and Immunity (2007) 8, 254–261 & 2007 Nature Publishing Group All rights reserved 1466-4879/07 $30.00 www.nature.com/gene

ORIGINAL ARTICLE Human lambda-1 (IFN-l1/IL-29) modulates the Th1/Th2 response

WJ Jordan1, J Eskdale2, S Srinivas2, V Pekarek2, D Kelner1, M Rodia1 and G Gallagher2 1Department of Oral Biology, New Jersey Dental School, Newark, NJ, USA and 2HUMIGEN, The Institute for Genetic Immunology, Hamilton, NJ, USA

Interferon lambda-1 (IFN-l1/IL-29) is a member of the Type-III interferon family, which contains three ligands: IFN-l1, 2 and 3. These three ligands use the same unique heterodimeric receptor composed of CRF2-12 (IFN-l-R1/IL-28Ra) and CRF2-4 (IL10-R-b) chains. Like their close relatives, the Type-I , IFN-l1, 2 and 3, promote the phosphorylation of STAT1 and STAT2, induce the ISRE3 complex, elevate OAS and MxA expression and exhibit antiviral activity in vitro. Their use of the IL10-R-b chain and their ability to phosphorylate STAT3, STAT4 and STAT5 suggested that they may also exhibit immunomodulatory activity; their antiviral action led us to hypothesize that this activity might be directed toward the Th1/Th2 system. Here, we have demonstrated that IFN-l1 altered the activity of Th cells in three separate experimental systems: (i) mitogen stimulation, (ii) mixed-lymphocyte reaction (MLR) and (iii) stimulation of naive T cells by -derived dendritic cells (mDC). In Con-A stimulation assays, the inclusion of IFN-l1 consistently led to markedly diminished levels of secreted (IL-13) with occasional coincident, modest elevation of secreted IFN-g. IL-13 secretion was 100-fold more sensitive to IFN-l1 than was IFN-g secretion. These observations were also made in the allogeneic two-way MLR. IFN-l1 was able to alter -mediated Th biasing and when naive T cells were exposed to allogeneic mDC that had been matured in the presence of IFN-l1, secreted IL-13 was again markedly and consistently reduced, whereas secreted IFN-g was largely unaltered. These functions were independent of IL-10. Our data support a hitherto unsuspected role for IFN-l1 in modulating the development of Th1 and Th2 cells, with an apparent emphasis on the diminution of IL-13 secretion. Genes and Immunity (2007) 8, 254–261. doi:10.1038/sj..6364382; published online 15 March 2007

