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T helper cell differentiation in immune response
T helper cell differentiation
in immune response
Steven 1 Reiner and Robert A Seder University
of Chicago,
Significant
progress
antigen-specific the network
Chicago
has been
CD4+
T helper
of cytokines,
the differentiation
and National
made
Current
or independence Opinion
molecules,
T helper
knockout mice and well characterized interdependence
understanding
cell responses.
accessory
of distinct
in
Institutes of Health,
cell
the
Bethesda,
maturation
Recent progress
centers
USA
of on
and cell types that shapes
subsets. Use of transgenic
and
in vivo models have helped clarify the of many of these complex factors.
in Immunology
1995,
7:360-366
Introduction
IL-1 2 in T-helper
development
Observations by Parish and Liew [l] in the 1970s indicated that cell-mediated and humoral immunity continue to alternate in reciprocal dominance over each other across a wide dose response of immunization. In the 1980s Mosmann, Co&an and colleagues [2] identified two types of lymphokine patterns produced by long-term cultured CD4+ T-helper (Th) clones that provide help for different arms of the immune system and that have profound counter-regulatory effects on each other. In vivo evidence for a regulatory role for the Thl and Th2 subsets was found shortly thereafier in a mouse model of leishmaniasis [3,4] and in humans with leprosy [5,6]. Two qualitatively opposing responses, such as cell-mediated and humoral immunity, from an immune system that is intrinsically bipotent can regulate a Eworable versus deleterious outcome in several infectious and autoimmune processes. A number of important in vitro and in viva observations have helped elucidate the differentiation process that directs a naive precursor Th (ThP) cell down a particular maturation pathway to become either a Thl or a Th2 cell. Prior understanding of T-helper differentiation was thoroughly reviewed a year ago by Seder and Paul [7] and the reader is encouraged to refer to this for a detailed history of the field. This review will focus on the newest developments that have emerged since February 1994 which have clarified the mechanisms of factors described previously and identified novel elements in the T-helper differentiation process. The discussion will generally be limited to murine models, with some reference to human studies when they have provided unique findings. In many instances Thl responses will be equated with interferon (IFN)-), production and Th2 responses with IL-4 production, although the T-helper cytokines within a given phenotype are not always coordinately regulated.
-IL-12, a heterodimeric cytokine produced primarily by phagocytic cells, induces IFN-y production by natural killer cells. Its role in directing Thl development in several in vitro and in vivo systems is now well established (see [8] for a review). Recent advances in IL-12 biology have elucidated some of the mechanisms by which it acts in the initiation and maintenance of immune responses in vivo. Several studies (see [8] for review) highlight the potency of IL-12 in selectively driving the differentiation of Thl development but the mechanism of action is less clear: some studies suggest that IL-12 can act directly on T cells to suppress IL-4 or increase IFN-y production, but some imply that the effects of IL-12 are mediated through the induction of IFN-y.
IL-12 directs Thl development in viva
The ability of IL-12 to induce Thl responses in vivo was initially established in studies of mice infected with L&mania major. Treatment of susceptible BALB/c mice with recombinant (r)IL-12 can inhibit the usual Th2 response and promote the successful outgrowth of Thl effector cells [9,10] and, when used in combination with parasite antigens as a vaccine, IL-12 preferentially directs the development of a protective Thl population [ll]. The past year has seen these findings amplified in a number of systems. Mice immunized with a hapten-protein conjugate plus IL-12 have dramatically reduced priming for cells producing IL-4, with a marked increase in priming for cells producing IFN-y [12”]. The importance of endogenous IL-12 is highlighted in murine models of candidiasis and toxoplasmosis, in which neutralization of IL-12 abrogated Thl development and impaired survival while promoting Th2-type responses [ 13**, 14**].
Abbreviations APC-antigen-presenting
360
cell; IFN-interferon;
Ig-immunoglobulin;
IL-interleukin;
r-recombinant;
0 Current Biology Ltd ISSN 0952-7915
TCR-T-cell
receptor; Th-T
helper.
T helper cell differentiation in immune response Reiner and Seder 361 IL-12 may not be essential for all Thl responses, nor sufficient to suppress all Th2 responses
Under some in vitro conditions, neutralization of IL-12 can still result in priming for IFN-y production [15]: recent in vivo data suggest that IL-12 p40 knockout mice have diminished Thl responses (M Gately, personal communication). We believe that IL-12 is most important in determining the magnitude of T h l responses during priming of undifferentiated cells. In toxoplasmosis, delayed use of antibody directed against IL-12 does not diminish Thl responses nor adversely affect survival, whereas neutralization of IL-12 early in infection clearly impairs both survival and IFN-y production [14"']. This suggests that some T h l responses in vivo require IL-12 for succesful initiation, but may become self-sustaining or independent of IL-12 once cells are committed to T h l maturation. An immune response generated in the absence of IL-12 may also provide sufficient IFN-y to mediate a Thl-associated outcome; studies with IL-12 p40 knockout mice will determine if this indeed occurs. In treatment of established Th2 responses, rlL-12 is often unable to suppress Th2 and/or induce T h l progression [9,16",17,18], perhaps analogous to how an established T h l response develops IL-12 independence [14"']. Even in some priming situations, such as candidiasis, rlL-12 is not sufficient to override the signals that favor Th2 priming or to cure susceptible animals [13°°]. These results suggest that IL-12 has limitations in downregulating initiation and/or maintenance of some Th2 responses. Effects of IL-12 on Thl induction are primarily IFN-y independent, in contrast to Th2 inhibition
One of the more perplexing questions in Th development is whether IL-12 exerts its effects directly on the T cell or requires induction of IFN-y to mediate Th differentiation. In vitro priming of T C R transgenic T cells with antigen presented by antigen-presenting cells (APCs) and rlL-12 promotes T h l induction even during neutralization of IFN-y [15]. In some systems, however, the effects of IL-12 in augmenting IFN- 3' induction are inhibited by IFN-~, neutralization [15,19]. In combination with optimal concentrations of rlL-4, rlL-12 does not diminish priming for IL-4 [15,20] although it does increase IFN-~/production [15]. When suboptimal concentrations of IL-4 are used, however, addition of IFN-y decreases priming for IL-4, suggesting that the effect oflL-12 on Th2 reduction may be indirect (working through IFN-y) whereas IFN- 3, augmentation may be a direct effect of IL-12 on naive precursor ThP cells. The following in vivo experiments highlight the complexity of these issues. During granuloma formation induced by schistosome eggs and during intestinal helminth expulsion (two prototype models of Th2 development) administration of rlL-12 diminished Th2-mediated responses, but many effects were negated with co-administration of anti-IFN-y antibody [18,21%22"]. In a murine model of graft-versus-host disease, rlL-12 suppressed
Th2-mediated responses in a manner dependent on IFN-y but augmented T h l differentiation even with simultaneous neutralization of IFN-y [17]. In mice treated with anti-IgD antibody, rlL-12 also suppressed splenic IL-4 transcription in an IFN-y dependent manner, although it augmented production of IFN-y in an IFN-y independent fashion [23]. These studies suggest that IL-12 diminishes Th2 cytokine production indirectly, via IFN-3'. In contrast, in IFN-y knockout mice infected with L. major, early IL-4 production was markedly curtailed by treatment with rlL-12 [16"]. In some instances, IL-12 may act directly on CD4 + T cells to suppress IL-4 production, but it is uncertain whether such cells are uncommitted precursors or pre-activated cells whose finite role is to influence Th precursors. We believe that most studies support the notion that the primary effect of IL-12 in inhibiting initiation of a Th2 response is indirect, through its induction of IFN-y, but that IL-12 can suppress IL-4 production directly in some CD4 + subpopulations. The role of these distinct CD4 + T cells is discussed below. Whether IFN-y is necessary for T h l responses induced by IL-12 (or even independent of IL-2) is equally perplexing. The priming for IFN-y production after protein immunization plus rlL-12 is unaffected by neutralization of IFN-y [12"']. In the anti-IgD and graft-versus-host disease models, T h l augmentation also occurred despite anti-IFN-y treatment [17,23]. IFN-y receptor knockout mice mount Thl responses to pseudorabies virus [24] and L. major [25°] even with simultaneous neutralization oflFN-y. Yet, IFN-~/gene knockouts display a default to the Th2 phenotype when challenged with L. major [26"], suggesting that IFN-y is required for T h l induction. Possible explanations for this discrepancy include a genetic difference in Th priming sensitivity between the two strains (R129 versus B6), another molecule existing for IFN-y reception in the receptor knockouts, promiscuous reception in the IFN-y knockout mice such that a Th2-inducing signal is transmitted via the IFN-y receptor, or that IFN-y receptor serves a negative regulatory function in homeostasis by associating or tying-up molecules with positive functions. The latter hypothesis could explain why the T h l response seen in the IFN-y receptor knockouts is, paradoxically, greater than that of wild-type mice [25°]. These results suggest that IL-12 can often direct Thl development in the absence of IFN-y (especially in those instances wherein pharmacologic rlL-12 is used) but that some priming situations require IFN-y to avert a Th2 default. Figure 1 represents our view of the role of cytokines in directing Th development.
