- Email: [email protected]
Acute induction and priming for cytokine production in lymphocytes
Cvtokine & Growth Factor Retie~*s Vol. 7. No. 2, pp. 123 132, 1996 Copyright C 1996 Elsevier Science Ltd. All rights reserved PII:
S1359-6101(96)00018-4
ELSEVIER
Printed in Great Britain. 1359-6101/96 $32.00+0.00
MINI REVIEW
Acute Induction and Priming for Cytokine Production in Lymphocytes
Giorgio Trinchieri*t, David Perittt and Franca Gerosat When T-lymphocytes (CD4 +, CD8 +, or TCR~6 +) and NK cells proliferate in vivo or in vitro in response to exposure to antigen or other stimuli, they often segregate into subsets with the ability to produce either type-1 [interferon-T (IFN-~) and interleukin-2 (IL-2)] or type-2 cytokines (IL-4, IL-5, IL-6 and IL-10). IL-12 induces the differentiation of type-I cytokine-producing T-cells primarily through its ability to prime them for high IFN-? production; however, paradoxically ILl2 also primes T-cells for high production of the type-2 cytokine IL-10. Priming of T-cells for IL-4 production requires the presence of IL-4, but it is maximally observed in cultures containing both IL-4 and IL-12. IL-12, in addition to priming T-cells for high IFN-? and IL-10 production, is also a potent acute inducer of expression of the IFN-y gene in T- and NK-cells, and, to a much lower extent, of the |L-10 gene. IL-4, which has a very powerful effect in priming T-cells for IL-4 production, does not appear to have a significant ability to directly activate the expression of the IL-4 gene. Thus, IL-12 and IL-4 affect the expression of type-1 and type-2 cytokine genes by two different mechanisms: an acute induction of gene expression which is rapid and reversible, and a priming of the genes to a highly responsive state to restimulation, a state that is stable and probably irreversible. Copyright ~ 1996 Elsevier Science Ltd. Key words: Acute induction" Priming" Cytokine production" Lymphocytes.
SUBSETS OF LYMPHOCYTES WITH DISTINCT CYTOKINE PRODUCTION PATTERNS
humoral responses, production of lgE and IgA, and activation of eosinophils and basophils. The differentiation of Th-cells toward a Th 1- or Th2-phenotype occurs early The immune response to infectious agents and to nominal during an immune response, and is influenced by many antigens is often characterized by a dominance of either interrelated factors, including the nature and the concell-mediated or humoral-type effector mechanisms [1], centration of the antigen, the anatomical localization of which has been attributed to a dichotomy in the cytokine the immune response, the nature of the antigen-preproduction pattern ofT-helper (Th) CD4+-cells [2]. Two senting cells (APC) and the cytokine milieu at the site of classes of Th-cells have been described: Thl-cells, which the immune response [3]. IL-4, produced by T-cell subsets produce interleukin-2 (IL-2), interferon-7 (IFN-7) and and possibly basophils, appears to be the most important lymphotoxin (LT) and favor cell-mediated immunity, cytokine responsible for Th2-cell generation [4], whereas delayed-type hypersensitivity, macrophage activation IL-12, produced by macrophages and professional APC, and production of opsonizing antibodies; and Th2-cells, such as dendritic cells and Langerhans cells [5-8], is which produce IL-4, IL-5, IL-6 and IL-10, and favor required for effective Thl-cell generation [9 12]. IL-12 acts at three different levels in favoring a Thl-response: (1) it acts as a proinflammatory cytokine inducing IFNt The Wistar Institute of Anatomy and Biology, Philadelphia, 7 from T- and NK-cells within a few hours after infection, U.S.A. and through IFN-?, it induces activation of phagocytic ~:Istituto di Immunologia e Malattie Infettive, Policlinico di cells for bacteriocidal activity and also for enhanced proBorgo Roma, 37100 Verona, Italy. * To whom correspondence should be addressed at: The Wistar duction of cytokines, including IL- 12 itself [6, 13-15]; (2) Institute, 3601 Spruce Street, Philadelphia, PA 19104, U.S.A. in the first few days of an immune response, IL-12 and Tel.: 215-898-3992; Fax: 215-898-2357; e-mail: trinch- IL-12-induced IFN-7 act on antigen-stimulated T-cells to ieri(a~wista.wistar.upenn.edu favor differentiation and proliferation of cells and a Thl123
124
G. Trinchieri et al.
phenotype [9-12]; (3) once differentiated Thl are generated, IL-12 is required for optimal cytokine production and, in certain cases, proliferation of Thl-cells [16]. The dichotomy in the cytokine production pattern is not limited to CD4 + Th-cells, but is also clearly demonstrated for CD8 + T-cells [17-20], for T-cells with a 76 T-cell receptor [21], and, to a certain extent, for natural killer cells [22]. Although the expression of Thl-type and Th2-type cytokines was initially considered to be mutually exclusive, Th-cells expressing both Thl- and Th2-cytokines (referred to as Th0-cells) have been identified both during differentiation of Th-cells [23] and in terminally differentiated cells [24]. Indeed, evidence for a stochastic acquisition of cytokine profile, suggesting independent regulation of each cytokine gene, was obtained during a mouse Th2-response to keyhole limpet hemocyanin (KLH) [25] and in human peripheral blood memory CD4 + T-cells [26, 27].
cells) is required for optimal IFN-7 production by resting PBL [14]. These accessory cells might provide costimulatory molecules for IFN-7 production. In murine spleen cells, it has been shown that IL-12 synergizes with TNF~ in inducing IFN-7 production [32, 33]. This synergistic effect of TNF-~ was not demonstrated with human lymphocytes, but antibodies to TNF-c~ or IL-1/~ efficiently inhibited IL-12-induced IFN-7 production suggesting that these two cytokines, endogenously produced in the PBL cultures, possibly by the class II positive accessory cells, act as costimulatory molecules for IFN-7 production together with IL-12 [34]. Another costimulatory signal possibly provided by the accessory cells is the B7 molecule, a ligand for the CD28 receptors on T-cells. Stimulation of T-cells with B7-transfected cells or with anti-CD28 antibodies, strongly synergized with IL-12 for induction of IFN-~ production [16, 28] and blockage of B7-CD28 interaction with the hybrid recombinant molecule CTLA4-Ig, significantly inhibited the ability of PBL to produce IFN-y in response to IL-12 [28]. These results suggest that TNF-~, IL-1//, and B7, possibly at IL-12 IS A POTENT INDUCER OF IFN-~, least in part, provided by the class-II accessory cells, are PRODUCTION IN BOTH RESTING AND ACTIVATED important costimulators for IFN-7 production in T-LYMPHOCYTES AND NK CELLS response to IL-12. The ability of IL-10 to inhibit IFN-7 production in T- and NK-cells is primarily due to its IL-12 induces IFN-~: production from resting and acti- ability to suppress IL-12 production, but also, in part, to vated NK- and T-cells, with a similar dose-response its ability to suppress expression of these costimulatory relationship and a half maximal activity at 3.5 pM [5, 14]. molecules on accessory cells [16, 28, 34]. Within T-cells, both T-cells with an ~/~ T-cell receptor Not only is IL-12 a potent inducer of IFN-7 (TCR) both CD4 + and CD8 +, and T-cells with 76 TCR production, but it is also most likely a required factor for are induced to produce IFN-y [14]. The induction of IFN- efficient IFN-7 production depending, in vivo and in vitro, by IL-12 is characterized by a powerful synergistic effect on accessory cells. When human PBMC were treated in with other IFN-y inducers, in particular IL-2 and phorbol vitro with stimuli, e.g.S, aureus, that were able to induce diesters [5, 14]. On T-cells, IL-12 also synergizes with production of IL-12, they rapidly produced large mitogenic lectins, with stimulation bf the TCR-CD3 com- amounts of IFN- 7. This production of IFN- 7 was almost plex by anti-CD3 antibodies or alloantigens [14], and completely inhibited by neutralizing antibodies against with stimulation of the CD28 receptor by anti-CD28 IL-12 [16]. Even when IFN- 7 inducers, which are not antibodies or its ligand B7 [16, 28]; and on NK-cells with known to stimulate IL-12 production, were used (e.g. ILstimulation by ligands of the CD16 receptor for IgG- 2 or anti-CD3 antibodies), the production of IFN- 7 from Fc (anti-CD 16 antibodies or immunocomplexes) and by PBMC was inhibited up to 80%, indicating that endogentarget cells [29]. IL-12 rapidly increases the tran- ously produced IL-12 is required for optimal IFN- 7 proscriptional rate of the IFN- 7 gene; however, when IL-12 duction [6]. However, if purified T- or NK-cells, in the and IL-2 are added together to the cells, most of the absence of IL-12 producing accessory cells, were stimusynergistic effects of the two inducers are observed at the lated to produce IFN-y (e.g. by IL-2 or anti-CD3 antiposttranscriptional level, with an increase of more than bodies), no inhibitory effect of anti-IL-12 antibodies two-fold of the half-life of the IFN-7 mRNA in the cells could be demonstrated [6]. treated with the two inducers together [30]. Both resting The ability of IL-12 to induce a rapid production of and activated NK- and T-cells are induced by IL-12 to IFN-y in vivo has been clearly shown in several experproduce IFN-7, although maximal IFN-y mRNA imental models of infectious diseases. A very informative accumulation is reached in 2-4 hr in activated T- or experimental model for the understanding of the role of NK-cells, and in 18-24 hr in resting peripheral blood IL-12 in inducing IFN-7 in vivo is provided by the endolymphocytes (PBL) [14]. Within PBL, IL-12 induces toxic shock in Bacille Calmette Gu6rin (BCG)-primed mRNA accumulation, as detected by in-situ hybrid- mice [13]. Several cytokines, in particular TNF-~ and ization, in a proportion of both NK- and T-cells [14], IFN-7, have been shown to be responsible for pathoalthough N K cells might be major contributors to the logical reactions which may lead to shock and death early production of IFN-7 in response to IL-12 or IL-2 observed in infection with Gram-negative bacteria and [31]. Although NK- and T-cells are the IFN-7 producers in response to endotoxins. Priming of mice with the aviin PBL preparations stimulated by IL-12, an accessory rulent BCG vaccine strain of Myeobacterium boris cell type (MHC class II positive, non-monocyte, non-B- increases the sensitivity of mice to the lethal effect of LPS
Cytokine Production in Lymphocytes and results in an efficient priming for cytokine production in response to LPS. Mice, injected with LPS, produced IL-12, which induced IFN-7 production, as demonstrated by the ability of neutralizing anti-IL-12 antibodies to suppress IFN- 7 production [13]. However, the concentration of biologically active IL-12 was similar in the serum of both BCG-primed or unprimed mice, reaching levels of 1 3 ng/ml at 3-6 hr after LPS injection, whereas IFN- 7 production was observed only in BCG-primed mice [13]. TNF-~ and other LPS-induced cofactors were required in cooperation with IL-12 to induce optimal IFN- 7 production. The priming effect of BCG on IFN- 7 production appears to be mostly due to its ability to increase TNF-~ production, which acts as a cofactor with LPS-induced IL-12 in inducing IFN- 7 production [13]. Neutralizing anti-IL-12 antibodies, in addition to inhibiting the in vivo LPS-induced IFN- 7 production, also protected mice from septic shock-induced death [13]. Thus, IL-12 is required for IFN- 7 production and lethality in an endotoxic shock model in mice. Injection of mice with a daily i.p. injection of 1 /~g of recombinant IL-12 induced high levels of serum IFN-7., but only starting 48 hr after the first injection [35]. This delayed response was probably due to the lack of appropriate costimulatory signals when only recombinant IL12 was injected. The injected IL-12 had a serum half-life of 3.3 hr [13], much longer than that of other cytokines, Although IL-4 has clearly been shown to be required for the differentiation of IL-4-producing T-cells [4, 36, 37], little information is available on the ability of IL-4 to directly upregulate its own gene by inducing acute production of IL-4. Induction of IL-4 from resting lymphocytes has been more difficult to detect than production of IL-2 or IFN-7; however, stimulation of human PBL with recombinations of anti-CD2, CD3, CD28 antibodies and/or IL-2 has been shown to result in the production of IL-4 within 2448 hr [38]. The presence of IL-4 during stimulation by antigen or anti-CD3 antibodies results in the induction of IL-4 gene expression only after 3-4 days during culture (A. Lichtman, personal communication), presumably when differentiation of IL4 producing Th2 cells is already taking place. Thus, unlike IL-12 that induces potent upregulation of the IFN-7 gene within a few hours of stimulation, IL-4 appears to be able to promote the generation of IL-4 producing cells, rather than to acutely induce the expression of the IL-4 gene.
