of HIV-infected patients [24,25] and from the gengiva of subjects with chronic adult periodontitis [26] and from normal human peritoneum [27]. While the functional role of Tc1 cells is well established, the in vivo functional meaning of Tc2 cells is still unclear. Murine Tc2 clones exhibited normal cytolytic potential and failed to provide cognate help for B cell antibody production [20]. However, at least in man, some of them can express the CD40 ligand (CD40L) [25], may have reduced cytolytic activity [28] and favor eosinophil activation and accumulation via the production of IL-5. Likewise, Tc2 clones gener-

235

ated from HIV-infected individuals showed reduced anti-HIV cytolytic activity and provided optimal polyclonal B-cell helper activity for production of immunoglobulin, including IgE, via the expression of CD40L [24,25,29]. It is also of interest that IL-4, in the absence of antigenic stimulation, induces an anergy-like state in differentiated Tc1 cells [30], thus, suggesting that Tc2 cells act as suppressor or anti-inflammatory cells through the production of ‘helper’ cytokines [31]. It is of note that, as for CD4 + T cell clones, CD30 and sCD30 are usually not detectable in Tc1 clones, but they were consistently found in Tc2 clones generated from HIV-infected individuals [32].

3. The ‘Th2 hypothesis’ in atopy As reported above, Th2 cells produce IL-4 and IL-13 which stimulate IgE and IgG4 antibody production, IL-5 which recruits and differentiates eosinophils and IL-10, which together with IL-4 and IL-13, inhibit several macrophage functions, thus explaining why the mast cell/eosinophil/IgEproducing B cell triad is involved in the pathogenesis of allergy [1]. Strong evidence has been accumulated to support this concept that Th2 cells play a central role in atopy. The study of cytokine profile of T-cell clones, specific for different antigens, has clearly shown that, in contrast to clones specific for bacterial antigens showing a prevalent Th1/Th0 phenotype, the great majority of allergen-specific T-cell clones generated from peripheral blood lymphocytes of atopic donors express a Th0/Th2 phenotype, with high production of IL-4 and IL-5 and no or low production of IFN-g [6,8]. A lot of evidence has been obtained that Th2like cells accumulate at level of target organs in allergic disorders, by using either cloning techniques or in situ hybridization. The majority of T-cell clones, generated from the conjunctival infiltrates of patients with vernal conjunctivitis, were found to develop into Th2 clones [33]. Using in situ hybridization, cells showing mRNA for Th2, but not Th1, cytokines were detected at the site of late phase skin reactions in skin biopsies from atopic patients, in mucosal bronchial biop-

236

E. Maggi / Immunotechnology 3 (1998) 233–244

sies or bronchoalveolar lavage (BAL) from patients with asthma [34 – 36] and after local allergen challenge in nasal mucosa of patients with allergen-induced rhinitis [37]. Likewise, increased levels of IL-4 and IL-5 were measured in the BAL of allergic asthmatics, whereas in nonallergic asthmatics, IL-2 and IL-5 predominated [38]. Inhaled allergens induce activation and recruitment of allergen-specific Th2 cells in the airway mucosa of patients with respiratory allergy. Biopsy specimens were obtained from the bronchial or nasal mucosa of patients with grass pollen-induced asthma or rhinitis 48 h after positive bronchial or nasal provocation test with allergen. High proportions of T cell clones, derived from stimulated airway mucosa of these patients, were specific for grass allergens and exhibited a Th2 profile [39]. Similarly, high proportions of Dermatophagoides pteronyssinus (DP)-specific Th2-like CD4 + T cell clones were generated from the skin of patients with atopic dermatitis (AD) taken after contact challenge with DP, suggesting that transcutaneous sensitization to aeroallergens may be essential in the induction of skin lesions in patients with AD [40]. As reported above, we have shown that CD30 is preferentially expressed by T-cell clones able to produce Th2-type cytokines [13]. No CD4 + CD30 + cells were detected in any of the nonatopic or atopic donors examined before the pollination season, whereas the majority of grass-sensitive donors showed small proportions of circulating CD4 + CD30 + cells (B 0.3%) during this period. When sorted into CD30 + and CD30 − cells, CD30 + , but not CD30 − , cells proliferated in response to Lol p 1 and exhibited the ability to produce IL-4 and IL-5 [13]. These findings demonstrate that grass allergen-reactive CD4 + CD30 + Th2 cells, can circulate in the peripheral blood of grass sensitive patients during the natural exposure to pollens. Finally the ‘Th2 hypothesis in atopy’ is confirmed by studies on the effect of allergen specific immunotherapy (IT) on the cytokine profile of T cells. Pollen IT did not affect the expression of Th2-type cytokine pattern in response to allergen exposure at level of allergen-induced late-phase cutaneous reactions, but mRNA expression of

Th1-type cytokines was enhanced [41]. In another study, successful IT was found to reduce IL-4 production by allergen-specific CD4 + T cells, whereas production of IFN-g was not affected [42]. Finally, both decreased production of IL-4 and increased production of IFN-g was observed in bee venom-sensitized patients treated with specific IT [43,44]. Although partially discordant, these reports support the concept that cytokine profiles of allergen-specific CD4 + T cells are changeable and can be manipulated by in vivo therapies.

4. Mechanisms involved in the regulation of Th2 development The mechanisms responsible for the preferential development of allergen-reactive Th2 cells in atopic subjects have not yet been completely clarified.