The role of IL-12 in costimulation
In addition to favoring the differentiation of Thl cells from a naive precursor, IL-12 acts as a second-signal growth factor for T h l , but not Th2 or (Th0), clones [27,28°]. This is consistent with the finding that both B7-1 and IL-12 are required for optimal activation of T h l clones [29] whereas naive T cells do not display
362
Lymphocyteactivation and effector functions Accessory cell
and it leaves open the posssibility that other factors apart from IL-10 and IFN-y regulate IL-12.
IL-10
IL-10 ~ . ~
f_.N
Macrophage (also dendritic cell, keratinocyte) /~----~Thl
IL-12
IL-4 + CD4 T cell (also mast cell,
Th2 IL-4
Va, CD8) © 1995 Current Opinion in Immunology
Fig. 1. The relationship between key cytokines during T-helper development. IL-12 and IL-4 are the most potent factors for priming Thl and Th2 responses by acting directly on the T cells to induce IFN-y and IL-4 production, respectively. IL-12 inhibition of IL-4 production appears, under many circumstances, to be indirect (through induction of IFN-7). In some instances, IL-12 is able to act directly on a population of early IL-4 producing cells that may have a distinct lineage from the naive Th precursors (ThP). IL-10 is derived from accessory cells (and possibly Th cells); although it acts to downregulate IL-12 production/'rhl augmentation, IL-12 has been found to induce IL-10 production thus providing a potential homeostatic feedback loop.
the same requirement for IL-12. The failure of IL-12 neutralization to reverse an established T h l response in vivo [14"°] stands in stark contrast to in vitro findings of IL-12's indispensability and suggests that either an in vivo T h l response is less terminally differentiated than T h l clones in vitro, or that other in vivo factors can act to supplant the lack o f IL-12, or 'optimal' augmentation in IFN-y production seen in vitro is an extreme state beyond in vivo relevance. Regulation of IL-12 IL-10, which was initially defined as an inhibitor of T h l cytokine production may act through inhibiting IL-12 production [28°] or downregulating B7-1 [30 °] on APCs (see Fig. 1). The observed effects o f IL-10 on T h l inhibition are intriguing given the finding in many in vivo systems that rlL-12 induces IL-10 [13°°,16",18,21°,23]. The source o f the IL-10 is probably not T cells as it is produced in severe combined immune deficient (SCID) mice [16°] and is not produced during the restimulation o f CD4 + T cells [22°]. The ability of IL-12 to increase the production of IL-10 may serve a homeostatic function, controlling a T h l response, perhaps by ameliorating pro-inflammatory effects that a prolonged response could elicit. In contrast to the negative regulatory effects of IL-10 on IL-12, IFN-y may give positive feedback for IL-12 induction as p40 m R N A is reduced when mice are treated with antibodies to IFN-T [21°]. This is not surprising given the ability of IFN-y to generally activate macrophages,
Several in vivo systems have used treatment with rlL-12 to induce T h l responses therapeutically. In murine models of AIDS, malaria, and L. donovani infection and histoplasmosis, the presence of rlL-12 effectively led to the protection o f the mice, whereas simultaneous neutralization of IFN- 3' abrogated protection ([31-33]; P Zhou et al., unpublished data; see also Note added in proot). In these instances, it is not clear whether neutralization of IFN-~, merely eliminates a key effector mechanism or actually impairs successful T h l development. Conversely, antibodies against IL-12 induced protection in the murine experimental autoimmune encephalomyelitis (EAE) system [34]. These studies highlight some o f the increasing applications of IL-12 and its blockade.
Costimulation and T-helper development Apart from the intense investigation o f the role o f IL-12 in Th development, an area that has grown considerably in the past year has been the role of costimulatory molecules, most notably the T cell activation receptors CD28 and CTLA-4 and their ligands B7-1 and B7-2, in the development of T h effector functions. The function o f these molecules in T-cell activation has recently been reviewed [35] and is covered in this issue (Sharpe, pp 389-395), but the following studies help ascribe more precise functions during T h differentiation. CD28 knockout mice have impaired Ig production, but preserved cytotoxic T lymphocyte and delayed type hypersensitivity responses [36]. Mice transgenic for soluble C T L A - 4 - I g molecule [37,38] have severe impairment in T-dependent B-cell responses and germinal center formation, whereas their T cells produce higher levels of IFN-y but lower levels of IL-4. In three in vivo models of Th2 development (anti-IgD administration, infection with Heligmosomoides polygyrus, and infection with L. major) C T L A - 4 - I g treatment had a profound effect in blocking the generation of IL-4 producing cells [39°,40°,41°°]. C T L A - 4 - I g administration to resistant C57BL/6 mice did not impair their ability to resist infection with L. major [41°°], and CD28 knockout mice on a resistant background have normal development of T h l responses and healing (SL Reiner, unpublished data). These results suggest that the initial interaction o f CD28 (or possibly CTLA-4) with its B7 ligand is probably required for priming Th2 responses but is non-essential for some T h l responses. The recent findings that B7-2 becomes rapidly upregulated on dendritic cells [42] and that both B7 ligands can be induced quickly on a wide variety of APCs bearing M H C class II [43] support their potential role at initiation of any immune response. Recent findings using selective blockade of B7-1 or B7-2 suggest unique roles for the B7 ligands in preferential Th development [44°]. Priming or restimulation
T
of T C R transgenic T cells leads to increased IL-4 production in the presence of anti-B7-1, but increased IFN-~ production in the presence of anti-B7-2. When anti-B7-1 was used in an experimental autoimmune encephalomyelitis model, disease severity was reduced (and IL-4 production was augmented), whereas the use of anti-B7-2 increased disease severity (and augmented IFN-~/ production) [44"]. A role for B7-1 in Thl development is supported by the finding that B7-1 costimulation increases the number of IL-2 m R N A positive cells among the T h l and Th0 clones but is less effective in increasing the number of Th2 cells that produce IL-4 [45°]. Given the finding that adequately primed Th2 responses" may become independent of costimulation [46"] it would be interesting to determine whether delayed blockade of anti-B7-2 following an adequately primed Th2 response can switch cells to a Thl phenotype by facilitating unopposed B7-1 costimulation. Despite these intriguing results, many of the mechanisms ofcostimulation and its blockade are still unresolved, especially those concerning the direct effects on Th cytokine signalling versus the indirect effects through induction or suppression of other cytokines, whether the blocking reagents being used have any agonistic effect on cytokine induction, and at what stage of Th initiation or maintenance specific costimulatory interactions are most important.
helper cell differentiation in immune responseReiner and Seder 363 CD8 effector populations can be primed into distinct patterns oflymphokine production analogous to Th cells [54"]. The role of these cells may be to augment, rather than initiate, Th development but in systems where pathogenesis favors initial antigen presentation through M H C class I, these cells may have primary influence as the first populations to become producers of IL-4 or IFN-y. Established Thl and Th2 lines from TCIL transgenic mice are long-lived and retain their cytokine phenotype after being adoptively transferred into normal recipients [55]. These cells may be an important source of priming cytokines during a chronic immune response that educates newly activated naive, precursor ThP cells during epitope spreading. Although the predominant source oflL-12 during in vivo immune responses appears to be macrophages, especially when infected by intracellular pathogens, dendritic cells (and keratinocytes) which may be initiators of immune responses in vivo also produce IL-12 [56",57]. Using dendritic cells as APCs to prime TC1L transgenic T cells, neutralization of endogenous IL-12 diminished IFN-y production, suggesting that much of the unprimed IFN-y produced without added rlL-12 is actually IL-12 dependent [56"]. As pathogen-stimulated macophages are the most prolific source of IL-12, it is possible that IL-12 derived from dendritic cells functions by initiating T h l responses that proceed in a more gradual manner.