THE ABILITY OF IL-12 AND Ik-4 TO INDUCE GENERATION OF T h l AND Th2 CELLS,
RESPECTIVELY, IS DUE IN PART, TO THEIR ABILITY TO PRIME T-CELLS FOR Thl-TYPE OR Th2-TYPE CYTOKINE PRODUCTION The requirement of IL-4 for the generation of IL-4 producing Th2 cells has been a well-established Concept for several years [4, 36, 37]. More recently, the role of IL12 for the efficient generation of IFN-7 production by Thl cells has become evident, and it has been proposed
125
that the balance between the levels of IL-4 and IL-12 early during an immune response may be responsible to bias the generation of Th2 and Thl cells, respectively [12],although the presence of other cytokines and various other factors regulating the immune response also play a major role. Furthermore, the synergistic/antagonistic interaction between IL-4 and IL-12 in regulating such responses is complex and not yet fully understood [39, 40]. Stimulation in vitro of peripheral blood lymphocytes (PBL) from atopic patients with allergens such as Dermatophagoides pteronyssinus Group 1 (Der p.1), results in the generation ofT-cell lines and clones with the high IL4 and low IFN-7 production typical of Th2-cells, whereas PBL stimulation with bacterial products (e.g. purified protein derivative, PPD) generates Thl-type T-cell lines and clones that produce IFN- 7 but not IL-4. When PBL are stimulated with Der p.I in the presence of IL-12, Tcell lines and clones are generated that exhibited a reduced ability to produce IL-4 and an increased ability to produce IFN-7 [9]. This Thl-inducing effect of IL-12 was not inhibited by anti-IFN-7, but was reduced by removal of NK cells from the PBL preparation. PPDspecific T-cell lines generated in the presence of antiIL-12 antibodies during the initial antigenic stimulation produced significant levels of IL-4, unlike the cell lines generated in the absence of antibodies, and gave rise to PPD-specific CD4 + cell clones showing a Th0/Th2phenotype rather than a Thl-phenotype [9]. These results indicate not only that IL-12 is able to facilitate proliferation and activation of Thl-cells in a memory response in vitro, but also that, as shown by the effect of anti-IL-12 antibodies, endogenously produced IL-12 is an obligatory factor for Thl generation. The ability of IL-12 to directly initiate Thl-cell development in na~'ve T-cells was shown by Hsieh et al. [10] who reported that nai've CD4 + T-cells derived from mice transgenic for an anti-ovalbumin TCR are induced by ovalbumin to develop into Thl-cells in the presence of IL- 12, whereas they develop into Th2-cells in the presence of IL-4. The effect of IL-4 in that system, is, however, dominant over that of IL-12. Since these early studies, the ability of IL-12 to induce Thl-cell generation was demonstrated in many models, in humans and in experimental animals, both in vitro and in vivo [8]. The originally reported dominance of IL-4 action over IL-12 [! 0] was observed to be a much more complex interaction, with the two cytokines antagonizing or synergizing each other, depending on the function analyzed [39-41]. The experimental systems used for studying Th response have not permitted determination of whether the different cytokines affecting Th-cell development induce differentiation of bipotential Th precursors or rather a selective priming and/or expansion of already committed Thl- and Th2-precursor cells [42-45]. This question is particularly relevant in human studies which have analyzed clonal expansion of memory Th-cells [9, 46]. However, once a Thl- or Th2 response has been established, it appears to be relatively stable, and no
126
G. Trinchieri et al.
factors capable of inducing qualitative changes in the cytokine profile of established murine or human T-cell clones have been reported, In the analysis of cytokine production from human Tcells stimulated with recall antigens (PPD) or allergens (Der p.1), the expansion of the small proportion of memory T-cells was first obtained in polyclonal T-cell cultures, from which single antigen-specific clones were obtained only after several weeks of culture of the polyclonal cell line [9, 46]. During this culture period, emergence of Th-cell subsets with characteristic cytokine production profiles could be due to differentiation of precursor Thcells, as well as to positive selection (growth advantage) of certain Th-subsets or negative selection (apoptosis, cytotoxicity, anti-proliferative effects) of other subsets, When human T-cell cultures were stimulated by a polyclonal stimulus affecting all T-cells, such as PHA or antiCD3, in the presence or absence of IL-12, similar results were obtained as in antigen-stimulated cultures, i.e. IFNproduction by the cells was enhanced and IL-4 production was almost completely abolished [47]. However, very different results were obtained when freshly isolated human peripheral blood T-cells were immediately cloned by limiting dilution in cultures stimulated by PHA and IL-2, in the presence or absence of IL-12 [19]. When restimulated with anti-CD3 and TPA after 5 weeks of culture, the clones generated in the presence of IL-12 produced on average five- to 20-fold higher levels of IFN7 than the clones generated in the absence of IL-12. This priming for IFN-7 production required the addition of IL-12 within the first week, but its presence for maximal priming was required only for 1 or 2 weeks [40]. Once the clones were established for 2 or 3 weeks, removal or addition of IL-12 from the culture medium did not significantly affect their ability to produce IFN- 7 [19]. Because the clonal efficiency in these experiments was close to 100%, the priming effect of IL-12 was not due to selection of high IFN-~-producing clones, but was exerted on each single T-cell, naive or memory. Furthermore, this effect was observed on both CD4 + and CD8 + cells, suggesting that IL-12 affects the differentiation of Thl-type clones from both subsets. Thus, the presence of IL-12 during the initial clonal proliferation of T-cells induces an irreversible priming for high IFN-7 production, which is maintained, even when the clones are cultured for several weeks in the absence of IL-12. However, unlike what is consistently observed in vivo and in polyclonal cultures and their derivative clones, the clones originated by limiting dilution in the presence or absence of IL-12 did not show any significant difference in their average ability to produce IL-4 [19, 47]. These results suggested that the ability of IL-12 to prime CD4 + cells for high IFN-7 production is due to a differentiation effect acting at the level ofCD4 + T-cell clone precursors, The ability of IL-12 to downregulate IL-4 production, however, was not observed at the clonal level, and is likely to be due to selective processes operative on polyclonal cultures and not to a direct effect on single clonal progenitors [19]. The nature of these mechanisms remains to
be investigated, although a possible selective proliferative effect of IL-12 on Thl clones or on IFN-~,-mediated negative selection against IL-4-producing clones can be postulated. Alternatively, the downregulation of IL-4 production might be a differentiation effect that requires cellular interaction or cell crowding (e.g. for the production Th2-suppressing factors such as I F N - J during the initial phase of proliferation of the T-cells; such interactions are not obtained in limiting dilution cultures, even in the presence of irradiated feeder cells. The ability of IL-12 to prime human T-cells for IFN7 production has also been suggested by experiments showing that na~'ve human cord blood T-cells are unable to produce IFN-7, but acquire this ability after a few days of culture in the presence of IL-12 [45]. However, selective effects and/or preferential proliferation of T-cell subsets could not be excluded in the polyclonal cultures of cord blood T-cells. That the mechanisms underlying the enhancing effect of IL-12 on IFN-7 and the inhibition of IL-4 production might be different is also suggested by data in the murine system, showing that the enhancement of IFN- 7 production is a direct effect of IL-12 on T-cells, whereas the inhibition of IL-4 production is due to an indirect effect on APC or on other cell types present in the APC preparations [48]. CD4 + and CD8 + clones obtained by limiting dilution in the presence of IL-12 produced significantly more IL10 than clones generated in the absence of IL- 12 [40]. We also observed that stimulation of human T-cell clones in the presence of IL-12 results in a several-fold increase in IL-10 production, in both high and low IL-10 producing clones (Peritt and Trinchieri, in preparation). However, in allergen-stimulated polyclonal T-cell culture, IL-12 was shown to downregulate both IL-10 and IL-4 [49]. These apparentlycontradictoryresultsare, however, consistent with the conclusion that IL-12 directly upregulates Thl cytokine production, but suppresses Th2 cytokine production by an indirect, possibly selective mechanism. These data also shed new light on the observation that IL-12 treatment in vivo induces the accumulation of IL10 mRNA [50]: although that finding was attributed to production of IL-10 by macrophages, the possibility that IL-12 also induces IL-10 production from T-cells must now be investigated. Figure 1 shows a typical limiting dilution cloning experiment [40] in which CD4 + and CD8 + clones were derived from unfractionated PBL from several donors and expanded in the presence, during the first 2 weeks of culture, of IL-12 or neutralizing antibodies anti-IL-12. After 4-5 weeks of culture, the clones were then stimulated with anti-CD3 antibodies and phorbol diesters, and cytokine production was evaluated. Clearly, the presence of IL-12 during the early 2 weeks of culture primed both the CD4 + and CD8 + clones for much higher production of both IL-10 and IFN- 7. The high variability in production of cytokines observed in human CD4 + T-cell clones expanded in the absence of exogenous IL-12 and IL-4 or in the presence of neutralizing antibodies against these two cytokines
Cytokine Production in Lymphocytes suggests that some of the cells were already primed in vivo for cytokine production. The cloning of sorted C D 4 5 R O - "naive" CD4+-cells and CD45RO + "mereory" CD4 +-cells supported this interpretation [40]. A high proportion of the clones generated from CD45RO ~ CD4+-cells in the presence of neutralizing antibodies to 1L-12 and IL-4 produced one or a combination of IFN7, IL-4 and IL-10, with a pattern of production that was not always consistent with the classical paradigm of Th 1and Th2-cells. When C D 4 5 R O - cells were cloned in the same conditions, the clones produced only negligible amounts of the three cytokines. However, in both populations, the presence of IL-12 during cloning endowed virtually all clones with the ability to produce high levels of IFN-7 and IL-10. The cytokine production pattern of clones derived from human CD4 + C D 4 5 R O - T-cells in the presence of IL-12 and/or IL-4 [40] is illustrated in Figure 2. Added IL-4 or endogenously produced IL-4 was necessary in the limiting dilution cultures to prime T-cell clones generated from C D 4 5 R O - cells for IL-4 production, whereas approximately half of the clones generated from CD45RO + cells produced IL-4, even when expanded in the absence of IL-4 and the requirement for 1L-4 in the generation of IL-4 producing cells was difficult to evaluate when total PBL were cloned, Although IL-12 is a major, and probably necessary, inducer o f a Thl response, it was also shown to potentiate IL-4 production and the development of Th2-cells from na~'ve CD4 + murine T-cells [51] and from neonatal CD4 + human T-cells [52], and to potentiate a Th2 response to Schistosoma mansoni in IFN- 7 knockout mice [53]. We showed [40] that IL-12 does not prevent IL-4 production from CD4 t clones derived from limiting dilutions of "nai've" adult peripheral blood C D 4 5 R O - cells and, in fact, significantly enhances the ability of IL-4 to prime the clones for high IL-4 production, thus extending the previous results [51-53] by demonstrating that IL-12 can enhance IL-4 production at the single clonal level via a differentiation effect. Furthermore, when T-cells were cloned in the simultaneous presence of IL-12 and IL-4, the IFN-7 priming effect of IL-12 was only partially, and often not significantly, inhibited by IL-4, whereas the priming for IL-10 production was reproducibly, and almost completely, blocked by IL-4 ([40] and Figure 2). Thus, paradoxically, IL-4 is more potent in inhibiting priming of Th cells for production of a Th2-type cytokine than for the typical Thl-type cytokine, IFN-7.