Keywords: IFN-l1; IL-29; IL-13; Th2; asthma

Introduction tional Society for Interferon and Cytokine Research. All three ligands can signal through the same heterodimeric The discovery of the CRF2-12 receptor chain through its receptor composed of CRF2-12 (IFN-l-R1/IL-28Ra) and similarity with the external domain of the interleukin CRF2-4 (IL10-R-b) chains. This receptor pair is not used (IL)-22 receptor alpha chain (G Gallagher, unpublished by any other known ligand, although the CRF2-4 chain is observations) and its pairing with an orphan ligand, also part of the IL-10, IL-22 and IL-26 receptors.4–7 described as ‘similar to type-one interferon’,1 led to the Binding of the IFN-l ligand to its receptor initiates discovery and characterization of the interferon lambda signaling reminiscent of the Type-I interferons. In (IFN-l) family members and their receptor in our particular, STAT2 is phosphorylated, ISGF3 is activated laboratories2 and in parallel elsewhere.3 This is a new and OAS and MxA are induced.2 These observations, family of interferons related to both the IL-10 family of in conjunction with the induction of the IFN-l1/2/3 ligands and the Type-I and Type-II interferon family. ligands by a range of viral infections and their ability to Although they share functional characteristics with rescue virally infected cells,2,3 have prompted a series of members of the intronless IFN-a family, the IFN-l genes studies that have further investigated the antiviral have an intron/exon structure identical to members of properties of these .8–21 However, the IFN-l the IL-10 family. ligands also induce the phosphorylation of STAT1, There are three ligands in the family: IFN-l1, 2 and 3 STAT3, STAT52,22 and STAT4.23 Phosphorylation of (known alternatively as IL-29, IL-28A and IL-28B, STAT1, STAT3, STAT5 in particular, suggests more respectively3). The ligands were termed ‘Type-III Inter- complex properties for the IFN-l ligand family; STAT3 ferons’ by the nomenclature committee of the Interna- is the signaling mechanism used by members of the IL-10 family (IL-10, IL-19, IL-20, etc.24–27), whereas STAT5 is used widely by cytokines, such as IL-2, IL-3 and Correspondence: Professor G Gallagher, HUMIGEN, The Institute granulocyte- colony stimulating factor for Genetic Immunology, 2439 Kuser Road, Hamilton, NJ (GM-CSF).28–32 STAT4 is the signature-signaling molecule 08690 3303, USA. E-mail: [email protected] of the Th1 response, a process which is complemented by 33–36 Received 9 October 2006; revised 21 January 2007; accepted 22 STAT1 signaling; thus, IFN-l1 may be able to alter January 2007; published online 15 March 2007 the Th1/Th2 balance. IFN-k1 downregulates the human Th2 response WJ Jordan et al 255 We, therefore, decided to investigate T-cell regulation tion than that required to increase IFN-g (Figure 1c). by IFN-l. Our data demonstrate that IFN-l1 is capable of Furthermore, the ability of IFN-l1 to modulate IL-13 downregulating IL-13 secretion (the Th2 response); in secretion was eliminated by proteinase-K treatment of some experimental environments, this is accompanied this ligand before its addition to the cultures, demon- by a coincident, and more modest, elevation of IFN-g strating the effect was not mediated by some lipopoly- secretion (the Th1 response). The ability of IFN-l to saccharide (LPS)-like contaminant in our commercial influence IL-13 secretion may be of direct relevance to (Peprotech, Rocky Hill, NJ, USA) IFN-l1 preparation the pathogenesis and treatment of asthma.37 (Figure 1d). Thus, IFN-l1 primarily inhibits the produc- tion of IL-13, although in some cases, perhaps reflecting intraindividual variation, elevated secretion of IFN-g Results may occur at higher IFN-l1 concentrations. However, IFN-l1 modulates cytokine secretion following mitogen even when effects on IFN-g are observed, IL-13 is much stimulation more sensitive to the effects of IFN-l1. We investigated whether IFN-l1 could influence T-cell responses. First, we examined the effect of IFN-l1 on the IFN-l1-mediated reduction of IL-13 secretion is apparently production of T-cell cytokines IFN-g (representative of independent of the actions of IL-10 Th1 responses) and IL-13 (representative of Th2 re- IL-10 is a multifunctional cytokine known for its sponses) following Con-A stimulation. The experimental immunomodulatory capabilities. The lambda interferons results are shown in Figure 1. IFN-l1 consistently use a receptor, which employs the IL-10-Rb chain, reduced IL-13 secretion (15/16 donors), often with no CRF2-4. We have recently demonstrated that IFN-l1 effect on IFN-g levels, across a range of concentrations of will elevate IL-10 levels in in vitro cultures of human Con A (Figure 1a). Occasionally (in only 5/16 donors), peripheral blood mononuclear cells (PBMCs) and that coincident-increased secretion of IFN-g was observed (as endogenous IL-10 can moderate IFN-l1-induced re- in Figure 1b). These effects were IFN-l1 dose-dependent. sponses.38 We wished to see whether the ability of IFN- Relatively high concentrations of IFN-l1 were required l1 to reduce IL-13 secretion was influenced by IL-10. to elevate IFN-g levels (as has been previously observed As shown in Figure 2a, the anti-IL-10 antibody elevated for its antiviral activities2,3,12). However, reduction of the secretion of IL-13, suggesting that endogenous IL-10 IL-13 consistently occurred at 100-fold lower concentra- in the cultures was controlling IL-13 secretion. When

a IL-13 (pg/ml) b IL-13 (pg/ml) c IL-13 (pg/ml) 300 600 1400

1200 200 400 1000 100 200 800 d IL-13 (pg/ml) 400 600 0.0 0.6 1.25 2.5 5.0 0 3 6 0 10 100 1000 Con-A (µg/ml) Days λ IFN- 1 (ng/ml) 300