Sources of cytokines that influence and augment Although other variables (such as antigen dose, type of APC and costimulatory signal) may favor Thl or Th2 development, no factor has been identified in the effective differentiation of these subsets that is more dominant than the effects oflL-12 and IL-4, respectively (reviewed in [7]). Intense interest over the past year has focused on determining the cellular sources of the IL-4 that primes Th2 responses (see Fig. 1). In the murine L. major model, there is a burst in IL-4 transcription from CD4 + cells four days a~er infection in both susceptible and resistant mice [47"]. Similar findings were made in the schistosome egg granuloma and intestinal helminth models [48,49]. In adoptive transfer studies with IL-4 knockout mice, CD4 + rather than CD4- cells are the priming source of IL-4 for Th2-mediated responses [50"]. A potential source for early IL-4 production by a CD4 + cell population includes the recently identified NKI.1 + CD4 + cell (reviewed in this issue by Bendelac, pp 367-374) which was the predominant source of IL-4 in one model of in vivo T-cell activation [51"]. A pre-activated T cell as priming source is also supported by studies in which MEL-14 low ('memory') cells were able to produce the IL-4 that drove the MEL-14 high ('naive') cells to become Th2-like [52"']. Another possible source of early IL-4 production was found among ~fi T cells from mice infeced with Nippostrongylus brasiliensis [53]. In listeriosis, "y6 T cells contained intracellular IFN-y early in infection, implicating a role in Th differentiation depending on the immune stimulus [53]. Related findings demonstrate that
Conclusions: genetic polymorphism and future directions Increasing interest lies in many of the in vitro and in vivo systems that have striking differences in Th outcome depending on the genetic background of the mouse. Murine leishmaniasis and candidiasis represent two well characterized examples, but genetic differences in T helper cell development are now known to play a role in the pathogenesis of autoimmune diabetes [58"], intestinal helminth infections [59"], and Lyme disease [60"]. The way in which a fixed antigen or set of antigens is capable of eliciting varied reponses in different genetic backgrounds is perplexing. One such example is that when BALB T cells are polyclonally activated in the presence of IL-4, Th2 priming is enhanced rather than supressed by the presence of IL-12 [61]. Results of experiments with T C R transgenic mice bred onto differing backgrounds indicate that some strains develop distinct patterns of lymphokine production when primed with the same antigen-APC, and that this difference segregates to the T-cell compartment [62"]. This could explain many earlier discrepancies noted from in vitro priming data using different systems (reviewed in [7]). The ability to reconstitute important cellular differences seen in disease models should pave the way for a more detailed molecular understanding of some of the factors that make certain strains have divergent priming patterns under fixed conditions. Common cytokine receptor usage (reviewed in this issue by Bolen, pp 306-311) and related transcription factor pathways
364
Lymphocyteactivationand effectorfunctions [63,64] are areas where minor polymorphism could regulate major differences in T-helper development. The past year has seen a number o f important advances concerning the selective development o f differing immune responses. In addition to a more detailed understanding o f the roles of IL-12 and costimulatory molecules in this process, we have gained a greater appreciation o f the complexities and subtleties o f the homeostatic network. Although the immune system often strives to mount an optimal T h l or Th2 response against certain pathogens, there are probably many instances in which a combination 0 f t h e two responses is more appropriate, and other instances where the failure to restrain a polarized response is frankly deleterious. We predict that the coming year will yield many advances in elucidating the cellular and molecular basis o f Th differentiation, both in differing genetic backgrounds and in response to a variety o f foreign and endogenous stimuli.
Note added in proof The study referred to in the text as P Zhou et al., unpublished data, has been accepted for publication [65].
Acknowledgements The authors wish to thank many colleagues for helpful discussions. SL R.einer is a recipient of a Clinical Investigator Award from the National Institutes of Health (AI01309) and a Burroughs Wellcome Fund New Investigator Award in Molecular Parasitology.
8.
Trinchieri G, Scott P: The role of interleukin 12 in the immune response, disease and therapy. Immunol Today 1994, 15:460-463.
9.
Heinzel FP, Schoenhaut DS, Rerko RM, Rosser LE, Gately MK: Recombinant interleukin 12 cures mice infected with Leishmania major. J Exp Med 1993, 177:1505-1509.
10.
Sypek JP, Chung CL, Mayor SEH, Subramanyam JM, Goldman SJ, Sieburth DS, Wolf SF, Schaub RG: Resolution of cutaneous leishmaniasis: interleukin 12 initiates a protective T helper type 1 immune response. J Exp Med 1993, 177:1797-1802.
11.
Afonso LCC, Scharton TM, Vieira LQ, Wysocka M, Trinchieri G, Scott P: The adjuvant effect of interleukin-12 in a vaccine against Leishmania major. Science 1994, 263:235-237.
12. ""
McKnight AJ, Zimmer G/, Fogelman I, Wolf SF, Abbas AK: Effectsof IL-12 on helper T cell-dependent immune responses in vivo. J Immunol 1994, 152:2172-2179. During protein immunization, IL-12 treatment enhanced interferon (IFN)y producing T cells and inhibited IL-4 secreting cells. The effect of IL-12 on increasing IFN-y production was not prevented by co-administration of a neutralizing antibody to IFN-y, supporting the view that IL-12 acts directly on precursor Th cells to cause Thl development. 13. ""
Romani L, Mencacci A, Tonnetti L, Spaccapelo R, Cenci E, Puccetti P, Wolf SF, Bistoni F: IL-12 is both required and prognostic in vivo for T helper type 1 differentiation in murine candidiasis. J Immunol 1994, 152:5167-5175. In contrast to the Leishmania major model, recombinant IL-12 cannot cure susceptible mice (although anti-lL-12 causes susceptibility). The genetics and probably the priming events of the low virulence Candida model (DBA/2 mice are susceptible/lh2 responders, BALB/c mice are resistant/Thl responders) are distinct from those involved in L. major infection. This work points to the complexity of innate immune signals and the limitations in IL-12 uses. 14. ""
Gazzinelli RT, Wysocka M, Hayashi S, Denkers EY, Hieny S, Caspar P, Trinchieri G, Sher A: Parasite-induced IL-12 stimulates early IFN-y synthesis and resistance during acute infection with Toxoplasma gondii. J Immunol 1994, 153:2533-2543. Important study illustrating that an IL-12 dependent interferon-y response becomes independent of IL-12 once maturation or the maintenance phase of the response is established.
References and recommended reading
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16. •
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34.
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38.
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21. •
22.
Oswald IF), Caspar P, Jankovic D, Wynn TA, Pearce EJ, Sher A: IL-12 inhibits Th2 cytokine responses induced by eggs of Schistosoma mansoni. J Immunol 1994, 153:1707-]713. In contrast to transcriptional analysis of unsorted tissue which shows that recombinant IL-12 causes an increase in IL-10 mRNA, restimulation of purified CD4 + cells shows no such increase, suggesting that the source of IL-10 is accessory cells. •
23.
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24.
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25. •
Swihart K, Froth U, Messmer N, Hug K, 8ehin R, Huang S, [:)el Giudice G, Aguet M, Louis JA: Mice from a genetically resistant background lacking the interferon y receptor are susceptible to infection with leishmania major but mount a polarized T helper cell 1-type CD4 + T cell response. J Exp Med 1995, 181:961-971. See annotation [26•1. 26. •
Wang Z-E, Reiner SL, Zheng S, Dalton DK, Locksley RM: CD4 + effector cells default to the Th2 pathway in interferon y-deflcient mice infected with Leishmania major. J Exp Med 1994, 179:1367-1371. Along with [25•], this paper provides perplexing results on dispensability versus necessity of interferon-y for T helper type 1 responses. Points out potential artifacts that can arise through use of mutant strains: unless the discrepancy is merely one of background genes, some feature of the deletion in one of these strains must explain the differences in outcome. 27.
Yanagida 1, Kato T, Igarashi O, Inoue T, Nariuchi H: Second signal activity of IL-12 on the proliferation and IL-2R expression of T helper cell-1 clone. J Immunol 1994, 152:4919-4928.
28. •
Kennedy MK, Picha KS, Shanebeck KD, Anderson DM, Grabstein KH: Interleukin-12 regulates the proliferation of Thl, but not Th2 or Th0, clones. Eur J Immunol 1994, 24:2271-2278. Suggests that one potential mechanism for IL-10's activity is the downregulation of IL-12 production by antigen-presenting cells. 29.
Murphy EE, Terres G, Macatonia SE, Hsieh C-S, Mattson J, Lanier L, W~/socka M, Trinchieri G, Murphy K, O'Garra A: B7 and interleukin 12 cooperate for proliferation and interferon-y production by mouse T helper clones that are unresponsive to B7 costimulation. J Exp Med 1994, 180:223-231.
Ding L, Linsley PS, Huang L-Y, Germain RN, Shevach EM: IL-10 inhibits macrophage costimulatory activity by selectively inhibiting the up-regulatlon of B7 expression. J Immunol 1993, 151:1224-1234. Suggests that another potential activity of IL-10 is inhibiting the production of interferon-y.
39. *
Lu P, Zhou XD, Chen S-J, Moorman M, Morris SC, Finkelman FD, Linsley P, Urban JF, Gause WC: CTLA-4 ligands are required to induce an in vlvo interleukin 4 response to a gastrointestinal nematode parasite. J Exp Med 1994, 180:693-698. Examines the role of costimulaton/signals mediated through CTLA-4 and its ligands on T helper cell immune responses in animals infected with Heligmosomoides polygyrus. Infected animals have increased T-cell derived IL-4 gene expression, but animals treated with CTLA-4-1g showed a striking decrease in IL-4 expression. These results support a role for costimulation through CTLA-4 and its ligands in Th2 cytokine expression. 40. •
Lu P, Zhou XD, Chen S-J, Moorman M, Schoneveld A, Morris S, Finkelman FD, Linsley P, Claassen E, Gause WC: Requirement of CTLA-4 counter receptors for IL.4 but not IL-10 elevations during a primary systemic in vivo immune response. J Immunol 1995, 154:1078-1087. Another model system that demonstrates the importance of costimulation for priming of IL-4 production. Points out that IL-4 and IL-10 are not coordinately regulated. 41. ••
1994, 153:4142-4148.