4.1. Cells Langherans cells (LC) in the skin and dendritic cells (DC) in the respiratory mucosa, represent the primary point of contact between the immune system and allergens coming through the skin or the respiratory airways, respectively. These cells are involved in allergen transport to regional lymphnodes where allergen presentation to allergen-specific CD4 + T cells occurs. It has been suggested that atopic patients with asthma have higher numbers of intraepithelial DC than non asthmatic subjects and that these cells (in the presence of allergen) can trigger T cells to release IL-4 and IL-5 [45]. Costimulatory signals, namely the interactions between CD28/CTLA and their ligands expressed on APC (CD80 and CD86), have been found to exert a clearcut effect on modulating the Th1 and/or the Th2 development [46]. Alterations of CD30L expression and/or activity seems to have some role in the development of allergen-specific Th2 responses in atopic individuals. Blocking of CD30L on APC may shift the in vitro differentiation of allergen-specific T cells from the Th0/Th2 to the Th0/Th1 profiles [47].

E. Maggi / Immunotechnology 3 (1998) 233–244

More recently, it was reported that varying either the density or the affinity of peptide for the MHC class II molecules could determine the type of T cell responses. High MHC class II-peptide density on the APC surface favored Th1-like responses, while low ligand densities favored Th2like responses [48]. Moreover, by using a panel of ligands with various class binding affinities, it was shown that stimulation with the highest affinity ligand resulted in IFN-g production and with ligand exerting lower MHC class II binding induced IL-4 secretion [49]. These findings suggest that the MHC binding affinity of antigenic peptides leads to differential interactions at the T cell-APC interface, which is crucial for the differential development of cytokine patterns in T cells. The role of T cell repertoire in determining the development of Th1- or Th2-type responses is still controversial. In Leishmania major infected mice, Th1 and Th2 cells displaying the same repertoire and recognizing the same peptide have been demonstrated, suggesting that cells with identical TCR can differentiate into either the Th1 or the Th2 phenotype [50]. However, evidence for the pivotal role of specific Vb-expressing T cell subsets in the stimulation of IgE production and increased airways responsiveness induced by ragweed allergen has been reported [51]. Thus, it cannot be excluded that the recognition of allergen by the TCR provides a direct signal driving T cells towards the production of IL-4 or alternatively, of IFN-g.

4.2. Soluble factors In both murine and human systems, IL-4 appears to be the most dominant factor in determining the likelihood for Th2 polarization in cultured cells [52,53]. Accordingly, IL-4-gene-targeted mice fail to generate mature Th2 cells in vivo and to produce IgE antibodies [54], suggesting that early IL-4 production by other cell types is involved. IFN-a, IL-12 and TGF-b, produced by macrophages and B cells, have been shown to play an important role in the induction of Th1 expansion [55]. IFN-g produced by T and NK cells promotes the differentiation of Th1 cells [52]. Likewise, IL-12, which is a powerful IFN-g in-

237

ducer [56] appears to be the most important natural initiator of Th1 responses by acting either directly or indirectly via the induction of IFN-g production [57–59]. Recent in vivo data from IL-12-deficient mice show that Th1 responses were impaired, even not completely lacking, the magnitude of DTH was substantially decreased and secretion of IL-4 was enhanced [60,61]. The effect of cytokines produced by macrophages and/ or B cells on the development of Th2 cells seems to be less critical. IL-10 has been shown to favor the development of Th2 cells, both in mouse and man. IL-1 is a selective co-factor for the growth of some murine Th2 clones and can favor the in vitro development of human Th2-like clones [62]. Recently, IL-6 produced by APC has been found as one of the Th2 skewing factors in the primary response [63]. The role of hormones in promoting the differentiation of Th cells or in favoring the shifting of differentiated Th cells from one to another cytokine profile has also been suggested. Glucocorticoids enhance Th2 activity and synergizes with IL-4, whereas dehydroepiandrostenone sulphate enhance Th1 activity [64]. Lastly, progesterone favors the in vitro development of human T cells producing Th2-type cytokines and promotes both IL-4/IL-5 production and membrane CD30 expression in established human Th1 clones [65]. It may represent one of the mechanisms involved in the Th1/Th2 switch occurring at the maternal-fetal interface in order to promote successful pregnancy [66]. One of the most intriguing point is the source of IL-4 in the primary response able to modulate Th2 differentiation. Possible candidates include mast cells and basophils, CD4 + NK1.1 + T- cell subset or the T helper cell themselves. Human mature mast cells and basophils produce IL-4 in response to several secretagogues [67,68], and more recently, it was found that activated human eosinophils can also release high IL-4 concentrations [69,70]. Thus, at least potentially, FcoR + non-T cells might be an amplification system for Th2 cells in vivo during allergic reactions and parasitic infestations. However, parasites or allergens are not able to crosslink FcgR and FcoR prior to parasite-specific IgG and IgE

238

E. Maggi / Immunotechnology 3 (1998) 233–244

antibodies are produced. In fact, mast cell-deficient mice develop normal Th2 responses [71] and only IL-4-producing T (but not IL-4-producing non-T) cells are able to reconstitute the antigenspecific IgE in IL-4 − / − mice [72]. The role of CD4 + NK1.1 + T cells in the development of Th2 responses has been suggested since: (i) They are selected by the nonpolymorphic MHC class I molecule, CD1 [73]; (ii) secrete large and transient amounts of IL-4 mRNA after intravenous injection of anti-CD3 antibody [74]; (iii) b2-microglobulin- and NK1.1 + T cell-deficient mice are unable to produce quickly IL-4 in response to anti-CD3 [75,76]. However, it is unlikely that all antigens promoting naive T cell differentiation into the Th2 pathway are capable of activating CD4 + NK1.1 + T cells. A more likely hypothesis is that the maturation of naive T cells into the Th2 pathway mainly depends on the levels of IL-4 production by naive T cells themselves at priming. This possibility is supported by several observations: (i) Low intensity signalling of TCR mediated by low peptide doses led to secretion of low levels of IL-4 by murine naive T cells [48]; (ii) naive T cells, recently activated in the presence of co-stimulatory signals, required two or more stimulation events to produce IL-4 and IL-5 and this cytokine secretion was blocked by anti-IL-4 mAb, suggesting a role for endogenous IL-4 produced by the naive T cells themselves [77]; (iii) human CD45RA + adult and neonatal T cells were found to develop into IL-4-producing cells in the absence of any source of IL-4 or anti-IL-4 antibodies [78,79]; (iv) high proportions of Th2 clones could be generated from single CD4 + ab + T cells isolated from thymus of small children [80]. A significant fraction of uncommitted T cells may be primed for a Th2 phenotype (independent of antigen and IL-4) if they are exposed to IL-2 and interact with accessory cells bearing the natural CD28 ligands B7-1 and B7-2. When stimulated by specific antigen, such primed Th2 precursor cells provide a source of IL-4 to promote Th2 immunity [81]. Thus, strong evidence suggests that the maturation of naive T cells into the Th2 pathway mainly depends upon the levels and the kinetics of autocrine IL-4 production at priming. Obviously, if