IFNγ (pg/ml) IFNγ (pg/ml) IFNγ (pg/ml) 5000 9000 110 200 Con λ-1 λ-1 PK +PK 3000 6000 90 3000 1000 70 0.0 0.6 1.25 2.5 5.0 0 3 6 0 10 100 1000 µ Con-A ( g/ml) Days IFN- 1 (ng/ml) Figure 1 Modulation of IL-13 and IFN-g by IFN-l1, following stimulation with Con-A. The ability of IFN-l1 to alter the development of Th cells under conditions of mitogen stimulation was examined using human PBMC. Cells were established in 96-well cultures as described and stimulated in the presence or absence of IFN-l1. (a) The secretion of IL-13 (upper panel) and IFN-g (lower panel) was determined by ELISA in the presence (black bars) or absence (white bars) of 1000 ng/ml of human IFN-l1 across a range of Con-A concentrations as shown, for 6 days. At least 50% reduction in IL-13 was observed, although IFN-g secretion was unaffected, representing the majority (15/16) of donor responses. (b) The secretion of IL-13 (upper panel) and IFN-g (lower panel) was determined by ELISA in the presence (black bars) or absence (white bars) of 1000 ng/ml of human IFN-l1 following stimulation with Con-A (5.0 mg/ml), for 3 or 6 days. At least 50% reduction in IL-13 was observed while IFN-g secretion was elevated at day 6, representing the minority (5/16) of donor responses. (c) Titration of IFN-l1 as shown in the presence of 5.0 mg/ml Con-A for 6 days demonstrates that IL-13 secretion (upper panel) is approximately 100-fold more sensitive to IFN- l1 than is IFN-g secretion (lower panel). (d) The ability of 1000 ng/ml IFN-l1(‘lÀ1’) to suppress the secretion of IL-13 in response to 5.0 mg/ ml Con-A (‘Con’) was destroyed by treatment with proteinase-K (‘lÀ1 þ PK’). Proteinase-K alone (‘PK’) was ineffective. Means71 standard deviation of triplicate wells are shown; data are representative of at least three independent donors. The different scales on the x axes reflect the variation in cytokine secretion by individual donors. Means71 standard deviation of triplicate wells are shown. (d) Representative of two independent donors.

Genes and Immunity IFN-k1 downregulates the human Th2 response WJ Jordan et al 256 a IL-13 (pg/ml) b IL-13 (pg/ml) a IL-13 (pg/ml) b IL-13 (pg/ml) 600 400 1500 1500

300 400 1000 1000 200 200 500 500 100

24 48 72 24 48 72 none α-IL-10 0.0 2.5 5.0 10.0 Hours Hours Con-A (µg/ml) Figure 2 IFN-l1 effects are independent of IL-10. The requirement IFNγ (pg/ml) IFNγ (pg/ml) for IL-10 to mediate the suppression of IL-13 by IFN-l1 was 2000 1500 established by treating cultures with the anti-IL-10 antibody ‘9D7’ 1500 (R&D Systems, Minneapolis, MN, USA). (a) The suppression of IL- 1000 13 secretion in the presence (black bars) or absence (white bars) of 1000 ng/ml IFN-l1 in the presence of 5.0 mg/ml Con-A was 1000 measured in the presence of no antibody (‘none’), or clone 9D7 500 500 (‘a-IL-10’). In both cases, IFN-l1 suppressed the production of IL-13. (b) In the presence of clone 9D7 and in the presence (black bars) or absence (white bars) of 1000 ng/ml IFN-l1, the suppression of IL-13 was observed across a range of Con-A levels. 2448 72 24 48 72 Hours Hours