127
T-cells and N K cells, is the ability of various stimuli, includingT-cell receptor stimulation and other stimuli or co-stimuli, including cytokines, acting alone or in combination, to rapidly induce gene expression and cytokine production. For example, as depicted on the top of Figure 3, I L-12, alone or in synergy with other stimuli, induces accumulation o f m R N A for IFN- 7 within a few hours of treatment of either resting or activated T- or NK-cells, followed by secretion of IFN- 7. This acute induction of IFN-7 subsides within a couple of days (or, in vivo, even within less than 12 hr [13]) and does not induce a permanent alteration in the ability of the cells to produce IFN- 7 in response to IL-12 or other stimuli. The phenomenon of priming of the cytokine genes is schematically illustrated on the bottom of Figure 3, and is quite different from acute induction. When T-cells (and NK-cells) are clonally expanded in the presence of IL-12 during the first few days of expansion, the clones are primed for high production of IFN-7 and IL-10, even when cultured for several weeks in the absence of IL-12 stimulated in its absence; conversely, the exposure o f T cells to IL-4 during clonal expansion induces priming for IL-4 production and generation of IL-4-producing cells. IL-12 is particularly potent both in mediating acute induction of the IFN- 7 gene and in stably priming it for the response to other stimuli; however, although IL-12 similarly primes the IL-10 gene, its ability to acutely induce this gene is modest and difficult to demonstrate. Analogously, whereas IL-4 is necessary and extremely potent for the priming of the IL-4 and the generation of IL-4 producing cells, its ability to acutely induce the expression of the IL-4 gene has not been demonstrated. The IL-2 effect on lymphokine production is different from that of IL-12 and IL-4; IL-2, alone or in synergy with other stimuli, is a potent inducer of acute expression of the several lymphokines, including IFN-;., IL-4 and IL-10; however, although the presence of IL-2 may be required in the priming phenomena of all three genes, I L2 by itself does not determine the specificity of the priming, that is instead directed by IL-12 and IL-4 [14, 31, 38]. As mentioned above and summarized in Table 1, IL-4 and IL-12 both antagonize and synergize in inducing priming of lymphokine genes: IL-4 almost completely abolishes the IL-12 priming for Ik-10 production, Table 1. Ability of IL-12 and/or IL-4 to acutely induce expression of cytokine genes or to prime them for activation in response to restimulation
ACUTE INDUCTION VERSUS PRIMING FOR CYTOKINE PRODUCTION From the studies of the generation of T h l - and Th2cells, including those of our group reviewed above, it is becoming apparent that production of lymphokines, both type-1 and type-2, can be regulated with two different mechanisms. The first mechanism, observed particularly in preactivated lymphocytes, but also in resting
Responding gene
Type of induction
IFN-7
Acute Priming Acute Priming Acute Priming
IL-10 IL-4
Inducing cytokines IL-12
IL-4
+ ++ + + ++ + +/++ + +
-++ +
IL-12+ IL-4 -4- + + ++ '~ + ,'++ ++
128
G. Trinchieri et al.
104
CD8(+)
clones
"
":
i[i,E...-.~,,,..w~,Z,11j • . . e
..,,= % I
10 2
~. ; V : ? , ~. T. ". .". % ": " ali~;~.. l l ,.t.;:..~ -|~.,~,, ..-.s. ~ ":..~,' ... •
1 0 ~.
S,i - , ~ , : . " "" |~,..., .... • • " • ."~ • I I!i l.% • • !,'°¢ it,
~edlw
10o
.
.
.-
• #~= | l i OIl I e d ~
~..
."
•
• •
"l--
I °
•
|
•
el
• •
•
lie
. . . . . . .~ . . . . . . . ~ . . . . . . ,
•
•
...... ,
......
.I
I 7 II
CD4(+)
clones
104-
1 0 s,
.• : ~ .
J.. II0
•
='•
•
-.'..~p
1
.,
.
i::',: ,:':
'" :
I~
10 o
-"
101
.
.
"~
. .l.,.q .
.
.
.
.
10 2
" .
~'
E
". °
~I,
" 0" II
•
."'_
.
......
.%,'-~ •
,J'~.../..o
...'..%.'~."
•"• 1 " " : "°° " . -
10 0
e w ,o % I P t
•
o•
10 =' :..,. 10"
•.'.~
• :. Z:,{t~',L~
• ~
(onti-lL-12
•
(onti-lL-
•
IL-12
•
IL-12
•
IL-12
•
IL-4
,oo'°°,
"
" II
"
• ",,l~
10 4
10 s
IL- 1 O, pg/ml Figure 1. IL-12 primes human CD4 ÷ and CD8 ÷ T-cell clones for production of IFN-v and IL-10. PBL from several donors were cloned in the presence of PHA, and the clones were expanded in the presence of feeder cells (irradiated autologous PBMC and RPMI-8866 cell line) and IL-2 (50 U/ml). Clonal growth efficiency was, on average, over 50%. After 4-5 weeks of culture, clones were washed in medium without cytokines, stimulated for 18 hr with soluble anti-CD3 antibodies and the phorbol diester TPA, and cytokine production in the supernatant was quantitated by radioimmunoassays. Each dot represents cytokine production from an individual clone. Red dots indicate clones expanded in the presence of IL-12 (2.5 ng/ml) in the first 2 weeks of culture; blue dots represent clones expanded in the presence of neutralizing anti-IL-12 antibodies,
I ,,z
+ anti-lL-4) 12)
(anti-lL-4) + IL-4 (onti-lL-12)
,oo'°°
,=
'
e
D 300.
......
10 ~
•
--I-
200"
300 200
100-
100
o" 1 " I - - ~ ' " 4.60 IL-4,
........
" 800 pg/ml
" 12'00
o ~ 0
='
...........
lO'OO ,L-IO,
2000
30100
pg/m,
Figure 2. Cytokine production by human CD4 + clones generated from sorted CD45RO- cells. CD45RO- cells were cloned by limiting dilution (PHA stimulation) in the presence or absence of IL-12 (2.5 ng/ml), IL-4 (50 U/ml), anti-IL-12 neutralizing mAb, anti-IL-4 neutralizing mAb or combinations thereof. After 4 weeks, the clones were washed, incubated for 24 hr in medium containing IL-2 (50 U/ml) only, washed again, and then stimulated for 18 hr with soluble anti-CD3 antibodies and TPA; cytokines were measured in the cell-free supernatant fluid by radioimmunoassay. The graphs indicate the m e a n + SE of the cytokine production of all clones analyzed in each cloning condition. The arbitrary threshold of cytokine production (broken lines) is drawn to facilitate the analysis of the results. Clonalefficiencywasapproximately50%,exceptinthepresence of anti-IL-4, where the efficiency was 20-30%.
Cytokine Production in Lymphocytes
129
Acute cytokine induction
4 Oh I
24h
48h I
IL-12 (+1- other co-stimuli)
Priming for c y t o k i n e p r o d u c t i o n ~--~
IL-2 +
i
c.,,.
I I 1 week 2 O~neither IL-12 nor IL-4)
I
II
IL-12
I
Cytokine production 5
I 3
I 4
I 5
I 6
t anti:CD3 + TPA (neither IL-12 nor IL-4)
IL-4
Figure 3. Acute induction vs. priming for cytokine production. The figure schematically depicts the characteristics of these two mechanisms of regulation of cytokine gene expression. The ability of IL-12, alone or in synergy with other co-stimuli, is presented as a typical example of acute induction. On both resting and activated T- and NK-cells, IL-12 rapidly induces accumulation of IFN-), m R N A and production of the protein; this induction subsides within 1 or 2 days without permanently altering the ability of the cells to respond to a second challenge (by IL-12 or other stimuli) with IFN- 7 production. The priming for cytokine production is observed when T-lymphocytes are expanded in the presence of IL-12 or IL-4 for the first 1-2 weeks of culture. The T-cells can then be expanded for several weeks in the absence of either IL-12 or IL-4. If the cells are then stimulated (e.g. by antiCD3 and TPA, with no requirement for the presence of either IL-12 or IL-4 during the restimulation), the cells that have been expanded in the presence of IL-12 produce much higher levels of both IFN- 7 and IL-10, and the cells that have been expanded in the presence of IL-4 produce much higher levels of IL-4. Thus, the exposure of the lymphocytes to either IL-12 or IL-4 in the first few days of clonal expansion determines a stable and likely reversible change (priming) in the ability of the lymphokine genes to be activated in response to a restimulation challenge.
130
G. Trinchieri et al.
whereas it only partially decreases the priming for IFN7; IL-12 potentiates, rather than inhibits, the IL-4 priming of T-cells from high IL-4 production, The priming of lymphokine genes represents a stable modification of the inducibility of the genes which is analogous to the stable phenotype in the pattern ofcytokine production typical of Th-subsets. Thus, it is likely that this priming mechanism plays a role in the determination of the Th-phenotype of activated T-cells. However, certain effects of IL-12 and IL-4 on Th generation are not observed when the ability of these cytokines to induce differentiation is analyzed at the single clonal level (for example, in this clonal analysis, the powerful ability of IL-12 to block IL-4 production is not reproduced and IL-12, paradoxically, induces T-cells priming for production of IL-10, a prevalently type-2 cytokine). Thus, although the priming of lymphokine genes is most likely the predominant mechanism by which IL-12 and IL-4 induce differentiation of Th-cells, the final generation of cells with T h l - and Th2-phenotype, both in vivo and in vitro, also depends on complex indirect effects of the cytokines, including selective mechanisms, in addition to a direct differentiative effect at the single cell level, The molecular mechanisms of both the acute induction and the priming effects, still remain mostly undetermined, The major signal transduction mechanisms for IL-4 and IL-12 have recently been elucidated, with the former cytokine inducing activation of STAT-6 [54] and the latter of STAT 1, 3 and 4 [55, 56]; however, the role of these transcription factors in the induction of expression of the IL-4, IFN-7 and IL-10 genes still remains to be elucidated. The priming effects may depend on the induction of a constitutive or facilitated expression of the transcription factors responsible for the expression of the lymphokine genes or in a stable alteration of the genes in a transcriptionally-prone conformation. For example, the IFN-7 gene has been shown to be differentially methylated in T h l - and Th2-clones [57]. Post-transcriptional mechanisms could also be responsible for the priming effect, The existence of alternative mechanisms of regulation of lymphokine gene expression adds a new complexity to the phenomenon of differentiation of Th-cells and other lymphocyte subsets. However, this new information also represents a challenge to address the molecular mechanisms responsible for determining the cytokine production pattern of lymphocyte subsets and will hopefully help to open the door to the understanding of these important mechanisms of regulation of the immune system. Acknowledgements: We would like to thank Ms Marion Kaplan
for preparing the manuscript. The experimental work described in this review was supported in part by NIH grants CA 10815, CA 20833, CA 32898 and AI 34412.