some signals trigger CD4 + NK1.1 + T cells or mastcell/basophils, they release rapidly high IL-4 amounts which contribute to the development of the Th2 pathway.

5. Possible genetic alterations favoring allergen-specific Th2 responses in atopic subjects The possibility that atopic subjects have a genetic dysregulation at level of IL-4 produced by Th cells is supported by several observations. First, CD4 + T cell clones from atopic individuals are able to produce noticeable amounts of IL-4 and IL-5 in response to bacterial antigens, such as PPD and streptokinase, that usually evoke responses with a restricted Th1-like cytokine profile in nonatopic individuals [82]. Second, T-cell clones generated from cord blood lymphocytes of newborns with atopic parents produce higher IL-4 concentrations than neonatal lymphocytes of newborns with nonatopic parents [83]. Moreover, large panels of Parietaria officinalis group 1 (Par o 1)-specific T-cell clones were generated from donors with low or high serum IgE levels and assessed for their profile of cytokine production and reactivity to two immunodominant Par o 1 peptides (p92 and p96). Interestingly, both p92and p96-specific T-cell clones generated from ‘high IgE’ donors produced remarkable amounts of IL-4 and low IFN-g. In contrast, T-cell clones generated from ‘low IgE’ donors showed a different profile of cytokine production: the majority of those specific for p96 produced high amounts of both IL-4 and IFN-g, whereas most p92-specific T cell clones showed a Th1-like profile (high IFN-g and low IL-4) (Parronchi et al., unpublished). Taken together, these data strongly suggest that allergen peptide ligand can influence the cytokine profile of Th cells; however, mechanisms underlying non-cognate regulation of IgE responsiveness are overwhelming. The role of CD8 + T cells in allergic sensitization is still unclear and probably complex. Some studies suggest a suppressive function for CD8 + T cells, associating IFN-g-producing CD8 + T cells with the inhibition of IgE production and consequent down-regulation of allergic sensitization

E. Maggi / Immunotechnology 3 (1998) 233–244

[84,85]. Recent data support the possible role of allergen-specific CD8 + T cells in controlling the Th response against allergens in humans. First, a nonapeptide expanded higher numbers of CD8 + T cell clones in ‘low’ rather than in ‘high’ IgE producers. In addition, lactalbumin expanded higher numbers of CD8 + T cell clones in nonatopic than in atopic milk-sensitive donors, suggesting that allergen-specific CD8 + T cells may play an important role (via IFN-g production ?) in preventing the differentiation of allergen-specific Th2 cells in nonatopic people. Regarding the role of the allergen peptide ligand, several studies have underlined the potential importance of MHC class II haplotype [86], but the data remain controversial. A gene (or genes) in the TCRa/d complex has been described to influence the development of a specific IgE response in allergic subjects [87] and a restricted usage of Va13 by T cell clones specific for Lolium perenne group 1 (Lol p 1), as well as an intra- and inter-individually restricted TCR-Vb usage in both Lol p 1and Poa pratensis group 9 (Poa p 9)-specific T cell lines, was also reported [88]. On the other hand, the evidence for a linkage of overall IgE to markers in chromosome 5q31.1, especially to the IL-4 gene [89] suggests that one or more polymorphisms exist in a coding region or more probably, a regulatory region of the IL-4 gene. Several studies have identified potential mechanisms governing the IL-4 gene expression in human and murine T cells. The entire IL-4 gene has been scanned for possible atopy-associated polymorphisms, based on the hypothesis that they may reside in transcriptional regulatory elements [90]. Analysis of the IL-4 promoter has revealed functionally important binding sites for several transcription factors, (including NF-AT, CCAAT box binding protein NF-Y, Oct 1, HMGI(Y), AP-1 members, NF-kB, and an unpurified factor termed PCC) [91–97] and silencer elements (that bind factors termed NRE) [98]. Moreover, the nuclear extracts from individuals with atopic dermatitis exhibited a higher affinity for a consensus P-element, suggesting that polymorphic residues in NFAT family members themselves or in their post-translationally modified versions may be involved [99]. More recently, it has been shown that

239

the proto-oncogene c-maf, a transcription factor, controls tissue-specific expression of IL-4. c-maf is expressed in Th2 but not Th1 clones and is induced during normal precursor cell differentiation along a Th2 but not Th1 lineage [100]. These data indicate that c-maf is a factor responsible for dictating Th2-selective IL-4 gene transcription and makes c-maf as an obvious candidate for atopy gene. Another good candidate for atopy gene is STAT6, which is a protein that binds to DNA sequences found in the promoters of IL-4-responsive genes [101]. The experiments on k.o. mice have shown that this member of STAT family of proteins is required for the development of Th2 cells [102,103]. Down-regulation of the mechanisms dampening IL-4 production can also be involved in the development of prevalent Th2 responses in atopy. IL-12 induces tyrosine phosphorylation and DNA-binding of STAT3 and STAT4 [104]. STAT-deficient mice have impaired Th1 development and show enhanced ability to develop Th2 cells [105]. A locus that controls the maintenance of IL-12 responsiveness and therefore favors the preferential development of Th1 cells has recently been described in B10.D2 mice [106]. This locus maps on a region of chromosome 11, which is synthetic with the locus on human chromosome 5q31.1, shown to be associated with elevated serum IgE levels [90]. Several other genes map within 5q31.1, including possible candidates which might influence IgE production. One of them is IRF1, whose gene product up-regulates IFN-a, which in turn down-regulates IgE production and inhibits Th2 cell development and IL12B, which encodes the b chain of IL-12, another down-regulator of Th2 cells. Either alterations of molecular mechanisms directly involved in the regulation of IL-4 gene expression, or deficient regulatory activity of cytokines responsible for inhibition of Th2-cell development or both, may account for the preferential Th2-type response in atopic people and also for the production by Th2 cells of the cytokines involved in the allergic inflammation. Therefore, it explains the persistent histological, pathophysiological and clinical aspects of allergic disorders.