l Figure 3 Modulation of IL-13 and IFN-g by IFN-l1 and IFN-l2, IFN- 1 was added in the presence of anti-IL-10 antibody, during the two-way MLR. (a) Two-way MLR were established using IL-13 secretion was reduced (Figure 2b), demonstrating untreated cells from two unrelated donors in the presence (black that the effects of IFN-l1 were independent of IL-10. bars) and absence (white bars) of 1000 ng/ml IFN-l1. The secretion of IL-13 (upper panel) and IFN-g (lower panel) at 24, 48 and 72 h IFN-l1 diminishes IL-13 and elevates IFN-g secretion in the was determined by ELISA. (b) As per panel a, except that 1000 ng/ two-way MLR ml IFN-l2 (gray bars) was used. Means71 standard deviation are We wished to determine whether IFN-l1 could influence shown, data are representative of seven independent experiments an assay that was dependent on T-cell receptor trigger- using different donor pairs. ing. Accordingly, we investigated the ability of IFN-l1to modulate Th1/Th2 cytokine secretion in the two-way mixed-lymphocyte reaction (MLR). As shown in used. The ability of IFN-l1 to influence these four T-cell Figure 3a, introduction of IFN-l1 led once again to a biasing experiments was examined. decrease in IL-13 secretion, with an accompanying As anticipated, Th1 biasing conditions led to higher increase in IFN-g secretion, at day 6 of the reaction (7/7 levels of IFN-g secretion, Th2 biasing to elevated IL-13 donors). Thus, IFN-l1 influences the developing T-cell secretion and Th0 conditions to an intermediate secretion response and the nature of the Th1/Th2 balance in this pattern (Figure 4a). When IFN-l1 was introduced to this system also. system (Figure 4b), IL-13 was markedly diminished in As far as is currently known, all three lambda each of the four test conditions, except in those of Th2 interferons use the same heterodimeric receptor.2,3 We biasing. This indicates that, although IFN-l1 is generally tested IFN-l2 in the MLR assay and showed that it able to diminish IL-13 secretion, it may not compete behaved similarly to IFN-l1, diminishing IL-13 and efficiently with IL-4’s ability to promote the IL-13 elevating IFN-g (Figure 3b), as expected. response under the conditions employed here. There was little, if any, effect on IFN-g secretion in these IFN-l1 diminishes IL-13 secretion in naive T cells following experiments as noted previously, even though the high- exposure to allogenic mDC est dose of IFN-l1 (1000 ng/ml; Figure 1c) was used. The ability of IFN-l1 to influence T-cell biasing was Notably, IFN-l1 did not appear to be able to replace IL- investigated. We developed dendritic cells (DC) from 12, as there was no elevation of IFN-g secretion in the peripheral CD14 þ (monocyte-derived den- Th0- or Th2-biasing conditions, nor could it augment IL- dritic cells, mDC), using GM-CSF and IL-4 (Peprotech) 12 effects in the Th1-biasing or no-biasing control. Thus, and used them to stimulate naive allogeneic T cells. IFN-l1 is able to alter the Th1/Th2 development of naive These T cells were then expanded in medium containing human T cells, under antigen-driven conditions, by IL-2 and stimulated to secrete cytokine by treatment with diminishing the development of Th2 cells and lowering phorbol myristate acetate (PMA) þ ionomycin, to indi- the secretion of IL-13. cate their overall Th1/Th2 development. At the time of exposing the T cells to the mDCs, three sets of conditions IFN-l1 alters the functional capability of mDC were introduced to influence the nature of the develop- The data presented above suggest that IFN-l1 may ing Th1/Th2 response: ‘Th1 biasing’, where IL-12 and directly affect developing T cells while they are interact- anti-IL-4 were introduced; ‘Th2 biasing’, where IL-4 and ing with the stimulating mDC or that it may alter mDC anti-IL-12 were introduced and ‘Th0 biasing’, where both function. We were interested in determining whether it anti-IL-4 and anti-IL-12 were introduced. A ‘no biasing’ was able to alter the stimulatory profile of mDC. First, we control in the absence of cytokines or antibodies was also induced mDC from peripheral blood monocytes using

Genes and Immunity IFN-k1 downregulates the human Th2 response WJ Jordan et al 257 a a Th2