Transient blockade of costimulation abrogates T helper type 2 development in susceptible BALB/c mice without affecting Th] development in these mice or in resistant C57BL/6 mice. Sustained blockade in BALB/c diminishes healing, suggesting a possible role for costimulation in maintenance of Thl responses. 42.
Inaba K, Witmer-Pack M, Inaba M, Hathcock KS, Sakuta H, Azuma M, Yagita H, Okumura K, Linsley PS, Ikehara S eta/.: The tissue distribution of the B7-2 costimulator in mice: abundant expression on dendritic cells in situ and during maturation in vitro. J Exp Med 1994, 180:1849-1860.
43.
Hathcock KS, Laszlo G, Pucillo C, Linsley P, Hodes RJ: Comparative analysis of B7-1 and B7-2 costimulatory ligands: expression and function. J Exp Med ]994, 180:631-640.
30. •
31.
Gazzinelli RT, Giese NA, Morse HC Ilk In vivo treatment with interleukin 12 protects mice from immune abnormalities observed during murlne acquired immunodeficiency syndrome (MAIDS). J Exp Med 1994, 180:2199-2208.
32.
SedegahM, Finkelman F, Hoffman SL: Interleukin 12 induction of interferon y-dependent protection against malaria. Proc Nat/ Acad Sci USA 1994, 91:10700-10702.
Corn/ DB, Reiner SL, Linsley PS, Locksley RM: Differential effects of blockade of CD28-B7 on the development of Thl or Th2 effector cells in experimental leishmanlasis. ] Immuno/
44. •
Kuchroo VK, Das MP, Brown JA, Ranger AM, Zamvil SS, Sobel RA, Weiner HL, Nabavi N, Glimcher LH: B7-1 and B7-2 costlmulatory molecules activate differentially the Thl/Th2 developmental pathways: application to autoimmune disease therapy. Cell 1995, 80:707-718. Selective inhibition of 87-1 or B7-2 affects the cytokine profile of T cells stimulated in vitro or in vivo. Shows that B7-1 is preferentially a costimulator of Thl, and that B7-2 favors the induction of Th2 cells. 45. •
Bucy RP, Panoskaltsis-Mortari A, Huang G-Q, Li J, Karr L, Ross M, Russell JH, Murphy KM, Weaver CT: Heterogeneity of single cell cytokine gene expression in clonal T cell populations. J Exp Med 1994, 180:1251-1262.
365
366
Lymphocyte activation and effector functions Examines single cell cytokine mRNA transcription and points out that increasing antigen dose in a clonal T helper type 1 (Thl) or Th2 population serves to increase the frequency of cytokine-positive cells, rather than to increase cytokine transcripts within a given cell. 46.
McKnight AJ, Perez VL, Shea CM, Gray GS, Abbas AK:
•
Costimulalor dependence of lymphokine secretion by naive and activated CD4 + T lymphocytes from TCR transgenic mice. J Immunol 1994, 152:5220-5225.
Suggests that costimulation is necessary for initiation of Th2 responses but that it can become dispensible if the Th2 response had been adequately primed. 47. "•
Reiner SL, Zheng S, Wang Z-E, Stowring L, Locksley RM: Leishmania promastigotes evade interleukin 12 (IL-12)
55.
Swain SL: Generation and in vivo persistence of polarized Thl and Th2 memory cells. Immunity 1994, 1:543-552.
56. •
Macatonia SE, Hosken NA, Litton M, Vieira P, Hsieh C-S, Culpepper IA, Wysocka M, Trinchieri G, Murphy KM, O'Garra
A: Dendritic cells produce intedeukin-12 and direct the development of Thl cells from naive CD4+ T cells. J Immunol 1995, 154:5071-5079. IL-12 derived from dendritic cells is responsible for inducing the production of a large portion of the IFN-y in the absence of other cytokines, and may serve to provide a homeostatic function in balancing newly primed immune responses. 57.
induction by macropbages and stimulate a broad range of cytokines from CD4 + T cells during initiation of infection. J Exp Med 1994, 179:447-456. The insect form of Leishmania does not induce IL-12, unlike the
I: IL-12 is expressed and released by human keratinocytes and epidermoid carcinoma cell lines. J Immunol 1994, 153:5366-5372.
mammalian form. A burst in IL-4 production and increase in IL-12 production are features common to both susceptible and resistant mice. Despite uniform priming by a combination of IL-4 and IL-12, most strains develop T helper type 1 (Thl) effectors but BALB/c mice develop Th2 effectors. The IL-4 burst is derived from CD4 + cells; this was also found in [48] (dealing with immune responses to schistosome egg) and [49] (heiminthic stimulation).
58. •
48.
Wynn TA, Eltoum I, Cheever AW, Lewis FA, Gause WC, Sher A: Analysis of cytokine mRNA expression during primary granuloma formation induced by eggs of Schistosoma mansoni. J Immunol 1993, 151:1430-1440.
•
Svetic A, Madden KB, Zhou XD, Lu P, Katona IM, Finkelman FD, Urban JF Jr, Gause WC: A primary intestinal
60. •
49.
helminthic infection rapidly induces a gut-associated elevation of Th2-associated cytokines and IL-3. J Immunol 1993, 150:3434-3441. 50. "•
Scott B, Liblau R, Degermann S, Marconi LA, Ogata L, Caton AI, McDevitt HO, Lo D: A role for non-MHC genetic polymorphism in susceptibility to spontaneous autoimmunity. Immunity 1994, 1:73-82. Highlights an emerging theme of Thl.-mediated pathology and Th2mediated protection in autoimmune syndromes. 59.
Matyniak IE, Reiner SL: T helper phenotype and genetic susceptibility in experimental Lyme disease. J Exp Med 1995, 181:1251-1254. Highlights frequency of T-helper polymorphism as cause of varying genetics of susceptibility. 61.
expression in T helper (Th) cells is not dependent on IL-4 from non-Th cells. J Exp Med 1994, 179:1349-1353. Suggests that an innate immune system component (mast cell or basophil) is not the priming element of Th2 responses; rather, it is the CD4 + cell, itself. Complements findings of an early IL-4 source seen in parasitic models ]47••,48,49].
62. •
Yoshimoto 1, Paul WE: CD41~n, NKI.lPOS T cells promptly •" produce interleukin 4 in response to in vivo cballenge with anti-CD3. J Exp Med 1994, 179:1285-1295. Ceils that produce early IL-4 in response to in vivo anti-CD3 stimulation are rich in CD4 + cells that have the phenotype CD44bright, CD45RB dull and NK1 .lp°s. These cells may provide a source of IL-4 that influences the development of Th2-1ike cells. 52. •"
Gollob KJ, Coffman RL: A minority subpopulation of CD4 + T' cells directs the development of naive CD4 + T cells into IL-4-secreting cells. J Immunol 1994, 152:5180-5188.
Another study in which CO4 + cells are subclassified and in which experiments demonstrate that MEL-14 low cells provide the priming doses of IL-4 necessary to make naive cells differentiate into Th2-1ike cells. 53.
Ferrick DA, Schrenzel MD, Mulvania I, Hsieh B, Ferlin WG, Lepper H: Differential production of interferon-y and inlerleukin-4 in response to Thl- and Th2-stimulating pathogens by ~i T cells in vivo. Nature 1995, 373:255-257.
54. •
Croft M, Carter L, Swain SL, Dutton RW: Generation of polarized antigen-specific CD8 effector populations: reciprocal action of interleukin (IL)-4 and IL-12 in promoting type 2 versus type 1 cytokine profiles. J Exp Med 1994,
180:1715-1728. This study confirms earlier work demonstrating that CD8 + T cells can be primed in vitro to produce IL-4 if IL-4 is present in the priming culture.
Else KJ, Finkelman FD, Maliszewski CR, Grencis RK: Cytokinemediated regulation of chronic intestinal helminth infection. J Exp Med 1994, 179:347-351.
Along with [60•], demonstrates that Th2 responses may be protective in certain infectious diseases, depending on the pathogenesis.
Schmitz J, Thiel A, K~hn R, Rajewsky K, M(Jller W, Assenmacher M, Radbruch A: Induction of interleukin 4 (IL-4)
51.
Aragane Y, Riemann H, Bhardwaj RS, Schwarz A, Sawada Y, Yamada H, Luger TA, Kubin M, Trinchieri G, Schwarz
Schmitt E, Hoehn P, Germann 1, ROde E: Differential effects of interleukln-12 on the development of naive mouse CD4 + cells. Eur J Immunol 1994, 24:343-347. Hsieh C-S, Macatonia SE, O'Garra A, Murphy KM: T cell
genetic background determines default T helper phenotype development in vitro. J Exp Med 1995, 181:713-721.
Interesting cellular model of varying cytokine profile among inbred strains in response to fixed priming. The T-cell compartment dictates the genotypic difference when antigen-presenting cells and T cells are crossed. 63.