240

E. Maggi / Immunotechnology 3 (1998) 233–244

6. New immunotherapeutical approaches in atopy Based on these new insights and the biotechnological advances, novel opportunities of treatment of allergic disorders can be hypothesized. It may be addressed to target allergen-specific T cells (allergen-specific immunotherapy-IT) or their effector molecules (non allergen-specific IT).

6.1. Allergen-specific approaches Anergy in allergen-specific Th2 cells in vitro was achieved by incubation of Dermatophagoides pteronyssinus group 1 (Der p 1)-specific Th2 clones with high doses of the relevant Der p 1-derived peptides in the absence of APC [107]. Der p1-specific T cell clones lost the ability to respond to subsequent stimulation with the intact antigen and APC [107], as well as to provide B-cell help for IgE production, even in the presence of exogenous IL-4 and IL-13 [108]. Thus, it is possible to use peptide-induced T cell anergy to treat human allergic diseases. Trials with bee venom phospholipase A2- or Fel d 1-derived peptides has shown good clinical efficacy with no adverse reactions [109]. Another strategy would be to change the cytokine profile of allergen-specific CD4 + T cells. It is reasonable that reversibility of polarized Th2 cell populations is lost after long-term stimulation [110]. However, memory CD4 + T cells can maintain the capacity to be further influenced to the opposite phenotype [111]. In man, memory cells previously committed to the Th2 pattern of cytokine secretion can be modulated to produce cytokines of the opposite phenotype [52,112]. Recently, successful specific IT in vivo, associated with changes in the cytokine profile of allergen-reactive Th2 cells, have been reported [41 – 44]. Thus, a potential approach should be to prime both naive and memory allergen-specific Th cells to select for prevalent Th1 phenotype. Up-regulation of allergen-specific Th1 responses was achieved in vitro by using cytokines, such as IFN-g, IFN-a [52,113] and IL-12 [58]. Several regulatory effects of Th1-inducing cytokines have also been demonstrated in vivo. (i) k.o. Mice for IFN-g receptor have impaired ability to resolve a

lung eosinophilic inflammatory response associated with infiltration of Th2 cells [114]; (ii) nebulized IFN-g decreases IgE production and normalizes airway function in a model of allergen sensitization [115]; (iii) IL-12 can block the antigen-induced airway hyperresponsiveness and pulmonary inflammation in vivo by suppressing Th2 cytokine expression [116]. These data provide evidence that the injection of selected allergen peptides plus Th1-inducing cytokines may represent the basis for a novel immunotherapeutic strategy. The density of antigenic peptide or its affinity for MHC class II molecules can affect the Th1/ Th2 responses; high MHC class II-peptide density favors Th1-like responses, while low ligand densities favour Th2-like responses [48]. The p28–40 analogues (alanine residues at positions 34 and 36 of the dominant T cell epitope of the group 2 mite allergen) were found able to alter the IFN-g/IL-4 ratio production in human Th0 cells by selectively enhancing IFN-g secretion [117]. Also replacement of the 21st residue arginine to lysine of the peptide fragment 18–31 from D. farinae group I allergen resulted in a significant increase in IFN-g production by a specific human Th0 clone [118]. Another novel approach is based on vaccination with plasmid vectors. These vaccines, called naked DNA, consist of a desired gene inserted into a plasmid which can enter cells near to the injection site, where it is transcribed and translated, causing expression of the gene product [119]. This treatment by-passes the problems associated with other vectors, induce the expression of antigens that resemble native epitopes and is constructed so that genes from several different allergens are included on the same plasmid. The gene protein enters the cells MHC class I pathway, thus resulting in the stimulation of CD8 + cytotoxic T cells alone.

6.2. Non allergen specific approaches Potential strategies are also based on targeting Th2 cells or the effector molecules produced as a consequence of activation of Th2 cells. This approach has become conceptually acceptable after the evidence that Th2 responses are not critical for survival and protection. Thus, modifying of

E. Maggi / Immunotechnology 3 (1998) 233–244

either Th2 cells or Th2-dependent effector molecules may be a reasonable form of immunotherapy in patients with severe atopic disorders. Since some transcription factors are critical for IL-4 production and/or Th2 cell development, such as the c-maf oncogene product, STAT6 and GATA3, they may be available targets for manipulating Th2 responses. Indeed mice knockout for STAT6 gene results in deficient Th2 responses [102,103]. IL-4 activity may be antagonized by soluble IL-4 receptors [120] or by a human IL-4 mutant protein [121]; the latter protein also antagonizes the biological activity of IL-13, as well as the IL-4-driven differentiation of Th2 cells in vitro [122]. The IL-5 activity can be antagonized by humanized antibodies to IL-5, which inhibits eosinophil infiltration and normalizes airway hyperreactivity in monkeys challenged with Ascaris suis [123]. Recently developed humanized anti-human IgE antibodies bind to IgE residues critical for receptor binding, not reacting with IgE bound to mast cells or basophils [115]. Interestingly, in housedust-sensitive mice IgE targeting cannot only block the IgE-mediated allergic inflammation, but also down-regulate Th2 responses and the subsequent infiltration of the eosinophils into the airways [115].