REFERENCES 1. Parish CR, Liew FY. Immune response to chemically modifled flagellin. 3. Enhanced cell-mediated immunity during
high and low zone antibody tolerance to flagellin. J Exp Med 1972, 135, 298-311. 2. Mosmann TR, Coffman RL. TH 1 and TH2 cells; different patterns oflymphokine secretion lead to different functional properties. Ann Rev Immunol 1989, 7, 145-173. 3. Seder RA, PaulWE. Acquisition of lymphokine-producing phenotype by CD4+ T cells. Ann Rev Immunol 1994, 12, 635-673. 4. Le Gros G, Ben-Sasson SZ, Seder R, Finkelman FD, Paul WE. Generation of interleukin 4 (IL-4)-producing cells in vivo and in vitro: IL-2 and IL-4 are required for in vitro generation of IL-4-producing cells. J Exp Med 1990, 172, 921-929. 5. Kobayashi M, Fitz L, Ryan M e t al. Identification and purification of Natural Killer cell stimulatory factor (NKSF), a cytokine with multiple biologic effects on human lymphocytes. J Exp Med 1989, 170, 827-846. 6. D'Andrea A, Rengaraju M, Valiante NM et al. Production of natural killer cell stimulatory factor (NKSF/IL-12) by peripheral blood mononuclear cells. J Exp Med 1992, 176, 1387-1398. 7. Macatonia SE, Hosken NA, Litton M et al. Dendritic cells produce IL-12 and direct the development of Thl cells from naive CD4 + T cells. JImmunol 1995, 154, 5071 5079. 8. Trinchieri G. Interleukin-12: a proinflammatory cytokine with immunoregulatory functions that bridge innate resistance and antigen-specific adaptive immunity. Ann Rev Immunol 1995, 13, 251-276. 9. Manetti R, Parronchi P, Giudizi MG et al. Natural killer cell stimulatory factor (NKSF/IL- 12) induces Th 1-type specific immune responses and inhibits the development of IL-4 producing Th cells. J Exp Med 1993, 177, 1199-1204. 10. Hsieh C, Macatonia, SE, Tripp CS, Wolf SF, O'Garra A, Murphy KM. Listeria-induced Thl development in eftTCR transgenic CD4 + T cells occurs through macrophage production of IL-12. Science 1993, 260, 547-549. 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, 235237. 12. Trinchieri G. Interleukin-12 and its role in the generation of TH1 cells. Immunol Today 1993, 14, 335-338. 13. Wysocka M, Kubin M, Vieira LQ et al. Interleukin-12 is required for interferon-7 production and lethality in lipopolysaccharide-induced shock in mice. Eur Jlmmuno11995, 25, 672-676. 14. Chan SH, Perussia B, Gupta JW et al. Induction of IFN-7 production by NK cell stimulatory factor (NKSF): Characterization of the responder cells and synergy with other inducers. J Exp Med 1991, 173, 869-879. 15. Kubin M, Chow JM, Trinchieri G. Differential regulation of interleukin-12 (IL-12), tumor necrosis factor-e, and ILlfl production in human myeloid leukemia cell lines and peripheral blood mononuclear cells. Blood 1994, 83, 18471855. 16. Murphy EE, Terres G, Macatonia SEet al. B7 and IL-12 cooperate forproliferationandIFN-Tproductionbymouse T helper clones that are unresponsive to B7 costimulation. J Exp Med 1994, 180, 223-231. 17. Erard F, Wild M, Garcia-Sanz JA, LeGros G. Switch of CD8 T cells to noncytolytic CD8 T cells that make Th2 cytokines and help B cells. Science 1993, 260, 1802-1803. 18. Seder RA, Boulay JL, Finkelman F et al. CD8 + T cells can be primed in vitro to produce IL-4. J. Immunol 1992, 148, 1652-1656. 19. Manetti R, Gerosa F, Giudizi MG et al. Interleukin-12 induces stable priming for interferon-7 (IFN-7) production during differentiation of human T helper (Th) cells and transient IFN-7 production in established Th2 cell clones. J Exp Med 1994, 179, 1273-1283.
Cytokine Production in Lymphocytes 20. Maggi E, Giudizi MG, Biagiotti R et al. Th2-1ike CD8 ÷ T cells showing B cell helper function and reduced cytolytic activity in human immunodeficiency virus type 1 infection. J Exp M e d 1994, 180, 489-495. 21. Ferrick DA, Schrenzel MD, Mulvania T, Hsieh B, Ferlin WG, Lepper H. Differential production of interferongamma and interleukin-4 in response to Thl- and Th2stimulating pathogens by gamma delta T cells in t,ivo. Nature 1995, 373, 255 257. 22. Warren HS, Kinnear BF, Phillips JH, Lanier LL. Production of IL-5 by human N K cells and regulation of I L-5 secretion by IL-4, IL-10, and IL-12. J Immunol 1995, 154, 5144-5152. 23. Kamogawa Y, Minasi LE, Carding SR, Bottomly K, Flaveil RA. The relationship of IL-4- and IFNT-producing T cells studied by lineage ablation of lL-4-producing cells. Cell 1993, 75, 985-995. 24. Firestein GS, Roeder WD, Laxer JA et al. A new murine CD4 ÷ T cell subset with an unrestricted cytokine profile. J lmmumd 1989, 143, 518-525. 25. Kelso A, Groves P, Troutt AB, Francis K. Evidence for the stochastic acquisition of cytokine profile by CD4 ~ T cells activated in a T helper type 2-like response in vivo. Eur J Immunol 1995, 25, 1168-1175. 26. Elson LH, Nutman TB, Metcalfe DD, Prussin C. Flow cytometric analysis for cytokine production identifies T helper 1, T helper 2, and T helper 0 cells within the human CD4 + CD27 lymphocyte subpopulation. Jlmmuno11995, 154, 4294-4301. 27. Picker LJ, Singh MK, Zdraveski Z et al. Direct demonstration of cytokine synthesis heterogeneity among human memory/effector T cells by flow cytometry. Blood 1995, 86, 1408-1419. 28. Kubin M, Kamoun M, Trinchieri G. Interleukin-12 synergizes with B7/CD28 interaction in inducing efficient proliferation and cytokine production of human T cells. J Exp Med 1994, 180, 211-222. 29. Aste-Amezaga M, D'Andrea A, Kubin M, Trinchieri G. Cooperation of natural killer cell stimulatory factor/interleukin-12 with other stimuli in the induction of cytokines and cytotoxic cell-associated molecules in human T and N K cells. Celllmmunol 1994, 156, 480-492. 30. Chan SH, Kobayashi M, Santoli D, Perussia B, Trinchieri G. Mechanisms of IFN- 7 induction by natural killer cell stimulatory factor (NKSF/IL-12): Role of transcription and m R N A stability in the synergistic interaction between N K S F and IL-2. J Imrnunol 1992, 148, 92 98. 31. Trinchieri G, Matsumoto-Kobayashi M, Clark SC, Sheehra J, London L, Perussia B. Response of resting human peripheral blood natural killer cells to interleukin2. J Exp M e d 1984, 160, 1147-1169. 32. Gazzinelli RT, Hieny S, Wynn TA, Wolf S, Sher A. Interleukin-12 is required for the T-lymphocyte independent induction of interferon-y by an intracellular parasite and induces resistance in T-deficient hosts. Proc Nail Acad Sci USA 1993,90, 6115 6119. 33. Tripp CS, W o l f S F , Unanue ER. Interleukin 12 and tumor necrosis factor alpha are costimulators of interferon gamma production by natural killer cells in severe combined immunodeficiency mice with listeriosis, and interleukin 10 is a physiologic antagonist. Proc Natl Acad Sci USA 1993.90, 3725-3729. 34. D'Andrea A, Aste-Amezaga M, Valinate NM, Ma X, Kubin M, Trinchieri G. Interleukin-10 inhibits human lymphocyte IFN-y production by suppressing natural killer cell stimulatory factor/interleukin-12 synthesis in accessory cells. J Exp M e d 1993, 178, 1041 1048. 35. Gately MK, Warrier RR, Honasoge Set al. Administration of recombinant IL-12 to normal mice enhances cytolytic lymphocyte activity and induces production of IFN-7 in t,iz~o, lnt lmmunol 1994, 6, 157-167.
131
36. Swain SL, Weinberg AD, English M, Hutson G. 1L-4 directs the development of Th2-1ike helper effectors. J Immunol 1990, 145, 3796-3806. 37. Seder RA, Paul WE, Davis MM, Fazekas de St Groth B. The presence of interleukin 4 during in vitro priming determines the lymphokine-producing potential of C D 4 + T cells from T cell receptor transgenic mice. J Exp Med 1992, 176, 1091-1098. 38. Van der Pouw-Kraan T, Van Kooten C, Rensuik I, Aarden L. Interleukin (IL)-4 production by human T cells; differential regulation of IL-4 vs IL-2 production. Eur .I Immunol 1992, 22, 1237-1241. 39. Trinchieri G, Scott P. Interleukin-12: a proinflammatory cytokine with immunoregulatory functions. Res hnmunol 1995, 146, 423-431. 40. Gerosa F, Paganin C, Peritt D et al. IL-12 primes human CD4 and CD8 T cell clones for high production of both IFN-7 and IL-10. J Exp Med 1996, 183, 2559-2570. 41. Seder RA, Gazzinelli R, Sher A, Paul WE. IL-12 acts directly on CD4 + T cells to enhance priming for IFNy production and diminishes IL-4 inhibition of such priming. Proc Natl A c a d S c i USA 1993, 90, 10188-10192. 42. Swain SL, McKenzie DT, Weinberg AD, Hancock W. Characterization of T helper 1 and 2 cell subsets in normal mice. Helper T cells responsible for IL-4 and IL-5 production are present as precursors that require priming before they develop into lymphokine-secreting cells. J Immunol 1988, 141, 3445-3455. 43. Street NE, Schumacher JH, Fong AT et al. Heterogeneity of mouse helper T cells: Evidence from bulk cultures and limiting dilution cloning for precursors of Thl and Th2 cells. J lmmunol 1993, 144, 1629 1639. 44. Gajewski TF, Joyce J, Fitch FW. Antiprolifertive effect of IFN-7 in immune regulation. III. Differential selection of Thl and Th2 murine helper T lymphocyte clones using recombinant IL-2 and recombinant IFN-y. J lmmuno11989, 143, 15 22. 45. Wu CY, Demeure C, Kiniwa M, Gately M, Delespesse G. IL-12 induces the production of IFN-gamma by neonatal human CD4 T cells. J lmmunol 1993. 151, 1938-1949. 46. Del Prete GF, De Carli M, Mastromauro C et al. Purified protein derivative of Mycobacterium tuberculosis and excretory-secretory antigen(s) of Toxocara canis expand in t,itro human T cells with stable and opposite (type 1 T helper or type 2 T helper) profile of cytokine production. J Clin Infest 1991, 88, 346-350. 47. Gerosa F, Trinchieri G. mechanisms o f T helper cell differentiation induced by interleukin-12. In Romagnani S, Del Prete G, Abbas AK, eds. Cytokines: Basic Principles and Practical Applications. Rome, Ares-Serono Symposia Publications, 1994, 251-263. 48. DeKruyff RH, Fang Y, Wolf SF, Umetsu DT. IL-12 inhibits IL-4 synthesis in keyhole limpet hemocyaninprimed CD4 * T cells through an effect on antigen-presenting cells. J Immunol 1995, 154, 2578 2587. 49. Marshall J, Secrist H, DeKruyff RH, Wolf SF, Umetsu DT. IL-12 inhibits the production of IL-4 and 1L-10 in allergen-specific human CD4 + T lymphocytes. J lmmunol 1995, 115, 111 117. 50. Finkelman FD, Madden KB, Cheever AW et al. Effects of interleukin 12 on immune responses and host protection in mice infected with intestinal nematode parasites. J Exp Med 1994, 179, 1563 1572. 51. Schmitt E, Hoehn P, Germann T, ROde E. Differential effects ofinterleukin-12on the development ofnaivemouse CD4 + T cells. Eur J Immunol 1994, 24, 343-347. 52. Wu CY, Demeure CE, Gately M e t al. In i;itro maturation of human neonatal CD4 T lymphocytes: I. Induction of IL4-producing cells after long-term culture in the presence of IL-4 plus either 1L-2 or 1L-12. J Immunol 1994, 152, 1141 1153.
