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Timing of IFN- Exposure during Human Dendritic Cell Maturation and Naive Th Cell Stimulation Has Contrasting Effects on Th1 Subset Generation: A Role for IFN--Mediated Regulation of IL-12 Family Cytokines and IL-18 in Naive Th Cell Differentiation¹

Taro Nagai^*,, Odile Devergne, Thomas F. Mueller, David L. Perkins, Jean Maguire van Seventer^2,* and Gijs A. van Seventer^2,*

^* Department of Environmental Health, Boston University School of Public Health, Boston, MA 02118; Tsukiyono Hospital, Gunma, Japan; Centre National de la Recherche Scientifique, Paris V University, Paris, France; and Laboratory of Molecular Immunology, Department of Medicine, Brigham and Women’s Hospital and Harvard Medical School, Boston, MA 02115

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Type I IFNs, IFN- and IFN-, are early effectors of innate immune responses against microbes that can also regulate subsequent adaptive immunity by promoting antimicrobial Th1-type responses. In contrast, the ability of IFN- to inhibit autoimmune Th1 responses is thought to account for some of the beneficial effects of IFN- therapy in the treatment of relapsing remitting multiple sclerosis. To understand the basis of the paradoxical effects of IFN- on the expression of Th1-type immune responses, we developed an in vitro model of monocyte-derived dendritic cell (DC)-dependent, human naive Th cell differentiation, in which one can observe both positive and negative effects of IFN- on the generation of Th1 cells. In this model we found that the timing of IFN- exposure determines whether IFN- will have a positive or a negative effect on naive Th cell differentiation into Th1 cells. Specifically, the presence of IFN- during TNF--induced DC maturation strongly augments the capacity of DC to promote the generation of IFN--secreting Th1 cells. In contrast, exposure to IFN- during mature DC-mediated primary stimulation of naive Th cells has the opposite effect, in that it inhibits Th1 cell polarization and promotes the generation of an IL-10-secreting T cell subset. Studies with blocking mAbs and recombinant cytokines indicate that the mechanism by which IFN- mediates these contrasting effects on Th1 cell generation is at least in part by differentially regulating DC expression of IL-12 family cytokines (IL-12 and/or IL-23, and IL-27) and IL-18.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Innate immune responses are initiated when specific pattern recognition receptors, including immune cell Toll-like receptors (TLRs),³ recognize unique pathogen-associated molecular patterns (danger signals) (1, 2). Activation of TLRs by pathogen-associated molecular patterns leads to the secretion of cytokines, including type I IFNs (IFN- and IFN-). In both humans and mice, there are several IFN- and one IFN- gene products, all of which signal through a common heterodimeric receptor, IFN-R1/R2 (3, 4). Type I IFNs are classically regarded as effector molecules of the innate immune response as a result of their direct antiviral properties. However, although they are products of innate immunity, IFN- can also regulate adaptive immunity. In particular, IFN- are well recognized for their ability to mature myeloid dendritic cells (DC) (5, 6, 7, 8) and to promote antimicrobial Th1-type immune responses that are characterized by the generation of CD4⁺ Th1 cells and CD8⁺ CTLs, which secrete high levels of IFN- (reviewed in Refs. 4, 9 , and 10).

IL-12 is a myeloid DC-derived cytokine that plays a well-established, critical role in inducing the differentiation of naive Th cells into IFN--secreting Th1 cells. An emerging paradigm of Th1-type immune responses envisions that while necessary, IL-12 alone is not sufficient for optimal Th1 polarization, and that such polarization may require multiple members of the IL-12 cytokine family (11, 12) as well as IL-18 (13). IL-12 is the prototype of the IL-12 family of cytokines (reviewed in Ref. 14), which includes IL-12, IL-23, and IL-27. Structurally, all three cytokines consist of related helical subunits (p35, p19, or p28) and related soluble cytokine receptor subunits (p40 or EBI3) that heterodimerize to form IL-12 p35/p40 (IL-12 p70), IL-23 p19/p40, or IL-27 p28/EBI3. Unlike p40 and EBI3, the helical IL-12 family chains are only secreted when associated in a heterodimer (14). Each IL-12 family cytokine has its own specific receptor (15). The IL-12R is composed of the IL-12R 1- and IL-12R 2-chains. IL-23 also uses the IL-12R 1-chain, and a second chain, IL-23R (16). To date, only one chain of the, presumed dimeric IL-27R has been identified, the IL-12R2 homologous, T cell cytokine receptor (WSX-1) (12, 17, 18).