Th0

Th1 1200 600 75 150 IL-12p70 (ng/ml) IL-10 (ng/ml) 400 200 250 500 b b i

400 200 30 60 400 200 1000 2000 IFNγ (ng/ml) IL-13 (ng/ml) ii Th0 Figure 5 Effect of IFN-l1 on the function of mDCs. (a) mDCs were derived from purified CD14 þ peripheral monocytes with GM- 400 200 37.5 75 CSF þ IL-4, then matured with LPS in the presence (black bars) or absence (white bars) of 1000 ng/ml IFN-l1. IL-12p70 and IL-10 secretion was measured by ELISA. (b) mDCs were derived from iii Th1 peripheral monocytes with GM-CSF þ IL-4 in the presence (black bars) or absence (white bars) of 1000 ng/ml IFN-l1 (as above). Cells 250 125 100 200 were washed free of exogenous IFN-l1, then matured with LPS (also in the absence of IFN-l1) and used to stimulate naive CD4 þ T iv cells from an unrelated donor; the secretion of IL-13 and IFN-g Th2 determined by ELISA. The mean71 standard deviation of triplicate wells is shown, data are representative of at least four (a) or six (b) 80 40 250 500 replicate experiments using different donor pairs. IFNγ (ng/ml) IL-13 (ng/ml) Figure 4 IFN-l1 inhibits the induction of Th2 cells during the developing T-cell response on allo-mDCs. (a) mDCs were derived from purified CD14 þ peripheral monocytes with GM-CSF þ IL-4 were grown and stimulated with PMA and ionomycin as and used to stimulate naive CD4 þ T cells from an unrelated donor. before and their IL-13 and IFN-g secretion determined. Three different T-cell biasing conditions were used: Th0 biasing As shown in Figure 5b, T cells, challenged (in the (‘Th0’), where a-IL-4 and a-IL-12 antibodies are added; Th1 biasing absence of IFN-l1) with allogeneic mDC (allo mDC) (‘Th1’), where IL-12 protein and a-IL-4 antibody are added and Th2 developed in the presence of IFN-l1, secreted much less biasing (‘Th2’), where IL-4 protein and a-IL-12 antibody are added. (b) mDCs were derived from purified CD14 þ peripheral monocytes IL-13 than those whose mDC had not been exposed to and used to stimulate naive CD4 þ T cells from an unrelated donor IFN-l1 during their development. IFN-g secretion was in the presence (black bars) or absence (white bars) of 1000 ng/ml not affected. Thus, IFN-l1 is capable of changing the IFN-l1. Four differing Th1/Th2 biasing conditions were considered: development of mDC in a way that results in their no biasing (panel i), Th0 biasing (panel ii), Th1 biasing (panel iii) causing diminished Th2 development without enhan- 7 and Th2 biasing (panel iv). Means 1 standard deviation of cing their capability to develop Th1 cells. triplicate wells are shown, data are representative of two separate experiments using different donor pairs. Different y axis scales are indicative of individual donor variation in cytokine secretion. Discussion Recently, our laboratory and others discovered and GM-CSF þ IL-4, then matured them either with LPS, IFN- presented the initial characterization of a new family of l1 or LPS þ IFN-l1. The ability of these cells to secrete cytokines, the ‘interferon lambda’ family, IFN-l1/2/3.2,3 IL-12 and IL-10 (two key mDC cytokines) was examined These cytokines (also known as IL-29, IL-28A and IL-28B, by enzyme-linked immunosorbent assay (ELISA) respectively) were recently defined as ‘Type-III’ inter- (Figure 5a). As expected, maturation of mDC with LPS ferons. They share many of the antiviral properties of the led to secretion of both IL-10 and IL-12. mDCs matured Type-I interferons and have subsequently garnered with LPS in the presence of IFN-l1 secreted approxi- particular interest as agents with activity against hepa- mately 20% less IL-10 than those not exposed to IFN-l1, titis .11,14,39 However, they may not be as efficient and the LPS-mediated IL-12 secretion was modestly antiviral agents as the Type-I interferons.3,12 In addition augmented by IFN-l1. In the absence of LPS (data not to their interferon-like antiviral properties, the lambda shown), IFN-l1 was unable to mature the mDCs, and interferons share a number of signaling and downstream neither IL-10 nor IL-12 was secreted with this cytokine events in common with the Type-I interferons. Notably, alone. IFN-l1 signaling involves the phosphorylation of Next, we considered the effect of IFN-l1 during the STAT2, considered a defining characteristic of Type-I induction phase of mDCs. Freshly isolated CD14 þ responses.40,41 Furthermore, interferon response genes human peripheral monocytes were established in cell such as MxA and 5030OAS are activated2 and subjected culture and developed to mDC in GM-CSF þ IL-4 in the to typical regulatory mechanisms.11 presence or absence of IFN-l1. Following the 6-day Although the IFN-l receptor is widely expressed,2,3 induction period, cells were washed, matured with LPS expression of the ligand is tightly controlled and takes (in the absence of IFN-l1) and allowed to stimulate naive place after viral infection2,17 or stimulation via many of T cells as before (again, in the absence of IFN-l1). the Toll-like receptors (TLRs),15 a process that is elevated Following antigen sensitization on these mDCs, T cells by IFN-a.42 Expression via stimulation of TLR 7, 8 and 9