132
G. Trinchieri et al.
53. Wynn TA, Jankovic D, Hieny S e t al. IL-12 exacerbates tyrosine phosphorylation of signal transducer and activator rather than suppresses T helper 2-dependent pathology in of transcription (Stat)3 and Stat4. J Exp Med 1995, 181, the absence of endogenous IFN- 7. J Immunol 1995, 1 5 4 , 1755-1762. 3999-4009. 56. Bacon CM, Petricoin III EF, Ortaldo JR et al. IL-12 54. Quelle FW, Shimoda K, Thierfelder W e t al. Cloning of induces tyrosine phosphorylation and activation of STAT4 murine Stat6 and human Stat6. Stat proteins that are tyroin human lymphocytes. Proc Natl Acad Sci USA 1995, 92, sine phosphorylated in responses to IL-4 and IL-3 but are 7307-731 l. not required for mitogenesis. Molec Cell Biol 1995, 15, 57. Young HA, Ghosh P, Ye J e t al. Differentiation of the T 3336-3343. helper phenotypes by analysis of the methylation state of 55. Jacobson NG, Szabo SJ, Weber-Nordt RM et al. Interthe IFN-7 gene. J l m m u n o l 1994, 153, 3603-3610. leukin 12 signaling in T helper type 1 (Thl) cells involves
S1359-6101(96)00018-4
ELSEVIER
Printed in Great Britain. 1359-6101/96 $32.00+0.00
MINI REVIEW
Acute Induction and Priming for Cytokine Production in Lymphocytes
Giorgio Trinchieri*t, David Perittt and Franca Gerosat When T-lymphocytes (CD4 +, CD8 +, or TCR~6 +) and NK cells proliferate in vivo or in vitro in response to exposure to antigen or other stimuli, they often segregate into subsets with the ability to produce either type-1 [interferon-T (IFN-~) and interleukin-2 (IL-2)] or type-2 cytokines (IL-4, IL-5, IL-6 and IL-10). IL-12 induces the differentiation of type-I cytokine-producing T-cells primarily through its ability to prime them for high IFN-? production; however, paradoxically ILl2 also primes T-cells for high production of the type-2 cytokine IL-10. Priming of T-cells for IL-4 production requires the presence of IL-4, but it is maximally observed in cultures containing both IL-4 and IL-12. IL-12, in addition to priming T-cells for high IFN-? and IL-10 production, is also a potent acute inducer of expression of the IFN-y gene in T- and NK-cells, and, to a much lower extent, of the |L-10 gene. IL-4, which has a very powerful effect in priming T-cells for IL-4 production, does not appear to have a significant ability to directly activate the expression of the IL-4 gene. Thus, IL-12 and IL-4 affect the expression of type-1 and type-2 cytokine genes by two different mechanisms: an acute induction of gene expression which is rapid and reversible, and a priming of the genes to a highly responsive state to restimulation, a state that is stable and probably irreversible. Copyright ~ 1996 Elsevier Science Ltd. Key words: Acute induction" Priming" Cytokine production" Lymphocytes.
SUBSETS OF LYMPHOCYTES WITH DISTINCT CYTOKINE PRODUCTION PATTERNS
humoral responses, production of lgE and IgA, and activation of eosinophils and basophils. The differentiation of Th-cells toward a Th 1- or Th2-phenotype occurs early The immune response to infectious agents and to nominal during an immune response, and is influenced by many antigens is often characterized by a dominance of either interrelated factors, including the nature and the concell-mediated or humoral-type effector mechanisms [1], centration of the antigen, the anatomical localization of which has been attributed to a dichotomy in the cytokine the immune response, the nature of the antigen-preproduction pattern ofT-helper (Th) CD4+-cells [2]. Two senting cells (APC) and the cytokine milieu at the site of classes of Th-cells have been described: Thl-cells, which the immune response [3]. IL-4, produced by T-cell subsets produce interleukin-2 (IL-2), interferon-7 (IFN-7) and and possibly basophils, appears to be the most important lymphotoxin (LT) and favor cell-mediated immunity, cytokine responsible for Th2-cell generation [4], whereas delayed-type hypersensitivity, macrophage activation IL-12, produced by macrophages and professional APC, and production of opsonizing antibodies; and Th2-cells, such as dendritic cells and Langerhans cells [5-8], is which produce IL-4, IL-5, IL-6 and IL-10, and favor required for effective Thl-cell generation [9 12]. IL-12 acts at three different levels in favoring a Thl-response: (1) it acts as a proinflammatory cytokine inducing IFNt The Wistar Institute of Anatomy and Biology, Philadelphia, 7 from T- and NK-cells within a few hours after infection, U.S.A. and through IFN-?, it induces activation of phagocytic ~:Istituto di Immunologia e Malattie Infettive, Policlinico di cells for bacteriocidal activity and also for enhanced proBorgo Roma, 37100 Verona, Italy. * To whom correspondence should be addressed at: The Wistar duction of cytokines, including IL- 12 itself [6, 13-15]; (2) Institute, 3601 Spruce Street, Philadelphia, PA 19104, U.S.A. in the first few days of an immune response, IL-12 and Tel.: 215-898-3992; Fax: 215-898-2357; e-mail: trinch- IL-12-induced IFN-7 act on antigen-stimulated T-cells to ieri(a~wista.wistar.upenn.edu favor differentiation and proliferation of cells and a Thl123
124
G. Trinchieri et al.
phenotype [9-12]; (3) once differentiated Thl are generated, IL-12 is required for optimal cytokine production and, in certain cases, proliferation of Thl-cells [16]. The dichotomy in the cytokine production pattern is not limited to CD4 + Th-cells, but is also clearly demonstrated for CD8 + T-cells [17-20], for T-cells with a 76 T-cell receptor [21], and, to a certain extent, for natural killer cells [22]. Although the expression of Thl-type and Th2-type cytokines was initially considered to be mutually exclusive, Th-cells expressing both Thl- and Th2-cytokines (referred to as Th0-cells) have been identified both during differentiation of Th-cells [23] and in terminally differentiated cells [24]. Indeed, evidence for a stochastic acquisition of cytokine profile, suggesting independent regulation of each cytokine gene, was obtained during a mouse Th2-response to keyhole limpet hemocyanin (KLH) [25] and in human peripheral blood memory CD4 + T-cells [26, 27].
cells) is required for optimal IFN-7 production by resting PBL [14]. These accessory cells might provide costimulatory molecules for IFN-7 production. In murine spleen cells, it has been shown that IL-12 synergizes with TNF~ in inducing IFN-7 production [32, 33]. This synergistic effect of TNF-~ was not demonstrated with human lymphocytes, but antibodies to TNF-c~ or IL-1/~ efficiently inhibited IL-12-induced IFN-7 production suggesting that these two cytokines, endogenously produced in the PBL cultures, possibly by the class II positive accessory cells, act as costimulatory molecules for IFN-7 production together with IL-12 [34]. Another costimulatory signal possibly provided by the accessory cells is the B7 molecule, a ligand for the CD28 receptors on T-cells. Stimulation of T-cells with B7-transfected cells or with anti-CD28 antibodies, strongly synergized with IL-12 for induction of IFN-~ production [16, 28] and blockage of B7-CD28 interaction with the hybrid recombinant molecule CTLA4-Ig, significantly inhibited the ability of PBL to produce IFN-y in response to IL-12 [28]. These results suggest that TNF-~, IL-1//, and B7, possibly at IL-12 IS A POTENT INDUCER OF IFN-~, least in part, provided by the class-II accessory cells, are PRODUCTION IN BOTH RESTING AND ACTIVATED important costimulators for IFN-7 production in T-LYMPHOCYTES AND NK CELLS response to IL-12. The ability of IL-10 to inhibit IFN-7 production in T- and NK-cells is primarily due to its IL-12 induces IFN-~: production from resting and acti- ability to suppress IL-12 production, but also, in part, to vated NK- and T-cells, with a similar dose-response its ability to suppress expression of these costimulatory relationship and a half maximal activity at 3.5 pM [5, 14]. molecules on accessory cells [16, 28, 34]. Within T-cells, both T-cells with an ~/~ T-cell receptor Not only is IL-12 a potent inducer of IFN-7 (TCR) both CD4 + and CD8 +, and T-cells with 76 TCR production, but it is also most likely a required factor for are induced to produce IFN-y [14]. The induction of IFN- efficient IFN-7 production depending, in vivo and in vitro, by IL-12 is characterized by a powerful synergistic effect on accessory cells. When human PBMC were treated in with other IFN-y inducers, in particular IL-2 and phorbol vitro with stimuli, e.g.S, aureus, that were able to induce diesters [5, 14]. On T-cells, IL-12 also synergizes with production of IL-12, they rapidly produced large mitogenic lectins, with stimulation bf the TCR-CD3 com- amounts of IFN- 7. This production of IFN- 7 was almost plex by anti-CD3 antibodies or alloantigens [14], and completely inhibited by neutralizing antibodies against with stimulation of the CD28 receptor by anti-CD28 IL-12 [16]. Even when IFN- 7 inducers, which are not antibodies or its ligand B7 [16, 28]; and on NK-cells with known to stimulate IL-12 production, were used (e.g. ILstimulation by ligands of the CD16 receptor for IgG- 2 or anti-CD3 antibodies), the production of IFN- 7 from Fc (anti-CD 16 antibodies or immunocomplexes) and by PBMC was inhibited up to 80%, indicating that endogentarget cells [29]. IL-12 rapidly increases the tran- ously produced IL-12 is required for optimal IFN- 7 proscriptional rate of the IFN- 7 gene; however, when IL-12 duction [6]. However, if purified T- or NK-cells, in the and IL-2 are added together to the cells, most of the absence of IL-12 producing accessory cells, were stimusynergistic effects of the two inducers are observed at the lated to produce IFN-y (e.g. by IL-2 or anti-CD3 antiposttranscriptional level, with an increase of more than bodies), no inhibitory effect of anti-IL-12 antibodies two-fold of the half-life of the IFN-7 mRNA in the cells could be demonstrated [6]. treated with the two inducers together [30]. Both resting The ability of IL-12 to induce a rapid production of and activated NK- and T-cells are induced by IL-12 to IFN-y in vivo has been clearly shown in several experproduce IFN-7, although maximal IFN-y mRNA imental models of infectious diseases. A very informative accumulation is reached in 2-4 hr in activated T- or experimental model for the understanding of the role of NK-cells, and in 18-24 hr in resting peripheral blood IL-12 in inducing IFN-7 in vivo is provided by the endolymphocytes (PBL) [14]. Within PBL, IL-12 induces toxic shock in Bacille Calmette Gu6rin (BCG)-primed mRNA accumulation, as detected by in-situ hybrid- mice [13]. Several cytokines, in particular TNF-~ and ization, in a proportion of both NK- and T-cells [14], IFN-7, have been shown to be responsible for pathoalthough N K cells might be major contributors to the logical reactions which may lead to shock and death early production of IFN-7 in response to IL-12 or IL-2 observed in infection with Gram-negative bacteria and [31]. Although NK- and T-cells are the IFN-7 producers in response to endotoxins. Priming of mice with the aviin PBL preparations stimulated by IL-12, an accessory rulent BCG vaccine strain of Myeobacterium boris cell type (MHC class II positive, non-monocyte, non-B- increases the sensitivity of mice to the lethal effect of LPS
Cytokine Production in Lymphocytes and results in an efficient priming for cytokine production in response to LPS. Mice, injected with LPS, produced IL-12, which induced IFN-7 production, as demonstrated by the ability of neutralizing anti-IL-12 antibodies to suppress IFN- 7 production [13]. However, the concentration of biologically active IL-12 was similar in the serum of both BCG-primed or unprimed mice, reaching levels of 1 3 ng/ml at 3-6 hr after LPS injection, whereas IFN- 7 production was observed only in BCG-primed mice [13]. TNF-~ and other LPS-induced cofactors were required in cooperation with IL-12 to induce optimal IFN- 7 production. The priming effect of BCG on IFN- 7 production appears to be mostly due to its ability to increase TNF-~ production, which acts as a cofactor with LPS-induced IL-12 in inducing IFN- 7 production [13]. Neutralizing anti-IL-12 antibodies, in addition to inhibiting the in vivo LPS-induced IFN- 7 production, also protected mice from septic shock-induced death [13]. Thus, IL-12 is required for IFN- 7 production and lethality in an endotoxic shock model in mice. Injection of mice with a daily i.p. injection of 1 /~g of recombinant IL-12 induced high levels of serum IFN-7., but only starting 48 hr after the first injection [35]. This delayed response was probably due to the lack of appropriate costimulatory signals when only recombinant IL12 was injected. The injected IL-12 had a serum half-life of 3.3 hr [13], much longer than that of other cytokines, Although IL-4 has clearly been shown to be required for the differentiation of IL-4-producing T-cells [4, 36, 37], little information is available on the ability of IL-4 to directly upregulate its own gene by inducing acute production of IL-4. Induction of IL-4 from resting lymphocytes has been more difficult to detect than production of IL-2 or IFN-7; however, stimulation of human PBL with recombinations of anti-CD2, CD3, CD28 antibodies and/or IL-2 has been shown to result in the production of IL-4 within 2448 hr [38]. The presence of IL-4 during stimulation by antigen or anti-CD3 antibodies results in the induction of IL-4 gene expression only after 3-4 days during culture (A. Lichtman, personal communication), presumably when differentiation of IL4 producing Th2 cells is already taking place. Thus, unlike IL-12 that induces potent upregulation of the IFN-7 gene within a few hours of stimulation, IL-4 appears to be able to promote the generation of IL-4 producing cells, rather than to acutely induce the expression of the IL-4 gene.