The responsiveness of Th cells to individual IL-12 family members appears to be developmentally regulated. IL-12 induces significant proliferation and IFN- secretion in both naive and memory activated human Th cells. In contrast, IL-23 preferentially induces proliferation and IFN- secretion in activated human memory Th cells, and it has little or no effect on naive Th cells (11). Finally, IL-27 promotes expansion in stimulated naive, but not memory, human Th cell populations, and it synergizes with IL-12 in inducing IFN- production only in naive Th cells (19). These observations together with numerous in vitro and in vivo studies involving overexpression or deficiency of specific IL-12 family cytokine chains and cytokine receptor chains (reviewed in Ref. 15) have revealed that IL-12, IL-23, and IL-27 are not redundant family members. Rather, it appears that the sequential contributions of IL-12, IL-23, and IL-27 at specific times during the induction and maintenance of a Th1 response may be required for optimal resolution of an immunological challenge.

Multiple sclerosis (MS) is a chronic inflammatory disease affecting the CNS. Overwhelming evidence indicates a primary role for autoimmune Th1 effector cells targeting the CNS in the pathogenesis of MS. Studies in the murine experimental allergic encephalomyelitis (EAE) model of MS have directly demonstrated IL-12 and IL-23 to be critical mediators of the disease (20, 21). IFN- therapy is a standard treatment for relapsing remitting (RR) MS. Initial in vitro studies by ourselves and others (22, 23) and later studies of MS patients suggest that the beneficial clinical effects of IFN- in MS are due, at least in part, to inhibition of Th1-type immune responses (reviewed in Ref. 24). Specifically, using an in vitro model of DC-dependent, naive Th cell differentiation, we found that IFN- can inhibit the generation of IFN--secreting Th1 cells (22) by suppressing CD40 ligand (CD40L)-induced DC IL-12 secretion (25). Studies of immune cells from individuals with MS support these in vitro findings, in that treatment with recombinant human (rh) IFN- is reported to decrease the number of circulating IFN--secreting Th cells (26) and also decrease IL-12 secretion in stimulated PBMC (24)

It remains an enigma how type I IFNs can have both positive and negative effects on the generation of IFN--secreting Th1 cells, as revealed by their divergent immunoregulatory functions in certain viral and autoimmune settings. We hypothesized that the timing of DC exposure to IFN- during an immune response is a crucial factor in determining whether IFN- will promote or inhibit Th1 cell generation. To test this hypothesis we developed an in vitro model of DC-dependent, human naive Th cell differentiation and examined how IFN- exposure during DC maturation and/or primary naive Th cell stimulation affects differentiation into Th1 cells. Our results demonstrate that maturation of immature monocyte-derived DC in the presence of IFN- generates DC that strongly promote polarization toward IFN--secreting Th1 cells, whereas exposure to IFN- during mature DC-mediated primary stimulation of naive Th cells significantly inhibits Th1 cell polarization. In addition, our data indicate that the mechanism by which IFN- mediates these contrasting effects is based on differential regulation by IFN- of DC IL-12 family cytokine and IL-18 secretion.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Antibodies

For Th cell stimulation, anti-CD3 mAb clone OKT3 (available as hybridoma from American Tissue Culture Collection, Manassas, VA) and anti-CD28 mAb clone 9.3 (a gift from Dr. C. June, University of Pennsylvania, Philadelphia, PA) were used. The following mAbs were used for the blocking experiments: anti-IL-12 p40 mAb clone B-P24 (IgG1) and anti-IL-12 p35 clone B-T21 (IgG1; Cell Sciences, Norwood, MA), anti-IL-18 (IgG1; MBL, Watertown, MA), MOPC21 (IgG1; ICN, Costa Mesa, CA), and anti-EBI3 mAbs 1A1 (IgG1) as previously described (27).

Cytokines

The following recombinant human cytokines were used: rhIL-12 p70, rhIL-4, and rhTNF- (all from BD PharMingen, San Diego, CA); rhGM-CSF (leukine; Immunex, Seattle, WA); rhIL-18 and rhIL-23 (both from R&D Systems, Minneapolis, MN); and rhIFN- (Avonex; Biogen, Cambridge, MA).