Rothman P, Kreider B, Azam M, Levy D, Wegenka U, Eilers A, Decker I-, Horn F, Kashleva H, lhle l, Schindler C: Cylokines and growth factors signal through tyrosine phosphorylation of a family of related transcription factors. Immunity 1994, 1:457~,68.
64.
Darnell JE Jr, Kerr iM, Stark GR: Jak.STAT pathways and transcriptional activation in response to IFNs and other extracellular signaling proteins. Science 1994, 264:1415-1421.
65.
Zhou P, Sieve MC, Bennett J, Kwon-Chung KJ, Tewari P, Gazzinelli RT, Sher A, Seder RA: It-2 prevents mortality in
mice infected ,with Histoplasma Capsulatum through induction of IFN-y, J Immunol 1995, in press: SL Reiner, Gwenn Knapp Center for Lupus and Immunology Research, Department of Medicine and Committee on Immunology, University of Chicago, 924 E 57th St, Chicago, IL 60637-5420, USA. R A Seder, Lymphokine Regulation Unit, National Institute for Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA.
in immune response
Steven 1 Reiner and Robert A Seder University
of Chicago,
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progress
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Chicago
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in Immunology
1995,
7:360-366
Introduction
IL-1 2 in T-helper
development
Observations by Parish and Liew [l] in the 1970s indicated that cell-mediated and humoral immunity continue to alternate in reciprocal dominance over each other across a wide dose response of immunization. In the 1980s Mosmann, Co&an and colleagues [2] identified two types of lymphokine patterns produced by long-term cultured CD4+ T-helper (Th) clones that provide help for different arms of the immune system and that have profound counter-regulatory effects on each other. In vivo evidence for a regulatory role for the Thl and Th2 subsets was found shortly thereafier in a mouse model of leishmaniasis [3,4] and in humans with leprosy [5,6]. Two qualitatively opposing responses, such as cell-mediated and humoral immunity, from an immune system that is intrinsically bipotent can regulate a Eworable versus deleterious outcome in several infectious and autoimmune processes. A number of important in vitro and in viva observations have helped elucidate the differentiation process that directs a naive precursor Th (ThP) cell down a particular maturation pathway to become either a Thl or a Th2 cell. Prior understanding of T-helper differentiation was thoroughly reviewed a year ago by Seder and Paul [7] and the reader is encouraged to refer to this for a detailed history of the field. This review will focus on the newest developments that have emerged since February 1994 which have clarified the mechanisms of factors described previously and identified novel elements in the T-helper differentiation process. The discussion will generally be limited to murine models, with some reference to human studies when they have provided unique findings. In many instances Thl responses will be equated with interferon (IFN)-), production and Th2 responses with IL-4 production, although the T-helper cytokines within a given phenotype are not always coordinately regulated.
-IL-12, a heterodimeric cytokine produced primarily by phagocytic cells, induces IFN-y production by natural killer cells. Its role in directing Thl development in several in vitro and in vivo systems is now well established (see [8] for a review). Recent advances in IL-12 biology have elucidated some of the mechanisms by which it acts in the initiation and maintenance of immune responses in vivo. Several studies (see [8] for review) highlight the potency of IL-12 in selectively driving the differentiation of Thl development but the mechanism of action is less clear: some studies suggest that IL-12 can act directly on T cells to suppress IL-4 or increase IFN-y production, but some imply that the effects of IL-12 are mediated through the induction of IFN-y.
IL-12 directs Thl development in viva
The ability of IL-12 to induce Thl responses in vivo was initially established in studies of mice infected with L&mania major. Treatment of susceptible BALB/c mice with recombinant (r)IL-12 can inhibit the usual Th2 response and promote the successful outgrowth of Thl effector cells [9,10] and, when used in combination with parasite antigens as a vaccine, IL-12 preferentially directs the development of a protective Thl population [ll]. The past year has seen these findings amplified in a number of systems. Mice immunized with a hapten-protein conjugate plus IL-12 have dramatically reduced priming for cells producing IL-4, with a marked increase in priming for cells producing IFN-y [12”]. The importance of endogenous IL-12 is highlighted in murine models of candidiasis and toxoplasmosis, in which neutralization of IL-12 abrogated Thl development and impaired survival while promoting Th2-type responses [ 13**, 14**].
Abbreviations APC-antigen-presenting
360
cell; IFN-interferon;
Ig-immunoglobulin;
IL-interleukin;
r-recombinant;
0 Current Biology Ltd ISSN 0952-7915
TCR-T-cell
receptor; Th-T
helper.
T helper cell differentiation in immune response Reiner and Seder 361 IL-12 may not be essential for all Thl responses, nor sufficient to suppress all Th2 responses
Under some in vitro conditions, neutralization of IL-12 can still result in priming for IFN-y production [15]: recent in vivo data suggest that IL-12 p40 knockout mice have diminished Thl responses (M Gately, personal communication). We believe that IL-12 is most important in determining the magnitude of T h l responses during priming of undifferentiated cells. In toxoplasmosis, delayed use of antibody directed against IL-12 does not diminish Thl responses nor adversely affect survival, whereas neutralization of IL-12 early in infection clearly impairs both survival and IFN-y production [14"']. This suggests that some T h l responses in vivo require IL-12 for succesful initiation, but may become self-sustaining or independent of IL-12 once cells are committed to T h l maturation. An immune response generated in the absence of IL-12 may also provide sufficient IFN-y to mediate a Thl-associated outcome; studies with IL-12 p40 knockout mice will determine if this indeed occurs. In treatment of established Th2 responses, rlL-12 is often unable to suppress Th2 and/or induce T h l progression [9,16",17,18], perhaps analogous to how an established T h l response develops IL-12 independence [14"']. Even in some priming situations, such as candidiasis, rlL-12 is not sufficient to override the signals that favor Th2 priming or to cure susceptible animals [13°°]. These results suggest that IL-12 has limitations in downregulating initiation and/or maintenance of some Th2 responses. Effects of IL-12 on Thl induction are primarily IFN-y independent, in contrast to Th2 inhibition
One of the more perplexing questions in Th development is whether IL-12 exerts its effects directly on the T cell or requires induction of IFN-y to mediate Th differentiation. In vitro priming of T C R transgenic T cells with antigen presented by antigen-presenting cells (APCs) and rlL-12 promotes T h l induction even during neutralization of IFN-y [15]. In some systems, however, the effects of IL-12 in augmenting IFN- 3' induction are inhibited by IFN-~, neutralization [15,19]. In combination with optimal concentrations of rlL-4, rlL-12 does not diminish priming for IL-4 [15,20] although it does increase IFN-~/production [15]. When suboptimal concentrations of IL-4 are used, however, addition of IFN-y decreases priming for IL-4, suggesting that the effect oflL-12 on Th2 reduction may be indirect (working through IFN-y) whereas IFN- 3, augmentation may be a direct effect of IL-12 on naive precursor ThP cells. The following in vivo experiments highlight the complexity of these issues. During granuloma formation induced by schistosome eggs and during intestinal helminth expulsion (two prototype models of Th2 development) administration of rlL-12 diminished Th2-mediated responses, but many effects were negated with co-administration of anti-IFN-y antibody [18,21%22"]. In a murine model of graft-versus-host disease, rlL-12 suppressed
Th2-mediated responses in a manner dependent on IFN-y but augmented T h l differentiation even with simultaneous neutralization of IFN-y [17]. In mice treated with anti-IgD antibody, rlL-12 also suppressed splenic IL-4 transcription in an IFN-y dependent manner, although it augmented production of IFN-y in an IFN-y independent fashion [23]. These studies suggest that IL-12 diminishes Th2 cytokine production indirectly, via IFN-3'. In contrast, in IFN-y knockout mice infected with L. major, early IL-4 production was markedly curtailed by treatment with rlL-12 [16"]. In some instances, IL-12 may act directly on CD4 + T cells to suppress IL-4 production, but it is uncertain whether such cells are uncommitted precursors or pre-activated cells whose finite role is to influence Th precursors. We believe that most studies support the notion that the primary effect of IL-12 in inhibiting initiation of a Th2 response is indirect, through its induction of IFN-y, but that IL-12 can suppress IL-4 production directly in some CD4 + subpopulations. The role of these distinct CD4 + T cells is discussed below. Whether IFN-y is necessary for T h l responses induced by IL-12 (or even independent of IL-2) is equally perplexing. The priming for IFN-y production after protein immunization plus rlL-12 is unaffected by neutralization of IFN-y [12"']. In the anti-IgD and graft-versus-host disease models, T h l augmentation also occurred despite anti-IFN-y treatment [17,23]. IFN-y receptor knockout mice mount Thl responses to pseudorabies virus [24] and L. major [25°] even with simultaneous neutralization oflFN-y. Yet, IFN-~/gene knockouts display a default to the Th2 phenotype when challenged with L. major [26"], suggesting that IFN-y is required for T h l induction. Possible explanations for this discrepancy include a genetic difference in Th priming sensitivity between the two strains (R129 versus B6), another molecule existing for IFN-y reception in the receptor knockouts, promiscuous reception in the IFN-y knockout mice such that a Th2-inducing signal is transmitted via the IFN-y receptor, or that IFN-y receptor serves a negative regulatory function in homeostasis by associating or tying-up molecules with positive functions. The latter hypothesis could explain why the T h l response seen in the IFN-y receptor knockouts is, paradoxically, greater than that of wild-type mice [25°]. These results suggest that IL-12 can often direct Thl development in the absence of IFN-y (especially in those instances wherein pharmacologic rlL-12 is used) but that some priming situations require IFN-y to avert a Th2 default. Figure 1 represents our view of the role of cytokines in directing Th development.