THE ABILITY OF IL-12 AND Ik-4 TO INDUCE GENERATION OF T h l AND Th2 CELLS,
RESPECTIVELY, IS DUE IN PART, TO THEIR ABILITY TO PRIME T-CELLS FOR Thl-TYPE OR Th2-TYPE CYTOKINE PRODUCTION The requirement of IL-4 for the generation of IL-4 producing Th2 cells has been a well-established Concept for several years [4, 36, 37]. More recently, the role of IL12 for the efficient generation of IFN-7 production by Thl cells has become evident, and it has been proposed
125
that the balance between the levels of IL-4 and IL-12 early during an immune response may be responsible to bias the generation of Th2 and Thl cells, respectively [12],although the presence of other cytokines and various other factors regulating the immune response also play a major role. Furthermore, the synergistic/antagonistic interaction between IL-4 and IL-12 in regulating such responses is complex and not yet fully understood [39, 40]. Stimulation in vitro of peripheral blood lymphocytes (PBL) from atopic patients with allergens such as Dermatophagoides pteronyssinus Group 1 (Der p.1), results in the generation ofT-cell lines and clones with the high IL4 and low IFN-7 production typical of Th2-cells, whereas PBL stimulation with bacterial products (e.g. purified protein derivative, PPD) generates Thl-type T-cell lines and clones that produce IFN- 7 but not IL-4. When PBL are stimulated with Der p.I in the presence of IL-12, Tcell lines and clones are generated that exhibited a reduced ability to produce IL-4 and an increased ability to produce IFN-7 [9]. This Thl-inducing effect of IL-12 was not inhibited by anti-IFN-7, but was reduced by removal of NK cells from the PBL preparation. PPDspecific T-cell lines generated in the presence of antiIL-12 antibodies during the initial antigenic stimulation produced significant levels of IL-4, unlike the cell lines generated in the absence of antibodies, and gave rise to PPD-specific CD4 + cell clones showing a Th0/Th2phenotype rather than a Thl-phenotype [9]. These results indicate not only that IL-12 is able to facilitate proliferation and activation of Thl-cells in a memory response in vitro, but also that, as shown by the effect of anti-IL-12 antibodies, endogenously produced IL-12 is an obligatory factor for Thl generation. The ability of IL-12 to directly initiate Thl-cell development in na~'ve T-cells was shown by Hsieh et al. [10] who reported that nai've CD4 + T-cells derived from mice transgenic for an anti-ovalbumin TCR are induced by ovalbumin to develop into Thl-cells in the presence of IL- 12, whereas they develop into Th2-cells in the presence of IL-4. The effect of IL-4 in that system, is, however, dominant over that of IL-12. Since these early studies, the ability of IL-12 to induce Thl-cell generation was demonstrated in many models, in humans and in experimental animals, both in vitro and in vivo [8]. The originally reported dominance of IL-4 action over IL-12 [! 0] was observed to be a much more complex interaction, with the two cytokines antagonizing or synergizing each other, depending on the function analyzed [39-41]. The experimental systems used for studying Th response have not permitted determination of whether the different cytokines affecting Th-cell development induce differentiation of bipotential Th precursors or rather a selective priming and/or expansion of already committed Thl- and Th2-precursor cells [42-45]. This question is particularly relevant in human studies which have analyzed clonal expansion of memory Th-cells [9, 46]. However, once a Thl- or Th2 response has been established, it appears to be relatively stable, and no
126
G. Trinchieri et al.
factors capable of inducing qualitative changes in the cytokine profile of established murine or human T-cell clones have been reported, In the analysis of cytokine production from human Tcells stimulated with recall antigens (PPD) or allergens (Der p.1), the expansion of the small proportion of memory T-cells was first obtained in polyclonal T-cell cultures, from which single antigen-specific clones were obtained only after several weeks of culture of the polyclonal cell line [9, 46]. During this culture period, emergence of Th-cell subsets with characteristic cytokine production profiles could be due to differentiation of precursor Thcells, as well as to positive selection (growth advantage) of certain Th-subsets or negative selection (apoptosis, cytotoxicity, anti-proliferative effects) of other subsets, When human T-cell cultures were stimulated by a polyclonal stimulus affecting all T-cells, such as PHA or antiCD3, in the presence or absence of IL-12, similar results were obtained as in antigen-stimulated cultures, i.e. IFNproduction by the cells was enhanced and IL-4 production was almost completely abolished [47]. However, very different results were obtained when freshly isolated human peripheral blood T-cells were immediately cloned by limiting dilution in cultures stimulated by PHA and IL-2, in the presence or absence of IL-12 [19]. When restimulated with anti-CD3 and TPA after 5 weeks of culture, the clones generated in the presence of IL-12 produced on average five- to 20-fold higher levels of IFN7 than the clones generated in the absence of IL-12. This priming for IFN-7 production required the addition of IL-12 within the first week, but its presence for maximal priming was required only for 1 or 2 weeks [40]. Once the clones were established for 2 or 3 weeks, removal or addition of IL-12 from the culture medium did not significantly affect their ability to produce IFN- 7 [19]. Because the clonal efficiency in these experiments was close to 100%, the priming effect of IL-12 was not due to selection of high IFN-~-producing clones, but was exerted on each single T-cell, naive or memory. Furthermore, this effect was observed on both CD4 + and CD8 + cells, suggesting that IL-12 affects the differentiation of Thl-type clones from both subsets. Thus, the presence of IL-12 during the initial clonal proliferation of T-cells induces an irreversible priming for high IFN-7 production, which is maintained, even when the clones are cultured for several weeks in the absence of IL-12. However, unlike what is consistently observed in vivo and in polyclonal cultures and their derivative clones, the clones originated by limiting dilution in the presence or absence of IL-12 did not show any significant difference in their average ability to produce IL-4 [19, 47]. These results suggested that the ability of IL-12 to prime CD4 + cells for high IFN-7 production is due to a differentiation effect acting at the level ofCD4 + T-cell clone precursors, The ability of IL-12 to downregulate IL-4 production, however, was not observed at the clonal level, and is likely to be due to selective processes operative on polyclonal cultures and not to a direct effect on single clonal progenitors [19]. The nature of these mechanisms remains to
be investigated, although a possible selective proliferative effect of IL-12 on Thl clones or on IFN-~,-mediated negative selection against IL-4-producing clones can be postulated. Alternatively, the downregulation of IL-4 production might be a differentiation effect that requires cellular interaction or cell crowding (e.g. for the production Th2-suppressing factors such as I F N - J during the initial phase of proliferation of the T-cells; such interactions are not obtained in limiting dilution cultures, even in the presence of irradiated feeder cells. The ability of IL-12 to prime human T-cells for IFN7 production has also been suggested by experiments showing that na~'ve human cord blood T-cells are unable to produce IFN-7, but acquire this ability after a few days of culture in the presence of IL-12 [45]. However, selective effects and/or preferential proliferation of T-cell subsets could not be excluded in the polyclonal cultures of cord blood T-cells. That the mechanisms underlying the enhancing effect of IL-12 on IFN-7 and the inhibition of IL-4 production might be different is also suggested by data in the murine system, showing that the enhancement of IFN- 7 production is a direct effect of IL-12 on T-cells, whereas the inhibition of IL-4 production is due to an indirect effect on APC or on other cell types present in the APC preparations [48]. CD4 + and CD8 + clones obtained by limiting dilution in the presence of IL-12 produced significantly more IL10 than clones generated in the absence of IL- 12 [40]. We also observed that stimulation of human T-cell clones in the presence of IL-12 results in a several-fold increase in IL-10 production, in both high and low IL-10 producing clones (Peritt and Trinchieri, in preparation). However, in allergen-stimulated polyclonal T-cell culture, IL-12 was shown to downregulate both IL-10 and IL-4 [49]. These apparentlycontradictoryresultsare, however, consistent with the conclusion that IL-12 directly upregulates Thl cytokine production, but suppresses Th2 cytokine production by an indirect, possibly selective mechanism. These data also shed new light on the observation that IL-12 treatment in vivo induces the accumulation of IL10 mRNA [50]: although that finding was attributed to production of IL-10 by macrophages, the possibility that IL-12 also induces IL-10 production from T-cells must now be investigated. Figure 1 shows a typical limiting dilution cloning experiment [40] in which CD4 + and CD8 + clones were derived from unfractionated PBL from several donors and expanded in the presence, during the first 2 weeks of culture, of IL-12 or neutralizing antibodies anti-IL-12. After 4-5 weeks of culture, the clones were then stimulated with anti-CD3 antibodies and phorbol diesters, and cytokine production was evaluated. Clearly, the presence of IL-12 during the early 2 weeks of culture primed both the CD4 + and CD8 + clones for much higher production of both IL-10 and IFN- 7. The high variability in production of cytokines observed in human CD4 + T-cell clones expanded in the absence of exogenous IL-12 and IL-4 or in the presence of neutralizing antibodies against these two cytokines
Cytokine Production in Lymphocytes suggests that some of the cells were already primed in vivo for cytokine production. The cloning of sorted C D 4 5 R O - "naive" CD4+-cells and CD45RO + "mereory" CD4 +-cells supported this interpretation [40]. A high proportion of the clones generated from CD45RO ~ CD4+-cells in the presence of neutralizing antibodies to 1L-12 and IL-4 produced one or a combination of IFN7, IL-4 and IL-10, with a pattern of production that was not always consistent with the classical paradigm of Th 1and Th2-cells. When C D 4 5 R O - cells were cloned in the same conditions, the clones produced only negligible amounts of the three cytokines. However, in both populations, the presence of IL-12 during cloning endowed virtually all clones with the ability to produce high levels of IFN-7 and IL-10. The cytokine production pattern of clones derived from human CD4 + C D 4 5 R O - T-cells in the presence of IL-12 and/or IL-4 [40] is illustrated in Figure 2. Added IL-4 or endogenously produced IL-4 was necessary in the limiting dilution cultures to prime T-cell clones generated from C D 4 5 R O - cells for IL-4 production, whereas approximately half of the clones generated from CD45RO + cells produced IL-4, even when expanded in the absence of IL-4 and the requirement for 1L-4 in the generation of IL-4 producing cells was difficult to evaluate when total PBL were cloned, Although IL-12 is a major, and probably necessary, inducer o f a Thl response, it was also shown to potentiate IL-4 production and the development of Th2-cells from na~'ve CD4 + murine T-cells [51] and from neonatal CD4 + human T-cells [52], and to potentiate a Th2 response to Schistosoma mansoni in IFN- 7 knockout mice [53]. We showed [40] that IL-12 does not prevent IL-4 production from CD4 t clones derived from limiting dilutions of "nai've" adult peripheral blood C D 4 5 R O - cells and, in fact, significantly enhances the ability of IL-4 to prime the clones for high IL-4 production, thus extending the previous results [51-53] by demonstrating that IL-12 can enhance IL-4 production at the single clonal level via a differentiation effect. Furthermore, when T-cells were cloned in the simultaneous presence of IL-12 and IL-4, the IFN-7 priming effect of IL-12 was only partially, and often not significantly, inhibited by IL-4, whereas the priming for IL-10 production was reproducibly, and almost completely, blocked by IL-4 ([40] and Figure 2). Thus, paradoxically, IL-4 is more potent in inhibiting priming of Th cells for production of a Th2-type cytokine than for the typical Thl-type cytokine, IFN-7.