Genes and Immunity IFN-k1 downregulates the human Th2 response WJ Jordan et al 258 is absolutely dependent on IRAK-4 signaling, whereas secretion from T cells stimulated by mDCs and we IFN-l1 can be transcribed following stimulation of the believe it is likely that the major role of IFN-l1 is on the cell via TLR 3 or 4 in an IRAK-4-independent manner.15 development of monocytes to intermediate DCs rather This expression following cell triggering by such a wide than on the final maturation of these cells. range of TLR molecules, together with the broad range of Further work is be necessary in this area. Taken STAT molecules activated, suggests a broader role for together, however, the diminished secretion of IL-13 by T IFN-l1, particularly in the response to intracellular cells stimulated with alloantigen in the presence of infection. mDCs induced with IFN-l1 supports the altered cyto- IFN-l1 is expressed in both mDC and plasmacytoid kine levels seen in the T-cell priming, MLR and Con-A dendritic cells (pDC). Following infection, IFN-l1 experiments (where IFN-l1 was present throughout the mRNA is present at approximately 100-fold greater experiment). This positions IFN-l1 as a new modulator levels in pDC compared with mDC.8 The expression of the Th1/Th2 system, whose overall function in our pattern in these cells resembles that of IFN-b rather than experiments is to diminish Th2 responses. The mechan- IFN-a. In pDCs, there is coordinated regulation of IFN-l1 isms that underlie this Th2 modulation by IFN-l1 are and IFN-b expression.8 Thus, there may be a role for IFN- currently unknown to us and will require further l1 in coordinating overall postinfection adaptive im- experimental effort to unravel. Interestingly, Type-I mune responses, both within DCs and in interfacing T interferons are known to be capable of diminishing the cells. The experiments reported here address this latter expression of IL-4-dependent genes through upregula- question. tion of SOCS-1.44 It may be that IFN-l1 is acting as a We have shown that IFN-l1 modulates the ability of Type-I interferon to effect the Th2 modulation we report T cells to secrete Th1 or Th2 cytokines. Both mitogen- here. Its ability to activate STAT423 and modestly enhance driven systems and MLRs showed strong skewing IL-12 secretion (this report) also suggests some possible against Th2 responses in the presence of IFN-l1. The direct, Th1 stimulating effects, although it may be that secretion of IL-13 was consistently and markedly the counterregulation of Th2 responses by these media- diminished, whereas that of IFN-g was enhanced, tors is more important, given the stronger and consistent although less frequently. These effects were dose- inhibition of IL-13 secretion by IFN-l1. responsive to IFN-l1 but importantly, the effect on IL- A recent report37 described diminished expression of 13 was stronger and more consistent. IFN-l1 diminished IFN-l1 mRNA in asthma patients following in vivo IL-13 levels at an approximately 100-fold lower concen- challenge with rhinovirus and in vitro challenge of their tration than that required to bring about elevation of leukocytes with LPS. IL-13 and the Th2 system are IFN-g. Furthermore, it was more consistent. For example, recognized as a key mediator in the pathogenesis of in the case of Con-A stimulation, the suppression of IL-13 bronchial asthma45,46 and its mechanism of action and was observed in 15/16 separate experiments, but the means of control are of interest as new therapeutic effect on IFN-g was seen in only 5/16 experiments (data targets.47 IL-13 mRNA and protein are expressed in the not shown). Further evidence to support a primary role lungs of allergen-challenged human volunteers48 and of IFN-l1 in downregulating Th2 responses rather than asthma patients,49 and its effect appears to be mediated in promoting Th1 responses is shown in Figure 4, where via elevating the expression of MHC class-II and CD40 IFN-l1 was unable to elevate IFN-g levels in the presence on CD11c-high alveolar DCs.50 In experimental asthma or absence of IL-12 and thereby being clearly distin- models, airway hyperreactivity accompanied by high guishable from the actions of IFN-g, notwithstanding its levels of IL-4 and IL-5 secretion can be induced in intact ability to phosphorylate STAT1 and STAT3.2 Thus, we but not IL-13À/À mice.51,52 Sensitized animals can be postulate that they combine with those data to provide a protected by treatment with IL-13Ra-chain constructs,53 picture of IFN-l1 activity that is more strongly anti-Th2 and although the IL-4Ra-chain may be involved, this is than pro-Th1. We do not yet know whether the disputed.46,49 Mice whose Th1 system is crippled by the modulation (or lack thereof) of IFN-g by IFN-l1is genetic disruption of T-bet expression develop sponta- consistent within the same individuals. Experiments neous asthma-like disease, which is accompanied by are currently underway to address this issue, which high numbers of CD44 and CD69 bright CD4 þ T cells; may indicate an important genetic variation between again, this condition can be ameliorated by the direct individuals. blockade of IL-13.54 In humans, allergen-specific T-cell When we examined direct effects on either the clones produce IL-13,55 and IL-13 will elevate mast-cell induction or maturation phases of mDC production, we proliferation;56 experimental anti-Th2 strategies which observed apparently distinct responses to IFN-l1. Its do not address IL-13 have failed.57 The IL-13 , which presence during the maturation phase (Figure 5a) led to is ethnically very diverse,58 is recognized as contributing the production of mDC with modestly enhanced IL-12 to asthma susceptibility.59,60 Thus, as elevated IL-13 is secretion, whereas its presence during the induction a recognized aspect of the Th2 response in asthma phase (Figure 5b) led to mDC with the ability to diminish patients, it may be the case that the diminished IFN-l1 IL-13 secretion from exposed allo T cells, with no effect response in these patients37 contributes directly to their on IFN-g production. At present, we can only speculate elevated IL-13 levels via the mechanism suggested by the on the potential role of this slight increase in IL-12 present data. production by mDCs. A recent report43 described the Recently, another new cytokine that affects Th2 inability of IFN-l1 to lead to the development of a responses has been described. IL-19 expression is mature DC phenotype, producing instead an intermedi- sensitive to IL-461 and is known to enhance developing ate phenotype, described as ‘tolerogenic’; our data would Th2 responses.62–64 IL-19 also alters the induction of not contradict this report. Clearly, however, it does not human mDC, such that they display elevated IL-10 interfere with the ability of IFN-l1 to inhibit IL-13 secretion with no effect on IL-12.65 We show here that