The role of IL-12 in costimulation
In addition to favoring the differentiation of Thl cells from a naive precursor, IL-12 acts as a second-signal growth factor for T h l , but not Th2 or (Th0), clones [27,28°]. This is consistent with the finding that both B7-1 and IL-12 are required for optimal activation of T h l clones [29] whereas naive T cells do not display
362
Lymphocyteactivation and effector functions Accessory cell
and it leaves open the posssibility that other factors apart from IL-10 and IFN-y regulate IL-12.
IL-10
IL-10 ~ . ~
f_.N
Macrophage (also dendritic cell, keratinocyte) /~----~Thl
IL-12
IL-4 + CD4 T cell (also mast cell,
Th2 IL-4
Va, CD8) © 1995 Current Opinion in Immunology
Fig. 1. The relationship between key cytokines during T-helper development. IL-12 and IL-4 are the most potent factors for priming Thl and Th2 responses by acting directly on the T cells to induce IFN-y and IL-4 production, respectively. IL-12 inhibition of IL-4 production appears, under many circumstances, to be indirect (through induction of IFN-7). In some instances, IL-12 is able to act directly on a population of early IL-4 producing cells that may have a distinct lineage from the naive Th precursors (ThP). IL-10 is derived from accessory cells (and possibly Th cells); although it acts to downregulate IL-12 production/'rhl augmentation, IL-12 has been found to induce IL-10 production thus providing a potential homeostatic feedback loop.
the same requirement for IL-12. The failure of IL-12 neutralization to reverse an established T h l response in vivo [14"°] stands in stark contrast to in vitro findings of IL-12's indispensability and suggests that either an in vivo T h l response is less terminally differentiated than T h l clones in vitro, or that other in vivo factors can act to supplant the lack o f IL-12, or 'optimal' augmentation in IFN-y production seen in vitro is an extreme state beyond in vivo relevance. Regulation of IL-12 IL-10, which was initially defined as an inhibitor of T h l cytokine production may act through inhibiting IL-12 production [28°] or downregulating B7-1 [30 °] on APCs (see Fig. 1). The observed effects o f IL-10 on T h l inhibition are intriguing given the finding in many in vivo systems that rlL-12 induces IL-10 [13°°,16",18,21°,23]. The source o f the IL-10 is probably not T cells as it is produced in severe combined immune deficient (SCID) mice [16°] and is not produced during the restimulation o f CD4 + T cells [22°]. The ability of IL-12 to increase the production of IL-10 may serve a homeostatic function, controlling a T h l response, perhaps by ameliorating pro-inflammatory effects that a prolonged response could elicit. In contrast to the negative regulatory effects of IL-10 on IL-12, IFN-y may give positive feedback for IL-12 induction as p40 m R N A is reduced when mice are treated with antibodies to IFN-T [21°]. This is not surprising given the ability of IFN-y to generally activate macrophages,
Several in vivo systems have used treatment with rlL-12 to induce T h l responses therapeutically. In murine models of AIDS, malaria, and L. donovani infection and histoplasmosis, the presence of rlL-12 effectively led to the protection o f the mice, whereas simultaneous neutralization of IFN- 3' abrogated protection ([31-33]; P Zhou et al., unpublished data; see also Note added in proot). In these instances, it is not clear whether neutralization of IFN-~, merely eliminates a key effector mechanism or actually impairs successful T h l development. Conversely, antibodies against IL-12 induced protection in the murine experimental autoimmune encephalomyelitis (EAE) system [34]. These studies highlight some o f the increasing applications of IL-12 and its blockade.
Costimulation and T-helper development Apart from the intense investigation o f the role o f IL-12 in Th development, an area that has grown considerably in the past year has been the role of costimulatory molecules, most notably the T cell activation receptors CD28 and CTLA-4 and their ligands B7-1 and B7-2, in the development of T h effector functions. The function o f these molecules in T-cell activation has recently been reviewed [35] and is covered in this issue (Sharpe, pp 389-395), but the following studies help ascribe more precise functions during T h differentiation. CD28 knockout mice have impaired Ig production, but preserved cytotoxic T lymphocyte and delayed type hypersensitivity responses [36]. Mice transgenic for soluble C T L A - 4 - I g molecule [37,38] have severe impairment in T-dependent B-cell responses and germinal center formation, whereas their T cells produce higher levels of IFN-y but lower levels of IL-4. In three in vivo models of Th2 development (anti-IgD administration, infection with Heligmosomoides polygyrus, and infection with L. major) C T L A - 4 - I g treatment had a profound effect in blocking the generation of IL-4 producing cells [39°,40°,41°°]. C T L A - 4 - I g administration to resistant C57BL/6 mice did not impair their ability to resist infection with L. major [41°°], and CD28 knockout mice on a resistant background have normal development of T h l responses and healing (SL Reiner, unpublished data). These results suggest that the initial interaction o f CD28 (or possibly CTLA-4) with its B7 ligand is probably required for priming Th2 responses but is non-essential for some T h l responses. The recent findings that B7-2 becomes rapidly upregulated on dendritic cells [42] and that both B7 ligands can be induced quickly on a wide variety of APCs bearing M H C class II [43] support their potential role at initiation of any immune response. Recent findings using selective blockade of B7-1 or B7-2 suggest unique roles for the B7 ligands in preferential Th development [44°]. Priming or restimulation
T
of T C R transgenic T cells leads to increased IL-4 production in the presence of anti-B7-1, but increased IFN-~ production in the presence of anti-B7-2. When anti-B7-1 was used in an experimental autoimmune encephalomyelitis model, disease severity was reduced (and IL-4 production was augmented), whereas the use of anti-B7-2 increased disease severity (and augmented IFN-~/ production) [44"]. A role for B7-1 in Thl development is supported by the finding that B7-1 costimulation increases the number of IL-2 m R N A positive cells among the T h l and Th0 clones but is less effective in increasing the number of Th2 cells that produce IL-4 [45°]. Given the finding that adequately primed Th2 responses" may become independent of costimulation [46"] it would be interesting to determine whether delayed blockade of anti-B7-2 following an adequately primed Th2 response can switch cells to a Thl phenotype by facilitating unopposed B7-1 costimulation. Despite these intriguing results, many of the mechanisms ofcostimulation and its blockade are still unresolved, especially those concerning the direct effects on Th cytokine signalling versus the indirect effects through induction or suppression of other cytokines, whether the blocking reagents being used have any agonistic effect on cytokine induction, and at what stage of Th initiation or maintenance specific costimulatory interactions are most important.
helper cell differentiation in immune responseReiner and Seder 363 CD8 effector populations can be primed into distinct patterns oflymphokine production analogous to Th cells [54"]. The role of these cells may be to augment, rather than initiate, Th development but in systems where pathogenesis favors initial antigen presentation through M H C class I, these cells may have primary influence as the first populations to become producers of IL-4 or IFN-y. Established Thl and Th2 lines from TCIL transgenic mice are long-lived and retain their cytokine phenotype after being adoptively transferred into normal recipients [55]. These cells may be an important source of priming cytokines during a chronic immune response that educates newly activated naive, precursor ThP cells during epitope spreading. Although the predominant source oflL-12 during in vivo immune responses appears to be macrophages, especially when infected by intracellular pathogens, dendritic cells (and keratinocytes) which may be initiators of immune responses in vivo also produce IL-12 [56",57]. Using dendritic cells as APCs to prime TC1L transgenic T cells, neutralization of endogenous IL-12 diminished IFN-y production, suggesting that much of the unprimed IFN-y produced without added rlL-12 is actually IL-12 dependent [56"]. As pathogen-stimulated macophages are the most prolific source of IL-12, it is possible that IL-12 derived from dendritic cells functions by initiating T h l responses that proceed in a more gradual manner.