DC generation and preparation for use as APC

Monocyte-derived mature DC were generated by standard 6-day culture of peripheral blood monocytes with 30 ng/ml of GM-CSF (2 x 10⁸ IU/mg) and IL-4 (10⁸ IU/mg) to produce immature DC as previously described (28), and additional 2-day maturation with either TNF- (400 U/ml) alone (DC-TNF-), or a combination of TNF- and IFN- (1000 U/ml; 5 ng/ml; DC-TNF-/IFN-). Mature DC were isolated and washed to remove any exogenously added cytokines. For stimulation of naive Th cells, mature DC were irradiated (2500 rad) and used as the APC for the superantigen, Staphylococcus enterotoxin A (SEA). For stimulation of isolated mature DC, DC were cocultured with either irradiated (2500 rad) murine L cells transfected with human CD40L (obtained from the American Type Culture Collection with permission from DNAX (Palo Alto, CA)) or irradiated control L cells (DT70; gift from Dr. L. Lanier, University of California, San Francisco, CA), at a ratio of 2:1 DC/CD40L or DT70. Mature DC stimulated with DT70 cells secrete barely detectable or, most often, undetectable levels of any of the cytokines examined in these studies (T. Nagai, O. Devergne, and G. van Seventer, unpublished observations).

In vitro model of human monocyte-derived DC-dependent, superantigen-mediated, naive Th cell differentiation

Human naive Th cells (CD4⁺CD45RA⁺RO^-), isolated from PBMC by negative selection as described previously (29), were activated by primary stimulation with the superantigen, SEA (1 µg/ml; Sigma-Aldrich, St. Louis, MO) and 2-day matured monocyte-derived DC (5:1 ratio, Th/DC). After 7 days, 1 million of the expanded Th cells were restimulated with coimmobilized anti-CD3 (clone OKT3; 1 µg/ml) and anti-CD28 mAb (clone 9.3; 1 µg/ml), in the absence of exogenous cytokine, and the Th cytokine secretion profile was determined by ELISA of 48-h culture supernatants. The regulatory effects of IFN- were studied by coculture with recombinant IFN- (1000 U/ml; 5 ng/ml) during the time of DC maturation and/or primary naive Th cell stimulation. Due to the difficult logistics of the large numbers of purified DC and CD4⁺ T cells required for these studies, the necessary culture period for DC generation, and the poor quality of thawed naive Th cells and peripheral blood monocytes, primary Th cell stimulation was performed with DC and Th cells derived from different donors. To eliminate any variation due to differences in the allostimulatory component of the Th cell response between donor combinations, each Th cell/DC donor combination was analyzed in parallel cultures of the different stimulatory conditions. Thus, in a given experiment all groups, both experimental and control, had Th cells from donor 1 stimulated by DC from donor 2, and statistical comparisons were always made between these groups.

The mAb blocking experiments (see Figs. 4 and 5) were performed by adding mAbs only in the primary stimulation, at a concentration of 10 µg/ml for each specific mAb. All mAbs, including the isotype control mAb, MOPC21, are of the mouse IgG1 isotype. In each culture condition, isotype control mAb was added to the various combinations of anti-IL-12 p35, anti-IL-12 p40, anti-EBI3, and anti-IL-18 mAbs to bring the final mAb concentration to either 40 µg/ml (Fig. 4) or 20 µg/ml (Fig. 5).

View larger version (26K): [in this window] [in a new window]   FIGURE 4. Functional differences between DC-TNF- and DC-TNF-/IFN- are revealed by the ability of anti-IL-18 mAb to block only DC-TNF-/IFN--dependent, Th1 cell IFN- and TNF- secretion. Blocking of DC-TNF--dependent (A–C) and DC-TNF-/IFN--dependent (D–F) Th1 cell cytokine secretion. Each of the indicated mAbs (10 µg/ml) was added to cultures at the start of primary stimulation of naive Th cells with DC, IFN-, and SEA (detailed in Fig. 1). Isotype control mAb was added to bring the total mAb concentration in each condition to 40 µg/ml. Anti-CD3/anti-CD28 restimulation (see Fig. 1) was performed in the absence of blocking mAb. Results are expressed as the percent inhibition of Th1 cell cytokine secretion, where secretion in the presence of isotype control mAb alone equals 100%. *, Significant blocking of cytokine secretion compared with blocking with isotype control mAb. n = 4–10 independent donor combinations.