Genes and Immunity IFN-k1 downregulates the human Th2 response WJ Jordan et al 259 IFN-l1 upregulated mDC IL-12 secretion with a corre- To examine the effect of IFN-l1 on the acquisition of sponding reduction of IL-10 secretion and consistently cytokine secreting phenotype by naive Tcells, mature diminished the Th2 response in several experimental DCs were generated as described above, washed and systems. This result complements our previously ob- seeded into 12-well plates along with naive Tcells (5 Â 105 served effect of IL-19 on mDC, where IL-10 was elevated naive CD4 þ T cells þ 5 Â 103 mDC in a final volume of and IL-12p70 was unaffected.66 Thus, these two cytokines 2 ml). In some experiments, antibodies and cytokines may represent a new and hitherto unexpected control were added to the MLR at this stage to direct the mechanism over mDC. With the present description of cytokine-producing phenotype of the developing T cells. modulation of Th1/Th2 responses by IFN-l1 against Th2 For Th2-biasing, IL-4 (10 ng/ml) and anti-IL-12 p70 and previous complementary reports on IL-19’s ability to (1 mg/ml) were added to the culture and for Th1-biasing, skew Th1/Th2 responses in favor of Th2, new doors may IL-12 p70 (10 ng/ml) and anti-IL-4 (1 mg/ml) were added. be opening on the regulation of the Th2 response that Experiments termed as ‘unbiased’ contained no cytokine may have subsequent impact on T-cell-dependent or antibody, whereas those defined as ‘Th0-type’-biased disorders, such as , , asthma (giving rise to an intermediate T-cell cytokine-secreting and atopic disease. phenotype based on IL-13/IFN-g levels) were induced using both anti-IL-12 and anti-IL-4. These MLRs were cultured for a total of 9 days, with 1 ml of supernatant Materials and methods being taken on days 3 and 6, being replaced with fresh medium containing IL-2 at 10 U/ml to expand reacting T Cells cells. Cells were then harvested, washed twice and rested Human PBMCs were isolated from anonymous buffy for 4 h before two more rounds of washing to remove any coats or volunteer donor peripheral blood by density- remaining antibody/cytokine. Cells were then resus- gradient centrifugation over ‘Histopaque-1077’ (Sigma, pended into 2 ml RPMI, 10% FCS containing PMA St Louis, MO, USA). ‘Untouched’ T cells were prepared (10 ng/ml) and ionomycin (1 mg/ml), transferred to from PBMC using the MACS system (Miltenyi Biotech, new 12-well plates and cultured for a further 18 h. Cells Auburn, CA, USA) and the ‘Pan isolation kit II’ and supernatant were taken for analysis to assess the (removing cells expressing CD14, CD16, CD19, CD36, development of cytokine development. CD56, CD123 and Glycophorin A). Monocytes were prepared through positive selection of CD14 þ cells Measurement of cytokine protein present within PBMC, again using the MACS system, Cytokine levels within supernatants were measured according to the manufacturer’s instructions. DCs were using ELISA. Paired antibodies consisting of purified created from positively selected CD14 þ monocytes captured and biotinylated secondary