Sources of cytokines that influence and augment Although other variables (such as antigen dose, type of APC and costimulatory signal) may favor Thl or Th2 development, no factor has been identified in the effective differentiation of these subsets that is more dominant than the effects oflL-12 and IL-4, respectively (reviewed in [7]). Intense interest over the past year has focused on determining the cellular sources of the IL-4 that primes Th2 responses (see Fig. 1). In the murine L. major model, there is a burst in IL-4 transcription from CD4 + cells four days a~er infection in both susceptible and resistant mice [47"]. Similar findings were made in the schistosome egg granuloma and intestinal helminth models [48,49]. In adoptive transfer studies with IL-4 knockout mice, CD4 + rather than CD4- cells are the priming source of IL-4 for Th2-mediated responses [50"]. A potential source for early IL-4 production by a CD4 + cell population includes the recently identified NKI.1 + CD4 + cell (reviewed in this issue by Bendelac, pp 367-374) which was the predominant source of IL-4 in one model of in vivo T-cell activation [51"]. A pre-activated T cell as priming source is also supported by studies in which MEL-14 low ('memory') cells were able to produce the IL-4 that drove the MEL-14 high ('naive') cells to become Th2-like [52"']. Another possible source of early IL-4 production was found among ~fi T cells from mice infeced with Nippostrongylus brasiliensis [53]. In listeriosis, "y6 T cells contained intracellular IFN-y early in infection, implicating a role in Th differentiation depending on the immune stimulus [53]. Related findings demonstrate that
Conclusions: genetic polymorphism and future directions Increasing interest lies in many of the in vitro and in vivo systems that have striking differences in Th outcome depending on the genetic background of the mouse. Murine leishmaniasis and candidiasis represent two well characterized examples, but genetic differences in T helper cell development are now known to play a role in the pathogenesis of autoimmune diabetes [58"], intestinal helminth infections [59"], and Lyme disease [60"]. The way in which a fixed antigen or set of antigens is capable of eliciting varied reponses in different genetic backgrounds is perplexing. One such example is that when BALB T cells are polyclonally activated in the presence of IL-4, Th2 priming is enhanced rather than supressed by the presence of IL-12 [61]. Results of experiments with T C R transgenic mice bred onto differing backgrounds indicate that some strains develop distinct patterns of lymphokine production when primed with the same antigen-APC, and that this difference segregates to the T-cell compartment [62"]. This could explain many earlier discrepancies noted from in vitro priming data using different systems (reviewed in [7]). The ability to reconstitute important cellular differences seen in disease models should pave the way for a more detailed molecular understanding of some of the factors that make certain strains have divergent priming patterns under fixed conditions. Common cytokine receptor usage (reviewed in this issue by Bolen, pp 306-311) and related transcription factor pathways
364
Lymphocyteactivationand effectorfunctions [63,64] are areas where minor polymorphism could regulate major differences in T-helper development. The past year has seen a number o f important advances concerning the selective development o f differing immune responses. In addition to a more detailed understanding o f the roles of IL-12 and costimulatory molecules in this process, we have gained a greater appreciation o f the complexities and subtleties o f the homeostatic network. Although the immune system often strives to mount an optimal T h l or Th2 response against certain pathogens, there are probably many instances in which a combination 0 f t h e two responses is more appropriate, and other instances where the failure to restrain a polarized response is frankly deleterious. We predict that the coming year will yield many advances in elucidating the cellular and molecular basis o f Th differentiation, both in differing genetic backgrounds and in response to a variety o f foreign and endogenous stimuli.
Note added in proof The study referred to in the text as P Zhou et al., unpublished data, has been accepted for publication [65].
Acknowledgements The authors wish to thank many colleagues for helpful discussions. SL R.einer is a recipient of a Clinical Investigator Award from the National Institutes of Health (AI01309) and a Burroughs Wellcome Fund New Investigator Award in Molecular Parasitology.
8.
Trinchieri G, Scott P: The role of interleukin 12 in the immune response, disease and therapy. Immunol Today 1994, 15:460-463.
9.
Heinzel FP, Schoenhaut DS, Rerko RM, Rosser LE, Gately MK: Recombinant interleukin 12 cures mice infected with Leishmania major. J Exp Med 1993, 177:1505-1509.
10.
Sypek JP, Chung CL, Mayor SEH, Subramanyam JM, Goldman SJ, Sieburth DS, Wolf SF, Schaub RG: Resolution of cutaneous leishmaniasis: interleukin 12 initiates a protective T helper type 1 immune response. J Exp Med 1993, 177:1797-1802.
11.
Afonso LCC, Scharton TM, Vieira LQ, Wysocka M, Trinchieri G, Scott P: The adjuvant effect of interleukin-12 in a vaccine against Leishmania major. Science 1994, 263:235-237.
12. ""
McKnight AJ, Zimmer G/, Fogelman I, Wolf SF, Abbas AK: Effectsof IL-12 on helper T cell-dependent immune responses in vivo. J Immunol 1994, 152:2172-2179. During protein immunization, IL-12 treatment enhanced interferon (IFN)y producing T cells and inhibited IL-4 secreting cells. The effect of IL-12 on increasing IFN-y production was not prevented by co-administration of a neutralizing antibody to IFN-y, supporting the view that IL-12 acts directly on precursor Th cells to cause Thl development. 13. ""
Romani L, Mencacci A, Tonnetti L, Spaccapelo R, Cenci E, Puccetti P, Wolf SF, Bistoni F: IL-12 is both required and prognostic in vivo for T helper type 1 differentiation in murine candidiasis. J Immunol 1994, 152:5167-5175. In contrast to the Leishmania major model, recombinant IL-12 cannot cure susceptible mice (although anti-lL-12 causes susceptibility). The genetics and probably the priming events of the low virulence Candida model (DBA/2 mice are susceptible/lh2 responders, BALB/c mice are resistant/Thl responders) are distinct from those involved in L. major infection. This work points to the complexity of innate immune signals and the limitations in IL-12 uses. 14. ""
Gazzinelli RT, Wysocka M, Hayashi S, Denkers EY, Hieny S, Caspar P, Trinchieri G, Sher A: Parasite-induced IL-12 stimulates early IFN-y synthesis and resistance during acute infection with Toxoplasma gondii. J Immunol 1994, 153:2533-2543. Important study illustrating that an IL-12 dependent interferon-y response becomes independent of IL-12 once maturation or the maintenance phase of the response is established.
References and recommended reading
15.
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16. •
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Macatonia SE, Hsieh C-S, Murphy KM, O'Garra A: Dendritic cells and macrophages are required for Thl development of CD4 + T cells from cc~ TCR transgenic mice: IL-12 substitution for macrophages to stimulate IFN-y production is I FN-y-dependent. Int Immunol 1993, 5:1119-1128.
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Leonard JP, Waldburger KE, Goldman SJ: Prevention of experimental autoimmune encephalomyelitis by antibodies against interleukin 12. J Exp Med 1995, 181:38]-386.
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June CH, Bluestone JA, Nadler LM, Thompson CB: The B7 and CD28 receptor families. Immuno/ Today 1994, 15:321-331.
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Shahinian A, Pfeffer K, Lee KP, K~ndig, Kishihara K, Wakeham A, Kawai K, Ohashi PS, Thompson CB, Mak TW: Differential T cell custimulatory requirements in CD28-deficient mice. Science 1993, 261:609-6] 2.
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Ronchese F, Hausmann 8, Hub•l• 5, Lane P: Mice transgenic for a soluble form of murlne CTLA-4 show enhanced expansion of antigen-specific CD4* T cells and defective antibody production in vivo. ] Exp Med 1994, 179:809-817.
38.
Lane P, Burdet C, Hubele S, Scheidegger D, M(iller U, McConnell F, Kosco-Vilbois M: B cell function in mice lransgenic for mCTLA4-H~I: lack of germinal centers correlated with poor affinity maturation and class switching despite normal priming of CD4 + T cells. ] Exp Meal 1994, 179:819-830.
21. •
22.
Oswald IF), Caspar P, Jankovic D, Wynn TA, Pearce EJ, Sher A: IL-12 inhibits Th2 cytokine responses induced by eggs of Schistosoma mansoni. J Immunol 1994, 153:1707-]713. In contrast to transcriptional analysis of unsorted tissue which shows that recombinant IL-12 causes an increase in IL-10 mRNA, restimulation of purified CD4 + cells shows no such increase, suggesting that the source of IL-10 is accessory cells. •
23.
Morris SC, Madden KB, Adamovicz )J, Gause WC, Hubbard 8R, Gately MK, Finkelman FD: Effects of IL-12 on in vivo cytokine gene expression and Ig isotype selection. J Immunol 1994, 152:1047-1056.
24.
Schijns VECJ, Haagmans BL, Rijke EO, Huang S, Aguet M, Horzinek MC: IFN y receptor-deficient mice generate antiviral Thl-characteristic cytokine profiles but altered antibody responses. J Immunol 1994, 153:2029-2037.
25. •
Swihart K, Froth U, Messmer N, Hug K, 8ehin R, Huang S, [:)el Giudice G, Aguet M, Louis JA: Mice from a genetically resistant background lacking the interferon y receptor are susceptible to infection with leishmania major but mount a polarized T helper cell 1-type CD4 + T cell response. J Exp Med 1995, 181:961-971. See annotation [26•1. 26. •
Wang Z-E, Reiner SL, Zheng S, Dalton DK, Locksley RM: CD4 + effector cells default to the Th2 pathway in interferon y-deflcient mice infected with Leishmania major. J Exp Med 1994, 179:1367-1371. Along with [25•], this paper provides perplexing results on dispensability versus necessity of interferon-y for T helper type 1 responses. Points out potential artifacts that can arise through use of mutant strains: unless the discrepancy is merely one of background genes, some feature of the deletion in one of these strains must explain the differences in outcome. 27.