View larger version (15K): [in this window] [in a new window]   FIGURE 5. Blocking with anti-p35, anti-p40, and anti-EBI3 mAbs during primary naive Th cell stimulation enhances Th cell IL-13 secretion. Effects of coculture with blocking mAbs on DC-TNF--dependent (A) and DC-TNF-/IFN--dependent (B) Th cell IL-13 secretion. Each of the indicated mAbs (10 µg/ml) was added to cultures at the start of primary stimulation of naive Th cells with the indicated DC, SEA, and IFN- (detailed in Fig. 1). Isotype control mAb was added to bring the total mAb concentration in each condition to 20 µg/ml. Anti-CD3/anti-CD28 restimulation (see Fig. 1) was performed in the absence of blocking mAb. Results are expressed as relative Th cell IL-13 secretion, where secretion in the presence of isotype control mAb alone equals 1. *, Significant augmentation of IL-13 secretion compared with blocking with isotype control mAb. n = 6–13 independent donor combinations.

ELISAs

An EBI3 ELISA, developed by us (27), was used to determine levels of combined free EBI3 plus IL-27 p28/EBI3. An IL-27 ELISA, recognizing only IL-27 p28/EBI3, was made available by Dr. R. Kastelein (DNAX, Palo Alto, CA) (12). All other ELISAs were performed using commercial reagents; levels of IFN-, TNF-, lymphotoxin (LT), IL-4, IL-5, IL-6, IL-10, IL-13, IL-12 p40, and IL-12 p70 were determined using OptEIA ELISA kits from BD PharMingen, and IL-18 was determined with an ELISA kit from MBL. The IL-12 p40 OPtEIA kit recognizes both free p40 and p35/p40.

Quantitative real-time PCR

Real-time PCR was performed using the ABI PRISM 7900HT Sequence Detection System (PE Applied BioSystems, Foster City, CA), and PCR products were detected by SYBR-Green, according to the manufacturer’s protocol. Each primer pair was rigorously tested to ensure 1) lack of primer-dimer formation, and 2) recognition of only specific human cytokine cDNA, and no cross-reactivity with cDNA species derived from either CD40L transfectants or murine spleen cells expressing transcripts for all cytokines chains under study. All primer pairs were extensively screened in validation experiments to ensure approximately equal amplification efficiencies, so as to allow accurate relative quantitation using comparative threshold cycle (C_T) methodology.

Statistics

Minitab version 10.2 for Windows (Minitab, State College, PA) was used for nonparametric analysis in a one-sample Wilcoxon test, and p 0.05 was considered significant.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Depending on the timing of exposure, IFN- can either promote or inhibit the generation of IFN--secreting Th1 cells

Type I IFNs (IFN-) can have paradoxical roles in regulating adaptive immune responses. Triggering of specific TLRs results in rapid secretion of type I IFNs (reviewed in Ref. 3) that can promote the generation of IFN--secreting Th1 cells from naive Th cells (reviewed in Refs. 4, 9, 10 , and 30). In contrast, sustained high levels of IFN-, such as might occur during chronic viremia or recombinant IFN- therapy in patients with MS, have an immunosuppressive effect that includes inhibition of Th1-inducing, DC IL-12 secretion (25, 31). To study the mechanisms involved in determining these opposing effects of IFN-, we established an in vitro model of DC-dependent differentiation of human naive Th cells into effector Th cells, in which the regulatory effects of IFN- were examined by adding recombinant IFN- (1000 U/ml; 5 ng/ml) during DC maturation and/or primary naive Th cell stimulation. In this model, human naive Th cells were stimulated with the superantigen, SEA, presented by DC matured in either the absence (DC-TNF-) or the presence (DC-TNF-/IFN-) of IFN-. After 7 days, Th cells were restimulated with coimmobilized anti-CD3 and anti-CD28 mAb in the absence of exogenous cytokine, and cytokine secretion profiles were determined by ELISA of 48-h culture supernatants.