detection antibo- through culturing in the presence of IL-4 and GM-CSF dies purchased from BD Pharmingen were used accord- (10 ng/ml, Peprotech) for 5–7 days and were matured in ing to the manufacturer’s recommendations. Standards the presence of LPS (100 ng/ml, Sigma) for 24 h. In some were purchased from Peprotech. The ELISA protocol experiments, IFN-l1 was added as indicated. Cells were was performed as described previously.61 then washed thrice, rested for 1 h and given a further round of washing to ensure removal of LPS and cytokines before use in functional assays. Acknowledgements Culture conditions This work was carried out in the laboratories of the Culture medium consisted of RPMI 1640 medium, 10% Department of Oral Biology, University of Medicine and fetal calf serum (FCS) supplemented with penicillin at Dentistry of New Jersey. Dr WJ Jordan was supported by m 100 IU/ml and streptomycin at 100 g/ml (all from a grant from NIDCR, NIH. Sigma). For dose–response experiments, whole PBMCs were cultured in 96-well, flat-bottomed plates (2 Â 105 in 200 ml) for 18 h over a range of IFN-l1 concentrations (Peprotech), as shown. For all other experiments, PBMCs References were cultured at 2 Â 106 cells in 2 ml in the presence or absence of human recombinant IFN-l1at1mg/ml. For 1 Sheppard P, Presnell SR, Fox B, Gilbert T, Haldeman B, antibody-blocking experiments, cells were preincubated Grant FJ. Interferon-like protein, Zcyto21. United States Patent Application #002003963 April 4, 2002, 2002. for 1 h with rat-anti-human-IL-10 (clone 9D7) or isotype 2 Kotenko SV, Gallagher G, Baurin VV, Lewis-Antes A, Shen M, control MAb (BD Pharmingen, San Jose, CA, USA) at a Shah NK et al. IFN-lambdas mediate antiviral protection concentration of 10 mg/ml before addition of IFN-l1. In through a distinct class II cytokine receptor complex. Nat some experiments, IL-10 (Peprotech) was added to Immunol 2003; 4: 69–77. cultures at 10 ng/ml. Supernatants were harvested and 3 Sheppard P, Kindsvogel W, Xu W, Henderson K, Schultsmeyer frozen at À801C for later cytokine analysis by ELISA. S, Whitmore TE et al. IL-28, IL-29 and their class II cytokine Whole PBMC two-way MLRs were performed in 24- receptor IL-28R. Nat Immunol 2003; 4: 63–68. well plates in a final volume of 2 ml using 1 Â106 PBMCs 4 Kotenko SV, Krause CD, Izotova LS, Pollack BP, Wu W, Pestka from each of two unrelated donors. Cells and super- S. Identification and functional characterization of a second chain of the interleukin-10 receptor complex. EMBO J 1997; 16: natants were harvested at the time points indicated for 5894–5903. later analysis of cytokine levels. Con-A stimulations were 5 Kotenko SV, Izotova LS, Mirochnitchenko OV, Esterova E, 6 performed in 24-well plates using 1 Â10 whole PBMCs Dickensheets H, Donnelly RP et al. Identification of the with a range of Con-A from 0–10 mg/ml in a final volume functional interleukin-22 (IL-22) receptor complex: the IL- of 2 ml. 10R2 chain (IL-10Rbeta) is a common chain of both the IL-10

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