Yanagida 1, Kato T, Igarashi O, Inoue T, Nariuchi H: Second signal activity of IL-12 on the proliferation and IL-2R expression of T helper cell-1 clone. J Immunol 1994, 152:4919-4928.
28. •
Kennedy MK, Picha KS, Shanebeck KD, Anderson DM, Grabstein KH: Interleukin-12 regulates the proliferation of Thl, but not Th2 or Th0, clones. Eur J Immunol 1994, 24:2271-2278. Suggests that one potential mechanism for IL-10's activity is the downregulation of IL-12 production by antigen-presenting cells. 29.
Murphy EE, Terres G, Macatonia SE, Hsieh C-S, Mattson J, Lanier L, W~/socka M, Trinchieri G, Murphy K, O'Garra A: B7 and interleukin 12 cooperate for proliferation and interferon-y production by mouse T helper clones that are unresponsive to B7 costimulation. J Exp Med 1994, 180:223-231.
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39. *
Lu P, Zhou XD, Chen S-J, Moorman M, Morris SC, Finkelman FD, Linsley P, Urban JF, Gause WC: CTLA-4 ligands are required to induce an in vlvo interleukin 4 response to a gastrointestinal nematode parasite. J Exp Med 1994, 180:693-698. Examines the role of costimulaton/signals mediated through CTLA-4 and its ligands on T helper cell immune responses in animals infected with Heligmosomoides polygyrus. Infected animals have increased T-cell derived IL-4 gene expression, but animals treated with CTLA-4-1g showed a striking decrease in IL-4 expression. These results support a role for costimulation through CTLA-4 and its ligands in Th2 cytokine expression. 40. •
Lu P, Zhou XD, Chen S-J, Moorman M, Schoneveld A, Morris S, Finkelman FD, Linsley P, Claassen E, Gause WC: Requirement of CTLA-4 counter receptors for IL.4 but not IL-10 elevations during a primary systemic in vivo immune response. J Immunol 1995, 154:1078-1087. Another model system that demonstrates the importance of costimulation for priming of IL-4 production. Points out that IL-4 and IL-10 are not coordinately regulated. 41. ••
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Transient blockade of costimulation abrogates T helper type 2 development in susceptible BALB/c mice without affecting Th] development in these mice or in resistant C57BL/6 mice. Sustained blockade in BALB/c diminishes healing, suggesting a possible role for costimulation in maintenance of Thl responses. 42.
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31.
Gazzinelli RT, Giese NA, Morse HC Ilk In vivo treatment with interleukin 12 protects mice from immune abnormalities observed during murlne acquired immunodeficiency syndrome (MAIDS). J Exp Med 1994, 180:2199-2208.
32.
SedegahM, Finkelman F, Hoffman SL: Interleukin 12 induction of interferon y-dependent protection against malaria. Proc Nat/ Acad Sci USA 1994, 91:10700-10702.
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44. •
Kuchroo VK, Das MP, Brown JA, Ranger AM, Zamvil SS, Sobel RA, Weiner HL, Nabavi N, Glimcher LH: B7-1 and B7-2 costlmulatory molecules activate differentially the Thl/Th2 developmental pathways: application to autoimmune disease therapy. Cell 1995, 80:707-718. Selective inhibition of 87-1 or B7-2 affects the cytokine profile of T cells stimulated in vitro or in vivo. Shows that B7-1 is preferentially a costimulator of Thl, and that B7-2 favors the induction of Th2 cells. 45. •
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365
366
Lymphocyte activation and effector functions Examines single cell cytokine mRNA transcription and points out that increasing antigen dose in a clonal T helper type 1 (Thl) or Th2 population serves to increase the frequency of cytokine-positive cells, rather than to increase cytokine transcripts within a given cell. 46.
McKnight AJ, Perez VL, Shea CM, Gray GS, Abbas AK:
•
Costimulalor dependence of lymphokine secretion by naive and activated CD4 + T lymphocytes from TCR transgenic mice. J Immunol 1994, 152:5220-5225.
Suggests that costimulation is necessary for initiation of Th2 responses but that it can become dispensible if the Th2 response had been adequately primed. 47. "•
Reiner SL, Zheng S, Wang Z-E, Stowring L, Locksley RM: Leishmania promastigotes evade interleukin 12 (IL-12)
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Swain SL: Generation and in vivo persistence of polarized Thl and Th2 memory cells. Immunity 1994, 1:543-552.
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induction by macropbages and stimulate a broad range of cytokines from CD4 + T cells during initiation of infection. J Exp Med 1994, 179:447-456. The insect form of Leishmania does not induce IL-12, unlike the
I: IL-12 is expressed and released by human keratinocytes and epidermoid carcinoma cell lines. J Immunol 1994, 153:5366-5372.
mammalian form. A burst in IL-4 production and increase in IL-12 production are features common to both susceptible and resistant mice. Despite uniform priming by a combination of IL-4 and IL-12, most strains develop T helper type 1 (Thl) effectors but BALB/c mice develop Th2 effectors. The IL-4 burst is derived from CD4 + cells; this was also found in [48] (dealing with immune responses to schistosome egg) and [49] (heiminthic stimulation).
58. •
48.
Wynn TA, Eltoum I, Cheever AW, Lewis FA, Gause WC, Sher A: Analysis of cytokine mRNA expression during primary granuloma formation induced by eggs of Schistosoma mansoni. J Immunol 1993, 151:1430-1440.
•
Svetic A, Madden KB, Zhou XD, Lu P, Katona IM, Finkelman FD, Urban JF Jr, Gause WC: A primary intestinal
60. •
49.
helminthic infection rapidly induces a gut-associated elevation of Th2-associated cytokines and IL-3. J Immunol 1993, 150:3434-3441. 50. "•
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expression in T helper (Th) cells is not dependent on IL-4 from non-Th cells. J Exp Med 1994, 179:1349-1353. Suggests that an innate immune system component (mast cell or basophil) is not the priming element of Th2 responses; rather, it is the CD4 + cell, itself. Complements findings of an early IL-4 source seen in parasitic models ]47••,48,49].
62. •
Yoshimoto 1, Paul WE: CD41~n, NKI.lPOS T cells promptly •" produce interleukin 4 in response to in vivo cballenge with anti-CD3. J Exp Med 1994, 179:1285-1295. Ceils that produce early IL-4 in response to in vivo anti-CD3 stimulation are rich in CD4 + cells that have the phenotype CD44bright, CD45RB dull and NK1 .lp°s. These cells may provide a source of IL-4 that influences the development of Th2-1ike cells. 52. •"
Gollob KJ, Coffman RL: A minority subpopulation of CD4 + T' cells directs the development of naive CD4 + T cells into IL-4-secreting cells. J Immunol 1994, 152:5180-5188.
Another study in which CO4 + cells are subclassified and in which experiments demonstrate that MEL-14 low cells provide the priming doses of IL-4 necessary to make naive cells differentiate into Th2-1ike cells. 53.
Ferrick DA, Schrenzel MD, Mulvania I, Hsieh B, Ferlin WG, Lepper H: Differential production of interferon-y and inlerleukin-4 in response to Thl- and Th2-stimulating pathogens by ~i T cells in vivo. Nature 1995, 373:255-257.
54. •
Croft M, Carter L, Swain SL, Dutton RW: Generation of polarized antigen-specific CD8 effector populations: reciprocal action of interleukin (IL)-4 and IL-12 in promoting type 2 versus type 1 cytokine profiles. J Exp Med 1994,
180:1715-1728. This study confirms earlier work demonstrating that CD8 + T cells can be primed in vitro to produce IL-4 if IL-4 is present in the priming culture.
Else KJ, Finkelman FD, Maliszewski CR, Grencis RK: Cytokinemediated regulation of chronic intestinal helminth infection. J Exp Med 1994, 179:347-351.
Along with [60•], demonstrates that Th2 responses may be protective in certain infectious diseases, depending on the pathogenesis.
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Aragane Y, Riemann H, Bhardwaj RS, Schwarz A, Sawada Y, Yamada H, Luger TA, Kubin M, Trinchieri G, Schwarz
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Interesting cellular model of varying cytokine profile among inbred strains in response to fixed priming. The T-cell compartment dictates the genotypic difference when antigen-presenting cells and T cells are crossed. 63.
Rothman P, Kreider B, Azam M, Levy D, Wegenka U, Eilers A, Decker I-, Horn F, Kashleva H, lhle l, Schindler C: Cylokines and growth factors signal through tyrosine phosphorylation of a family of related transcription factors. Immunity 1994, 1:457~,68.
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Darnell JE Jr, Kerr iM, Stark GR: Jak.STAT pathways and transcriptional activation in response to IFNs and other extracellular signaling proteins. Science 1994, 264:1415-1421.
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