127
T-cells and N K cells, is the ability of various stimuli, includingT-cell receptor stimulation and other stimuli or co-stimuli, including cytokines, acting alone or in combination, to rapidly induce gene expression and cytokine production. For example, as depicted on the top of Figure 3, I L-12, alone or in synergy with other stimuli, induces accumulation o f m R N A for IFN- 7 within a few hours of treatment of either resting or activated T- or NK-cells, followed by secretion of IFN- 7. This acute induction of IFN-7 subsides within a couple of days (or, in vivo, even within less than 12 hr [13]) and does not induce a permanent alteration in the ability of the cells to produce IFN- 7 in response to IL-12 or other stimuli. The phenomenon of priming of the cytokine genes is schematically illustrated on the bottom of Figure 3, and is quite different from acute induction. When T-cells (and NK-cells) are clonally expanded in the presence of IL-12 during the first few days of expansion, the clones are primed for high production of IFN-7 and IL-10, even when cultured for several weeks in the absence of IL-12 stimulated in its absence; conversely, the exposure o f T cells to IL-4 during clonal expansion induces priming for IL-4 production and generation of IL-4-producing cells. IL-12 is particularly potent both in mediating acute induction of the IFN- 7 gene and in stably priming it for the response to other stimuli; however, although IL-12 similarly primes the IL-10 gene, its ability to acutely induce this gene is modest and difficult to demonstrate. Analogously, whereas IL-4 is necessary and extremely potent for the priming of the IL-4 and the generation of IL-4 producing cells, its ability to acutely induce the expression of the IL-4 gene has not been demonstrated. The IL-2 effect on lymphokine production is different from that of IL-12 and IL-4; IL-2, alone or in synergy with other stimuli, is a potent inducer of acute expression of the several lymphokines, including IFN-;., IL-4 and IL-10; however, although the presence of IL-2 may be required in the priming phenomena of all three genes, I L2 by itself does not determine the specificity of the priming, that is instead directed by IL-12 and IL-4 [14, 31, 38]. As mentioned above and summarized in Table 1, IL-4 and IL-12 both antagonize and synergize in inducing priming of lymphokine genes: IL-4 almost completely abolishes the IL-12 priming for Ik-10 production, Table 1. Ability of IL-12 and/or IL-4 to acutely induce expression of cytokine genes or to prime them for activation in response to restimulation
ACUTE INDUCTION VERSUS PRIMING FOR CYTOKINE PRODUCTION From the studies of the generation of T h l - and Th2cells, including those of our group reviewed above, it is becoming apparent that production of lymphokines, both type-1 and type-2, can be regulated with two different mechanisms. The first mechanism, observed particularly in preactivated lymphocytes, but also in resting
Responding gene
Type of induction
IFN-7
Acute Priming Acute Priming Acute Priming
IL-10 IL-4
Inducing cytokines IL-12
IL-4
+ ++ + + ++ + +/++ + +
-++ +
IL-12+ IL-4 -4- + + ++ '~ + ,'++ ++
128
G. Trinchieri et al.
104
CD8(+)
clones
"
":
i[i,E...-.~,,,..w~,Z,11j • . . e
..,,= % I
10 2
~. ; V : ? , ~. T. ". .". % ": " ali~;~.. l l ,.t.;:..~ -|~.,~,, ..-.s. ~ ":..~,' ... •
1 0 ~.
S,i - , ~ , : . " "" |~,..., .... • • " • ."~ • I I!i l.% • • !,'°¢ it,
~edlw
10o
.
.
.-
• #~= | l i OIl I e d ~
~..
."
•
• •
"l--
I °
•
|
•
el
• •
•
lie
. . . . . . .~ . . . . . . . ~ . . . . . . ,
•
•
...... ,
......
.I
I 7 II
CD4(+)
clones
104-
1 0 s,
.• : ~ .
J.. II0
•
='•
•
-.'..~p
1
.,
.
i::',: ,:':
'" :
I~
10 o
-"
101
.
.
"~
. .l.,.q .
.
.
.
.
10 2
" .
~'
E
". °
~I,
" 0" II
•
."'_
.
......
.%,'-~ •
,J'~.../..o
...'..%.'~."
•"• 1 " " : "°° " . -
10 0
e w ,o % I P t
•
o•
10 =' :..,. 10"
•.'.~
• :. Z:,{t~',L~
• ~
(onti-lL-12
•
(onti-lL-
•
IL-12
•
IL-12
•
IL-12
•
IL-4
,oo'°°,
"
" II
"
• ",,l~
10 4
10 s
IL- 1 O, pg/ml Figure 1. IL-12 primes human CD4 ÷ and CD8 ÷ T-cell clones for production of IFN-v and IL-10. PBL from several donors were cloned in the presence of PHA, and the clones were expanded in the presence of feeder cells (irradiated autologous PBMC and RPMI-8866 cell line) and IL-2 (50 U/ml). Clonal growth efficiency was, on average, over 50%. After 4-5 weeks of culture, clones were washed in medium without cytokines, stimulated for 18 hr with soluble anti-CD3 antibodies and the phorbol diester TPA, and cytokine production in the supernatant was quantitated by radioimmunoassays. Each dot represents cytokine production from an individual clone. Red dots indicate clones expanded in the presence of IL-12 (2.5 ng/ml) in the first 2 weeks of culture; blue dots represent clones expanded in the presence of neutralizing anti-IL-12 antibodies,
I ,,z
+ anti-lL-4) 12)
(anti-lL-4) + IL-4 (onti-lL-12)
,oo'°°
,=
'
e
D 300.
......
10 ~
•
--I-
200"
300 200
100-
100
o" 1 " I - - ~ ' " 4.60 IL-4,
........
" 800 pg/ml
" 12'00
o ~ 0
='
...........
lO'OO ,L-IO,
2000
30100
pg/m,
Figure 2. Cytokine production by human CD4 + clones generated from sorted CD45RO- cells. CD45RO- cells were cloned by limiting dilution (PHA stimulation) in the presence or absence of IL-12 (2.5 ng/ml), IL-4 (50 U/ml), anti-IL-12 neutralizing mAb, anti-IL-4 neutralizing mAb or combinations thereof. After 4 weeks, the clones were washed, incubated for 24 hr in medium containing IL-2 (50 U/ml) only, washed again, and then stimulated for 18 hr with soluble anti-CD3 antibodies and TPA; cytokines were measured in the cell-free supernatant fluid by radioimmunoassay. The graphs indicate the m e a n + SE of the cytokine production of all clones analyzed in each cloning condition. The arbitrary threshold of cytokine production (broken lines) is drawn to facilitate the analysis of the results. Clonalefficiencywasapproximately50%,exceptinthepresence of anti-IL-4, where the efficiency was 20-30%.
Cytokine Production in Lymphocytes
129
Acute cytokine induction
4 Oh I
24h
48h I
IL-12 (+1- other co-stimuli)
Priming for c y t o k i n e p r o d u c t i o n ~--~
IL-2 +
i
c.,,.
I I 1 week 2 O~neither IL-12 nor IL-4)
I
II
IL-12
I
Cytokine production 5
I 3
I 4
I 5
I 6
t anti:CD3 + TPA (neither IL-12 nor IL-4)
IL-4
Figure 3. Acute induction vs. priming for cytokine production. The figure schematically depicts the characteristics of these two mechanisms of regulation of cytokine gene expression. The ability of IL-12, alone or in synergy with other co-stimuli, is presented as a typical example of acute induction. On both resting and activated T- and NK-cells, IL-12 rapidly induces accumulation of IFN-), m R N A and production of the protein; this induction subsides within 1 or 2 days without permanently altering the ability of the cells to respond to a second challenge (by IL-12 or other stimuli) with IFN- 7 production. The priming for cytokine production is observed when T-lymphocytes are expanded in the presence of IL-12 or IL-4 for the first 1-2 weeks of culture. The T-cells can then be expanded for several weeks in the absence of either IL-12 or IL-4. If the cells are then stimulated (e.g. by antiCD3 and TPA, with no requirement for the presence of either IL-12 or IL-4 during the restimulation), the cells that have been expanded in the presence of IL-12 produce much higher levels of both IFN- 7 and IL-10, and the cells that have been expanded in the presence of IL-4 produce much higher levels of IL-4. Thus, the exposure of the lymphocytes to either IL-12 or IL-4 in the first few days of clonal expansion determines a stable and likely reversible change (priming) in the ability of the lymphokine genes to be activated in response to a restimulation challenge.
130
G. Trinchieri et al.
whereas it only partially decreases the priming for IFN7; IL-12 potentiates, rather than inhibits, the IL-4 priming of T-cells from high IL-4 production, The priming of lymphokine genes represents a stable modification of the inducibility of the genes which is analogous to the stable phenotype in the pattern ofcytokine production typical of Th-subsets. Thus, it is likely that this priming mechanism plays a role in the determination of the Th-phenotype of activated T-cells. However, certain effects of IL-12 and IL-4 on Th generation are not observed when the ability of these cytokines to induce differentiation is analyzed at the single clonal level (for example, in this clonal analysis, the powerful ability of IL-12 to block IL-4 production is not reproduced and IL-12, paradoxically, induces T-cells priming for production of IL-10, a prevalently type-2 cytokine). Thus, although the priming of lymphokine genes is most likely the predominant mechanism by which IL-12 and IL-4 induce differentiation of Th-cells, the final generation of cells with T h l - and Th2-phenotype, both in vivo and in vitro, also depends on complex indirect effects of the cytokines, including selective mechanisms, in addition to a direct differentiative effect at the single cell level, The molecular mechanisms of both the acute induction and the priming effects, still remain mostly undetermined, The major signal transduction mechanisms for IL-4 and IL-12 have recently been elucidated, with the former cytokine inducing activation of STAT-6 [54] and the latter of STAT 1, 3 and 4 [55, 56]; however, the role of these transcription factors in the induction of expression of the IL-4, IFN-7 and IL-10 genes still remains to be elucidated. The priming effects may depend on the induction of a constitutive or facilitated expression of the transcription factors responsible for the expression of the lymphokine genes or in a stable alteration of the genes in a transcriptionally-prone conformation. For example, the IFN-7 gene has been shown to be differentially methylated in T h l - and Th2-clones [57]. Post-transcriptional mechanisms could also be responsible for the priming effect, The existence of alternative mechanisms of regulation of lymphokine gene expression adds a new complexity to the phenomenon of differentiation of Th-cells and other lymphocyte subsets. However, this new information also represents a challenge to address the molecular mechanisms responsible for determining the cytokine production pattern of lymphocyte subsets and will hopefully help to open the door to the understanding of these important mechanisms of regulation of the immune system. Acknowledgements: We would like to thank Ms Marion Kaplan
for preparing the manuscript. The experimental work described in this review was supported in part by NIH grants CA 10815, CA 20833, CA 32898 and AI 34412.