7. Concluding remarks There is a general consensus that the Th-cell population contains functionally distinct subsets defined by the patterns of cytokines they produce in response to different types of antigenic stimulation. Although Th1 and Th2 cells were first identified by in vitro analysis of murine T-cell clones, strong evidence now exists for similar subsets in vivo, in mice, rats and humans. These two extremely polarized forms of the specific cellular immune response, evoked by intracellular parasites and gastrointestinal nematodes, respectively, provide a useful model for explaining not only the different types of protection but also the patho-

241

genic mechanisms of several immunopathological disorders. Studies on mechanisms involved in the development of polarized Th1 or Th2 responses have demonstrated that such a development is regulated by either environmental factors, including dose of antigen and nature of immunogen, or other undefined factors in the genetic background, mainly operating at level of the so-called ‘natural immunity’ (co-stimulatory molecules expressed by APC, APC-released cytokines, etc.). Th1-dominated responses are very effective in eradicating infectious agents, including those hidden within the cell; however, if the Th1 response is not effective or excessively prolonged, it may become dangerous for the host, due to both the activity of cytotoxic cytokines and the strong activation of phagocytic cells. In contrast, Th2 responses are not sufficiently protective against the majority of infectious agents, but they provide the best possible protection against some gastrointestinal nematodes. Th2 cells are indeed able to make the life of these complex microrganisms in the body unpleasant and at the same time, they inhibit phagocyte activity, thus limiting attempts to destroy large parasites through Th1-mediated responses that may be harmful to the host itself. Thus, Th2 responses should also be regarded as an important down-regulatory mechanism for exaggerated and/or excessively prolonged Th1 responses. Clearcut evidence suggest that allergen-reactive Th2 cells play an essential role in the activation and/or recruitment of IgE antibody-producing B cells, mast cells and eosinophils, the cellular triad involved in allergic inflammation. Th2 cells provide B cell help for IgE isotype with at least two signals: soluble IL-4 and a T–B cell-to-cell physical interaction, occurring between the CD40L expressed on the activated Th cell and the CD40 molecule on the B cell. The Th2 cellderived IL-4 induces germ line o expression on the B cell, whereas the CD40L/CD40 interaction is required for the expression of productive mRNA and for the synthesis of IgE protein. Other soluble factors produced by both T cells and non-T cells have also been shown to play negative or positive regulatory effects on the human IgE synthesis.

242

E. Maggi / Immunotechnology 3 (1998) 233–244

Th2 cells represent the polarized arm of the effector specific response that plays some role in the protection against nematodes and also act as cross-regulatory cells for chronic and/or excessive Th1-mediated responses. Th2 cells are generated from precursor naive Th cells when they encounter the specific antigen in an IL-4-containing microenvironment. However, the source of IL-4 required at the initiation of response for the development of naive Th cells into Th2 effectors is still unknown. The more likely possibility is that the maturation into the Th2 pathway mainly depends upon the levels and the kinetics of IL-4 production by naive Th cells themselves at priming. The question of how these Th2 cells are selected in atopic patients is also unclear. Both the nature of the TCR signalling provided by the allergen peptide ligand and a disregulation of IL-4 production likely concur to determine the Th2 profile of allergen-specific Th cells, but the genetic disregulation of IL-4 production is certainly overwhelming. Several gene products selectively expressed in Th2 cells or selectively controlling the expression of IL-4 have recently been described. This allows the suggestion that the up-regulation of genes controlling IL-4 expression and/or abnormalities of regulatory mechanisms of Th2 development and/or function may be responsible for Th2 responses against common environmental allergens in atopic people. These findings provide exciting opportunities for the development of novel immunotherapeutic strategies for atopic diseases.

References [1] Romagnani S. Curr Opin Immunol 1994;6:838–46. [2] Mosmann TR, Coffman RL. Annu Rev Immunol 1989;7:145 – 73. [3] Romagnani S. J Clin Immunol 1995;15:121–9. [4] Romagnani S. Annu Rev Immunol 1994;12:227–57. [5] Seder RA, Paul WE. Annu Rev Immunol 1994;12:635– 74. [6] Kapsenberg ML, Wierenga EA, Bos JD, Jansen HM. Immunol Today 1991;12:392–5. [7] Del Prete GF, De Carli M, Mastromauro C, Macchia D, Biagiotti R, Ricci M, Romagnani S. J Clin Invest 1991;88:346 – 51.

[8] Parronchi P, Macchia D, Piccinni M-P, Biswas P, Simonelli C, Maggi E, Ricci M, Ansari AA, Romagnani S. Proc Natl Acad Sci USA 1991;88:4538 – 43. [9] Romagnani S. Immunol Today 1991;12:256 – 7. [10] Del Prete GF, De Carli M, Ricci M, Romagnani S. J Exp Med 1991;174:809 – 13. [11] Del Prete GF, De Carli M, Lammel RM, D’Elios MM, Daniel KC, Giusti B, Abbate R, Romagnani S. Blood 1995;86:250 – 7. [12] Smith CA, Gruss H-J, Davis T, Anderson D, Farrah T, Baker E, Sutherland GR, Brannan CI, Copeland NG, Jenkins NA, Grabstein KH, Glinaik B, McAllister IB, Fanslow W, Alderson M, Falk B, Gimpel S, Gillis S, Din WS, Goodwin R, Armitage RJ. Cell 1993;73:1349 – 60. [13] Del Prete G-F, De Carli M, Almerigogna F, Daniel KC, D’Elios MM, Zancuoghi D, Vinante E, Pizzolo G, Romagnani S. FASEB J 1995;9:81 – 6. [14] Triebel F, Jitsukawa S, Baixeras E, Roman-Roman S, Genevee C, Viegas-Pequignot E, Hercend T. J Exp Med 1990;171:1393– 405. [15] Annunziato F, Manetti R, Tomasevic L, Giudizi M-G, Biagiotti R, Gianno` V, Germano P, Mavilia C, Maggi E, Romagnani S. FASEB J 1996;10:769 – 75. [16] Assenmacher M, Scheffold A, Schmitz J, Segura Checa JA, Miltenyi S, Radbruch A. Eur J Immunol 1996;26:263 – 7. [17] Rogge L, Barberis L, Passini N, Presky DH, Gubler U, Sinigaglia F. J Exp Med 1997;185:825 – 33. [18] Seder RA, Boulay J-L, Finkelman F, Barbier S, BenSasson SZ, Le Gros GG, Paul WE. J Immunol 1992;148:1652– 6. [19] Erard F, Wild M-T, Garcia-Sanz JA, Le Gros G. Science 1993;260:1802– 5. [20] Sad S, Marcotte R, Mosmann TR. Immunity 1995;2:271 – 9. [21] Croft M, Carter L, Swain SL, Dutton RW. J Exp Med 1994;180:1715– 28. [22] Coyle AJ, Erard F, Bertrand C, Walti S, Pircher H, Le Gros G. J Exp Med 1995;181:1229 – 33. [23] Salgame P, Abrams JS, Clayberger K, Goldstein H, Convitt J, Modlin RL, Bloom BR. Science 1991;254:279 – 81. [24] Maggi E, Giudizi M-G, Biagiotti R, Annunziato F, Manetti R, Piccinni M-P, Parronchi P, Sampognaro S, Giannarini L, Zuccati G, Romagnani S. J Exp Med 1994;180:489 – 95. [25] Paganelli R, Scala E, Ansotegui IJ, Ausiello CM, Halapi E, Fanales-belasio E, D’Offizi G, Mezzaroma I, Pandolfi F, Fiorilli M, Aiuti F. J Exp Med 1995;181:423 – 8. [26] Wassenaar A, Reinhardus C, Abraham-Inpijin L, Kievits F. Immunology 1996;87:113 – 8. [27] Birkhofer A, Rehbock J, Fricke H. Eur J Immunol 1996;26:957 – 60. [28] Maggi E, Mazzetti M, Ravina A, Manetti R, De Carli M, Annunziato F, Piccinni MP, Carbonari M, Pesce AM, Del Prete G, Romagnani S. Science 1994;265:244 – 8.

E. Maggi / Immunotechnology 3 (1998) 233–244 [29] Cronin DC, Stack R, Fitch FW. J Immunol 1995;154:3118– 27. [30] Sad S, Mosmann TR. J Exp Med 1995;182:1505–15. [31] Seder RA, Le Gros G. J Exp Med 181;1995:5–7. [32] Manetti R, Annunziato F, Biagiotti R, Giudizi M-G, Piccinni M-P, Giannarini L, Sampognaro S, Parronchi P, Vinante F, Pizzolo G, Maggi E, Romagnani S. J Exp Med 1994;180:2407–12. [33] Maggi E, Biswas P, Del Prete GF, Parronchi P, Macchia D, Simonelli C, Emmi L, De Carli M, Tiri A, Ricci M, Romagnani S. J Immunol 1991;146:1169–74. [34] Kay AB, Ying S, Varney V, Gaga M, Durham SR, Moqbel R, Wardlaw AJ, Hamid Q. J Exp Med 1991;173:775 – 8. [35] Hamid Q, Azzawi M, Ying S, Moqbel R, Wardlaw AJ, Corrigan CJ, Bradley B, Durham SR, Collins JV, Jeffery PK, Quint DJ, Kay AB. J Clin Invest 1991;87:1541–6. [36] Robinson DS, Hamid Q, Ying S, Tsicopoulos A, Barkans J, Bentley AM, Corrigan CJ, Durham SR, Kay AB. New Engl J Med 1992;326:295–304. [37] Bradding P, Feather IH, Wilson S, Bardin PG, Heusser CH, Holgate ST, Howarth PH. J Immunol 1993;151:3853– 60. [38] Walker C, Bode E, Boer L, Hansel TT, Blaser K, Virchow J-C. Am Rev Respir Dis 1992;146:109–15. [39] Del Prete GF, De Carli M, D’Elios MM, Maestrelli P, Ricci M, Fabbri L, Romagnani S. Eur J Immunol 1993;23:1445 – 9. [40] van Reijsen FC, Bruijnzeel-Koomen CAFM, Kalthoff FS, Maggi E, Romagnani S, Westland JKT, Mudde GC. J Allergy Clin Immunol 1992;90:184–92. [41] Varney VA, Hamid Q, Gaga M, Ying S, Jacobson M, Frew AJ, Kay AB, Durham SR. J Clin Invest 1993;92:644 – 51. [42] Secrist H, Chelen CJ, Wen Y, Marshall JD, Umetsu DT. J Exp Med 1993;178:2123–30. [43] Jutel M, Pichler WJ, Skrbic D, Urwyler A, Dahinden C, Muller UR. J Immunol 1995;154:4187–94. [44] McHugh SM, Deighton J, Stewart AG, Lachmann PJ, Ewan PW. Clin Exp Allergy 1995;25:828–38. [45] Schon-Hegrad MA, Oliver J, McMenamin PG, Holt PG. J Exp Med 1995;173:1345–56. [46] Gause WC, Halvorson MJ, Lu P, Greenwald R, Linsley P, Urban JF, Finkelman FD. Immunol Today 1997;18:115 – 20. [47] Del Prete GF, De Carli M, D’Elios MM, Daniel KC, Smith CA, Thomas E, Romagnani S. J Exp Med 1995;182:1 – 7. [48] Pfeiffer C, Stein J, Southwood S, Ketelaar H, Sette A, Bottomly K. J Exp Med 1995;181:1569–74. [49] Kumar V, Bhardwaj V, Soares L, Alexander J, Sette A, Sercarz E. Proc Natl Acad Sci USA 1995;92:9510–4. [50] Reiner SL, Wang Z-E, Hatam F, Scott P, Locksley RM. Science 1993;259:1457–60. [51] Renz H, Saloga J, Bradley KL, Loader JE, Greenstein JL, Larsen G, Gelfand EW. J Immunol 1993;151:1907– 17.