REFERENCES 1. Parish CR, Liew FY. Immune response to chemically modifled flagellin. 3. Enhanced cell-mediated immunity during
high and low zone antibody tolerance to flagellin. J Exp Med 1972, 135, 298-311. 2. Mosmann TR, Coffman RL. TH 1 and TH2 cells; different patterns oflymphokine secretion lead to different functional properties. Ann Rev Immunol 1989, 7, 145-173. 3. Seder RA, PaulWE. Acquisition of lymphokine-producing phenotype by CD4+ T cells. Ann Rev Immunol 1994, 12, 635-673. 4. Le Gros G, Ben-Sasson SZ, Seder R, Finkelman FD, Paul WE. Generation of interleukin 4 (IL-4)-producing cells in vivo and in vitro: IL-2 and IL-4 are required for in vitro generation of IL-4-producing cells. J Exp Med 1990, 172, 921-929. 5. Kobayashi M, Fitz L, Ryan M e t al. Identification and purification of Natural Killer cell stimulatory factor (NKSF), a cytokine with multiple biologic effects on human lymphocytes. J Exp Med 1989, 170, 827-846. 6. D'Andrea A, Rengaraju M, Valiante NM et al. Production of natural killer cell stimulatory factor (NKSF/IL-12) by peripheral blood mononuclear cells. J Exp Med 1992, 176, 1387-1398. 7. Macatonia SE, Hosken NA, Litton M et al. Dendritic cells produce IL-12 and direct the development of Thl cells from naive CD4 + T cells. JImmunol 1995, 154, 5071 5079. 8. Trinchieri G. Interleukin-12: a proinflammatory cytokine with immunoregulatory functions that bridge innate resistance and antigen-specific adaptive immunity. Ann Rev Immunol 1995, 13, 251-276. 9. Manetti R, Parronchi P, Giudizi MG et al. Natural killer cell stimulatory factor (NKSF/IL- 12) induces Th 1-type specific immune responses and inhibits the development of IL-4 producing Th cells. J Exp Med 1993, 177, 1199-1204. 10. Hsieh C, Macatonia, SE, Tripp CS, Wolf SF, O'Garra A, Murphy KM. Listeria-induced Thl development in eftTCR transgenic CD4 + T cells occurs through macrophage production of IL-12. Science 1993, 260, 547-549. 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, 235237. 12. Trinchieri G. Interleukin-12 and its role in the generation of TH1 cells. Immunol Today 1993, 14, 335-338. 13. Wysocka M, Kubin M, Vieira LQ et al. Interleukin-12 is required for interferon-7 production and lethality in lipopolysaccharide-induced shock in mice. Eur Jlmmuno11995, 25, 672-676. 14. Chan SH, Perussia B, Gupta JW et al. Induction of IFN-7 production by NK cell stimulatory factor (NKSF): Characterization of the responder cells and synergy with other inducers. J Exp Med 1991, 173, 869-879. 15. Kubin M, Chow JM, Trinchieri G. Differential regulation of interleukin-12 (IL-12), tumor necrosis factor-e, and ILlfl production in human myeloid leukemia cell lines and peripheral blood mononuclear cells. Blood 1994, 83, 18471855. 16. Murphy EE, Terres G, Macatonia SEet al. B7 and IL-12 cooperate forproliferationandIFN-Tproductionbymouse T helper clones that are unresponsive to B7 costimulation. J Exp Med 1994, 180, 223-231. 17. Erard F, Wild M, Garcia-Sanz JA, LeGros G. Switch of CD8 T cells to noncytolytic CD8 T cells that make Th2 cytokines and help B cells. Science 1993, 260, 1802-1803. 18. Seder RA, Boulay JL, Finkelman F et al. CD8 + T cells can be primed in vitro to produce IL-4. J. Immunol 1992, 148, 1652-1656. 19. Manetti R, Gerosa F, Giudizi MG et al. Interleukin-12 induces stable priming for interferon-7 (IFN-7) production during differentiation of human T helper (Th) cells and transient IFN-7 production in established Th2 cell clones. J Exp Med 1994, 179, 1273-1283.
Cytokine Production in Lymphocytes 20. Maggi E, Giudizi MG, Biagiotti R et al. Th2-1ike CD8 ÷ T cells showing B cell helper function and reduced cytolytic activity in human immunodeficiency virus type 1 infection. J Exp M e d 1994, 180, 489-495. 21. Ferrick DA, Schrenzel MD, Mulvania T, Hsieh B, Ferlin WG, Lepper H. Differential production of interferongamma and interleukin-4 in response to Thl- and Th2stimulating pathogens by gamma delta T cells in t,ivo. Nature 1995, 373, 255 257. 22. Warren HS, Kinnear BF, Phillips JH, Lanier LL. Production of IL-5 by human N K cells and regulation of I L-5 secretion by IL-4, IL-10, and IL-12. J Immunol 1995, 154, 5144-5152. 23. Kamogawa Y, Minasi LE, Carding SR, Bottomly K, Flaveil RA. The relationship of IL-4- and IFNT-producing T cells studied by lineage ablation of lL-4-producing cells. Cell 1993, 75, 985-995. 24. Firestein GS, Roeder WD, Laxer JA et al. A new murine CD4 ÷ T cell subset with an unrestricted cytokine profile. J lmmumd 1989, 143, 518-525. 25. Kelso A, Groves P, Troutt AB, Francis K. Evidence for the stochastic acquisition of cytokine profile by CD4 ~ T cells activated in a T helper type 2-like response in vivo. Eur J Immunol 1995, 25, 1168-1175. 26. Elson LH, Nutman TB, Metcalfe DD, Prussin C. Flow cytometric analysis for cytokine production identifies T helper 1, T helper 2, and T helper 0 cells within the human CD4 + CD27 lymphocyte subpopulation. Jlmmuno11995, 154, 4294-4301. 27. Picker LJ, Singh MK, Zdraveski Z et al. Direct demonstration of cytokine synthesis heterogeneity among human memory/effector T cells by flow cytometry. Blood 1995, 86, 1408-1419. 28. Kubin M, Kamoun M, Trinchieri G. Interleukin-12 synergizes with B7/CD28 interaction in inducing efficient proliferation and cytokine production of human T cells. J Exp Med 1994, 180, 211-222. 29. Aste-Amezaga M, D'Andrea A, Kubin M, Trinchieri G. Cooperation of natural killer cell stimulatory factor/interleukin-12 with other stimuli in the induction of cytokines and cytotoxic cell-associated molecules in human T and N K cells. Celllmmunol 1994, 156, 480-492. 30. Chan SH, Kobayashi M, Santoli D, Perussia B, Trinchieri G. Mechanisms of IFN- 7 induction by natural killer cell stimulatory factor (NKSF/IL-12): Role of transcription and m R N A stability in the synergistic interaction between N K S F and IL-2. J Imrnunol 1992, 148, 92 98. 31. Trinchieri G, Matsumoto-Kobayashi M, Clark SC, Sheehra J, London L, Perussia B. Response of resting human peripheral blood natural killer cells to interleukin2. J Exp M e d 1984, 160, 1147-1169. 32. Gazzinelli RT, Hieny S, Wynn TA, Wolf S, Sher A. Interleukin-12 is required for the T-lymphocyte independent induction of interferon-y by an intracellular parasite and induces resistance in T-deficient hosts. Proc Nail Acad Sci USA 1993,90, 6115 6119. 33. Tripp CS, W o l f S F , Unanue ER. Interleukin 12 and tumor necrosis factor alpha are costimulators of interferon gamma production by natural killer cells in severe combined immunodeficiency mice with listeriosis, and interleukin 10 is a physiologic antagonist. Proc Natl Acad Sci USA 1993.90, 3725-3729. 34. D'Andrea A, Aste-Amezaga M, Valinate NM, Ma X, Kubin M, Trinchieri G. Interleukin-10 inhibits human lymphocyte IFN-y production by suppressing natural killer cell stimulatory factor/interleukin-12 synthesis in accessory cells. J Exp M e d 1993, 178, 1041 1048. 35. Gately MK, Warrier RR, Honasoge Set al. Administration of recombinant IL-12 to normal mice enhances cytolytic lymphocyte activity and induces production of IFN-7 in t,iz~o, lnt lmmunol 1994, 6, 157-167.
131
36. Swain SL, Weinberg AD, English M, Hutson G. 1L-4 directs the development of Th2-1ike helper effectors. J Immunol 1990, 145, 3796-3806. 37. Seder RA, Paul WE, Davis MM, Fazekas de St Groth B. The presence of interleukin 4 during in vitro priming determines the lymphokine-producing potential of C D 4 + T cells from T cell receptor transgenic mice. J Exp Med 1992, 176, 1091-1098. 38. Van der Pouw-Kraan T, Van Kooten C, Rensuik I, Aarden L. Interleukin (IL)-4 production by human T cells; differential regulation of IL-4 vs IL-2 production. Eur .I Immunol 1992, 22, 1237-1241. 39. Trinchieri G, Scott P. Interleukin-12: a proinflammatory cytokine with immunoregulatory functions. Res hnmunol 1995, 146, 423-431. 40. Gerosa F, Paganin C, Peritt D et al. IL-12 primes human CD4 and CD8 T cell clones for high production of both IFN-7 and IL-10. J Exp Med 1996, 183, 2559-2570. 41. Seder RA, Gazzinelli R, Sher A, Paul WE. IL-12 acts directly on CD4 + T cells to enhance priming for IFNy production and diminishes IL-4 inhibition of such priming. Proc Natl A c a d S c i USA 1993, 90, 10188-10192. 42. Swain SL, McKenzie DT, Weinberg AD, Hancock W. Characterization of T helper 1 and 2 cell subsets in normal mice. Helper T cells responsible for IL-4 and IL-5 production are present as precursors that require priming before they develop into lymphokine-secreting cells. J Immunol 1988, 141, 3445-3455. 43. Street NE, Schumacher JH, Fong AT et al. Heterogeneity of mouse helper T cells: Evidence from bulk cultures and limiting dilution cloning for precursors of Thl and Th2 cells. J lmmunol 1993, 144, 1629 1639. 44. Gajewski TF, Joyce J, Fitch FW. Antiprolifertive effect of IFN-7 in immune regulation. III. Differential selection of Thl and Th2 murine helper T lymphocyte clones using recombinant IL-2 and recombinant IFN-y. J lmmuno11989, 143, 15 22. 45. Wu CY, Demeure C, Kiniwa M, Gately M, Delespesse G. IL-12 induces the production of IFN-gamma by neonatal human CD4 T cells. J lmmunol 1993. 151, 1938-1949. 46. Del Prete GF, De Carli M, Mastromauro C et al. Purified protein derivative of Mycobacterium tuberculosis and excretory-secretory antigen(s) of Toxocara canis expand in t,itro human T cells with stable and opposite (type 1 T helper or type 2 T helper) profile of cytokine production. J Clin Infest 1991, 88, 346-350. 47. Gerosa F, Trinchieri G. mechanisms o f T helper cell differentiation induced by interleukin-12. In Romagnani S, Del Prete G, Abbas AK, eds. Cytokines: Basic Principles and Practical Applications. Rome, Ares-Serono Symposia Publications, 1994, 251-263. 48. DeKruyff RH, Fang Y, Wolf SF, Umetsu DT. IL-12 inhibits IL-4 synthesis in keyhole limpet hemocyaninprimed CD4 * T cells through an effect on antigen-presenting cells. J Immunol 1995, 154, 2578 2587. 49. Marshall J, Secrist H, DeKruyff RH, Wolf SF, Umetsu DT. IL-12 inhibits the production of IL-4 and 1L-10 in allergen-specific human CD4 + T lymphocytes. J lmmunol 1995, 115, 111 117. 50. Finkelman FD, Madden KB, Cheever AW et al. Effects of interleukin 12 on immune responses and host protection in mice infected with intestinal nematode parasites. J Exp Med 1994, 179, 1563 1572. 51. Schmitt E, Hoehn P, Germann T, ROde E. Differential effects ofinterleukin-12on the development ofnaivemouse CD4 + T cells. Eur J Immunol 1994, 24, 343-347. 52. Wu CY, Demeure CE, Gately M e t al. In i;itro maturation of human neonatal CD4 T lymphocytes: I. Induction of IL4-producing cells after long-term culture in the presence of IL-4 plus either 1L-2 or 1L-12. J Immunol 1994, 152, 1141 1153.
132
G. Trinchieri et al.
53. Wynn TA, Jankovic D, Hieny S e t al. IL-12 exacerbates tyrosine phosphorylation of signal transducer and activator rather than suppresses T helper 2-dependent pathology in of transcription (Stat)3 and Stat4. J Exp Med 1995, 181, the absence of endogenous IFN- 7. J Immunol 1995, 1 5 4 , 1755-1762. 3999-4009. 56. Bacon CM, Petricoin III EF, Ortaldo JR et al. IL-12 54. Quelle FW, Shimoda K, Thierfelder W e t al. Cloning of induces tyrosine phosphorylation and activation of STAT4 murine Stat6 and human Stat6. Stat proteins that are tyroin human lymphocytes. Proc Natl Acad Sci USA 1995, 92, sine phosphorylated in responses to IL-4 and IL-3 but are 7307-731 l. not required for mitogenesis. Molec Cell Biol 1995, 15, 57. Young HA, Ghosh P, Ye J e t al. Differentiation of the T 3336-3343. helper phenotypes by analysis of the methylation state of 55. Jacobson NG, Szabo SJ, Weber-Nordt RM et al. Interthe IFN-7 gene. J l m m u n o l 1994, 153, 3603-3610. leukin 12 signaling in T helper type 1 (Thl) cells involves