243

[52] Maggi E, Parronchi P, Manetti R, Simonelli C, Piccinni M-P, Santoni-Rugiu F, De Carli M, Ricci M, Romagnani S. J Immunol 1992;148:2142– 7. [53] Swain SL, Weinberg AD, English M, Huston G. J Immunol 1990;145:3796 – 806. [54] Kopf M, Le Gros G, Bachmann M, Lamers MC, Bluthmann H, Kohler G. Nature 1993;362:245 – 8. [55] Romagnani S. Immunol Today 1992;13:379 – 81. [56] Chehimi J, Trinchieri G. J Clin Immunol 1995;14:149 – 61. [57] Hsieh CS, Macatonia SE, Tripp CS, Wolf SF, O’Garra A, Murphy KM. Development of TH1 CD4 + T cells through IL-12 produced by Leisteria-induced macrophages. Science 1993;260:547 – 9. [58] Manetti R, Parronchi P, Giudizi MG, Piccinni MP, Maggi E, Trinchieri G, Romagnani S. J Exp Med 1993;177:1199– 204. [59] Manetti R, Gerosa F, Giudizi MG, Biagiotti R, Parronchi P, Piccinni MP, Sampognaro S, Maggi E, Romagnani S, Trinchiei G. J Exp Med 1994;179:1273 – 83. [60] Thierfelder S, Mocikat R, Mysliwietz J, Lindhofer M, Kremmer E. Eur J Immunol 1995;25:2242 – 6. [61] Seder RA, Paul WE, Davis MM, Fazekas de St. Groth B. J Exp Med 1992;176:1091– 8. [62] Manetti R, Barak V, Piccinni M-P, Sampognaro S, Parronchi P, Maggi E, Dinarello CA, Romagnani S. Res Immunol 1994;145:93 – 100. [63] Rincon M, Anguita J, Nakamura T, Fikrig E, Flavell RA. J Exp Med 1997;185:461 – 9. [64] Rook GAW, Hernandez-Pando R, Lightman SL. Immunol Today 1994;15:301 – 3. [65] Piccinni M-P, Giudizi M-G, Biagiotti R, Beloni L, Giannarini L, Manetti R, Annunziato F, Livi C, Romagnani S, Maggi E. J Immunol 1995;155:128 – 33. [66] Wegmann TG, Lin H, Guilbert L, Mosmann TR. Immunol Today 1993;14:353 – 6. [67] Brunner T, Heusser CH, Dahinden CA. J Exp Med 1993;177:605 – 11. [68] Bradding P, Feather IH, Howarth PH, Meuller R, Roberts JA, Britten K, Bews JPA, Hunt TC, Okayama Y, Heusser CH, Bullock GR, Church MK, Holgate ST. J Exp Med 1992;176:1381– 2. [69] Nonaka M, Nonaka R, Woolley K, Adelroth E, Miura K, Okhawara Y, Glibetic M, Nakano K, O’Birne P, Dolovich J, Jordana M. J Immunol 1995;155:3234 – 44. [70] Nakajima H, Gleich GJ, Kita H. J Immunol 1996;156:4859– 66. [71] Wershil BK, Theodos CM, Galli SJ, Titus RG. J Immunol 1994;152:4563– 71. [72] Schmitz. J, Thiel A, Kuhn R, Rajewsky K, Muller W, Assenmacher M, Radbruch A. J Exp Med 1994;179:1349– 53. [73] Bendelac A, Lantz O, Quimby ME, Yewdell JW, Bennink JR, Butkiewicz RR. Science 1995;268:863 – 5. [74] Yoshimoto T, Paul WE. J Exp Med – 95. [75] Yoshimoto T, Bendelac A, Watson C, Hu-Li J, Paul WE. Science 1995;270:1845– 7.

E. Maggi / Immunotechnology 3 (1998) 233–244

244

[76] Yoshimoto T, Bendelac A, Hu-Li J, Paul WE. Proc Natl Acad Sci USA 1995;92:11931–4. [77] Croft M, Swain SL. J Immunol 1995;154:4269–82. [78] Demeure CE, Yang LP, Byun DG, Ishihara H, Vezzio N, Delespesse G. Eur J Immunol 1995;25:2722–5. [79] Yang L-P, Demeure D-G, Vezzio CE, Delespesse G. Eur J Immunol 1995;12:3517–20. [80] Mingari MC, Maggi E, Cambiaggi A, Annunziato F, Schiavetti F, Manetti R, Moretta L, Romagnani S. Eur J Immunol 1996;26:1083–7. [81] Brinkmann V, Kinzel B, Kristofic C. J Immunol 1996;156:4100– 6. [82] Parronchi P, De Carli M, Manetti R, Simonelli C, Piccinni M-P, Macchia D, Maggi E, Del Prete G-F, Ricci M, Romagnani S. Eur J Immunol 1992;22:1615–20. [83] Piccinni M-P, Beloni L, Giannarini L, Livi C, Scarselli G, Romagnani S, Maggi E. Eur J Immunol 1996;26:2293–8. [84] McMenamin C, Holt PG. J Exp Med 1993;178:889–99. [85] Renz H, Lack G, Saloga J, Schwinzer R, Bradley K, Loader J, Kupfer A, Larsen GL, Gelfand EW. J Immunol 1994;152:351 – 60. [86] Marsh DG, Lockhart A, Holgate ST. The genetics of asthma. Oxford: Blackwell Scientific, 1993. [87] Moffatt MF, Hill MR, Cornelis F, Schou C, Faux JA, Young RP, James AL, Ryan G, le Souef P, Musk AW, Hopkin JM, Cookson WOCM. Lancet 1994;343:1597– 600. [88] Mohapatra SS, Mohapatra S, Yang M, Anari AA, Parronchi P, Maggi E, Romagnani S. Immunology 1994;81:15 – 20. [89] Marsh DG, Neely JD, Breazeale DR, Ghosh B, Freidhoff LR, Ehrlich-Kautzky E, Schou C, Krishnaswamy G, Beaty TH. Science 1994;264:1152–6. [90] Rosenwasser LJ, Klemm DJ, Dresback JK, Inamura H, Mascali JJ, Klinnert M, Borish L. Clin Exp Allergy 1995;25:74 – 8. [91] Abe E, de Waal-Malefyt R, Matsuda I, Arai K, Arai N. Proc Natl Acad Sci USA 1992;89:2864–8. [92] Chuvpilo S, Schomberg C, Gerwig R, Heinfling A, Reeves R, Grummt F, Serfling E. Nucleic Acids Res 1993;21:5694 – 704. [93] Szabo SJ, Gold JS, Murphy TL, Murphy KM. Mol Cell Biol 1993;13:4793 –805. [94] Todd MD, Grusby MJ, Lederer JA, Lacy E, Lichtman AH, Glimcher LH. J Exp Med 1993;177:1663–74. [95] Lederer JA, Liou JS, Todd MD, Glimcher LH, Lichtman AH. J Immunol 1994;152:77–86. [96] Hodge MR, Rooney JW, Glimcher LH. J Immunol 1995;154:6397– 405. [97] Rooney JW, Hoey T, Glimcher LH. Immunity 1995;2:473 – 83. [98] Li-Weber M, Eder A, Ktaf-Czepa H, Krammer PH. J Immunol 1996;148:1913–8. [99] Chan SC, Brown MA, Willcox TM, Li SH, Stevens SR, Tara D, Hanifin JM. J Dermatol Invest 1996;106:1131–6.

.

.

[100] Ho CI, Hodge MR, Rooney JW, Glimcher LH. Cell 1996;85:973 – 83. [101] Lederer JA, Liou JS, Kim S, Rice N, Lichtman AH. J Immunol 1996;156:56 – 63. [102] Takeda K, Tanaka T, Shi W, Matsumoto M, Minami M, Kashiwamura SI, et al. Nature 1996;380:627 – 30. [103] Shimoda K, van Deursen J, Sangster MY, Sarawar SR, Carson RT, Tripp RA, et al. Nature 1996;380:630 – 2. [104] Bacon CM, McVicar DW, Ortaldo JR, Rees RC, O’Shea JJ, Johnston JA. J Exp Med 1995;181:399 – 404. [105] Kaplan MH, Schindler U, Smiley ST, Grusby MJ. Immunity 1996;4:313 – 9. [106] Gorham JD, Guler ML, Steen RG, Mackey AJ, Daly MJ, Frederick K, et al. Proc Natl Acad Sci USA 1996;93:12467– 72. [107] Lord CJM, Lamb JR. Clin Exp Allergy 1996;26:756 – 65. [108] Fasler S, Averso G, Terr A, Thestrup-Pedersen K, de Vries JE, Yssel Y. J Immunol 1995;155:4199– 206. [109] Norman PS, Ohman JL, Long AA. J Allergy Clin Immunol 1994;93:231 – 5. [110] Murphy E, Shibuya K, Hosken N, Openshaw P, Maino V, Davis K, et al. J Exp Med 1996;183:901 – 14. [111] Bradley LM, Dalton DK, Croft M. J Immunol 1996;157:1350– 8. [112] Parronchi P, De Carli M, Manetti R, Simonelli C, Sampognaro S, Piccinni MP, Macchia D, Maggi E, Del Prete GF, Romagnani S. J Immunol 1992;149:2977– 82. [113] Parronchi P, Mohapatra S, Sampogna¸ro S, Giannarini L, Wahn U, Chong P, Mohapatra S, Maggi E, Renz H, Romagani S. J Immunol 1996;26:697 – 703. [114] Coyle AJ, Tsuyuki S, Bertrand C, Huang S, Aguet M, Alkan SS, Anderson GP. J Immunol 1996;156:2680– 5. [115] Lack G, Renz H, Saloga J, Bradley K, Loader J, Leung DYM, Gelfand EW. J Immunol 1994;152:2546– 54. [116] Gavett SH, O’Hearn DJ, Li X, Huang S-K, Finkelman FD, Wills-Karp M. J Exp Med 1995;182:1527 – 36. [117] Tsitoura DC, Verhoef A, Gelder CM, O’Heir R, Lamb JR. J Immunol 1996;157:2160 – 5. [118] Matsuoka T, Kohrogi H, Ando M, Nishimura Y, Matsushita S. J Immunol 1996;157:4837 – 43. [119] Tang D, DeVit M, Johnston SA. Nature 1992;365:152 – 4. [120] Renz H, Enssle K, Lauffer L, Kurrle R, Gelfand EW. Int Arch Allergy Appl Immunol 1995;106:46 – 54. [121] Aversa G, Punnonen J, Cocks BG, de Waal Malefyt R, Vega F Jr., Zurawski SM, et al. J Exp Med 1993;178:2213– 8. [122] Sornasse T, Larenas PV, Davis KA, de Vries JE, Yssel H. Differentiation and stability of T Helper 1 and 2 cells derived from naive human neonatal CD4 + T cells, analyzed at single-cell level. J Exp Med 1996;184:473 – 83. [123] Egan RW, Athwahl D, Chou C-C, Emtage S, Jehn C-H, Kung TT, et al. Int Arch Allergy Appl Immunol 1995;107:321 – 2.