Fig. 1 depicts the comparative abilities of DC-TNF- and DC-TNF-/IFN- to promote Th cell expansion and cytokine secretion in the absence or the presence of IFN- during primary stimulation. Th cell expansion was unaffected by DC exposure to IFN- during maturation (Fig. 1A), whereas addition of exogenous IFN- during primary stimulation dramatically reduced the Th cell expansion induced by either DC-TNF- or DC-TNF-/IFN-. Both DC-TNF- and DC-TNF-/IFN- promoted the generation of Th1-like cells, as indicated by high level Th cell IFN-, TNF-, and LT secretion (Fig. 1B), with DC-TNF-/IFN- inducing significantly more IFN- secretion than DC-TNF-. In contrast to this Th1-promoting effect of IFN- exposure during DC maturation, exposure during the primary stimulation significantly inhibited the acquisition of a Th1 cytokine secretion profile regardless of the stimulating DC type (Fig. 1B), by suppressing Th cell secretion not only of IFN-, but also of TNF- and LT. Similar inhibitory results were obtained with 1 ng/ml IFN- (200 U/ml; data not shown). Interestingly, although secretion of the Th2 cytokines IL-4, IL-5, and IL-6 was not detected under any condition, both DC-TNF- and DC-TNF-/IFN- induced the secretion of IL-13, with the highest levels of IL-13 occurring in DC-TNF-/IFN--stimulated Th cells. Similar to Th1 cytokine secretion, IL-13 secretion was inhibited by IFN- coculture during the primary stimulation. Lastly, secretion of the immunosuppressive cytokine, IL-10 was also induced by both mature DC types. Contrary to the other measured Th cell cytokines, IFN- addition did not alter Th cell secretion of IL-10.

View larger version (21K): [in this window] [in a new window]   FIGURE 1. Effects of IFN- on human Th cell expansion and Th1 differentiation. Naive Th cells were stimulated with SEA (1 µg/ml) and DC (1:5 ratio, DC/Th cells) matured in either the absence (DC-TNF-) or the presence (DC-TNF-/IFN-) of IFN-. Exogenous IFN- was added during primary stimulation at a concentration of 5 ng/ml (1000 U/ml), as indicated. One million Th cells were restimulated with coimmobilized anti-CD3 and anti-CD28 mAbs (1 µg/ml each) in the absence of exogenous cytokine. A, Relative Th cell expansion was determined on day 7, before restimulation; B, Th cytokine secretion was determined by ELISA of culture supernatants harvested 48 h after the initiation of anti-CD3/anti-CD28 restimulation. *, Significant differences (p 0.05) between conditions due to IFN- exposure. n = 11–20 independent donor combinations.

IFN- differentially regulates DC expression of IL-12 family members and IL-18

We hypothesized that IFN- influences the generation of IFN--secreting Th1 cells at least in part by regulating DC cytokine secretion. Consequently, we examined the effects of IFN- on the expression of DC cytokines known to promote Th cell IFN- secretion, the IL-12 family cytokines and IL-18. To mimic the DC:naive Th cell interaction occurring in the DC-dependent, naive Th cell differentiation model, isolated DC were stimulated with a combination of CD40L-transfected L cells, SEA, and low dose (1 ng/ml) IFN-. We reasoned that in the Th cell differentiation model, exposure of DC to IFN- precedes Th cell CD40L expression (and, thus, DC CD40 signaling) by at least 18 h. Accordingly, DC-TNF- and DC-TNF-/IFN- either were not pretreated or were pretreated for 18 h with IFN- before initiating CD40L/IFN-/SEA stimulation at 0 h.

Levels of DC-secreted IL-12 p40, IL-12 p35/p40 (p70) heterodimer, EBI3, IL-27 p28/EBI3 heterodimer, and IL-18 were determined by ELISA in 48-h culture supernatants (Fig. 2). An IL-23 (p19/p40 heterodimer) ELISA was not available due to the lack of a commercial anti-p19 Ab. In summary, DC-TNF- secreted significantly more IL-12 p40, IL-12 p70, EBI3, and IL-27 than DC-TNF-/IFN-, whereas DC-TNF-/IFN- secreted more IL-18 (Fig. 2, A–E, respectively). In addition, for both DC-TNF-/IFN- and DC-TNF-, IFN- pretreatment down-regulated IL-12 p40 and p70 secretion, but significantly augmented IL-27 secretion. Lastly, it should be noted that IFN- pretreatment alone did not induce expression in either DC type of any of the cytokines examined (data not shown).

View larger version (20K): [in this window] [in a new window]   FIGURE 2. Secreted cytokine levels in CD40L/IFN-/SEA-stimulated DC-TNF- and DC-TNF-/IFN-, in the absence or the presence of IFN- pretreatment. DC, matured as detailed in Fig. 1, were cultured for 18 h with either medium alone or medium containing IFN- (5 ng/ml) before initiation of stimulation (0 h) with CD40L-transfected L cells (2:1 ratio, DC/CD40L cells), IFN- (1 ng/ml), and SEA (1 µg/ml). A–E, Accumulated levels of, respectively, secreted IL-12 p40, IL-12 p70, EBI3, IL-27, and IL-18 were determined by ELISA of 48-h culture supernatants. *, Significant differences (p 0.05) between conditions due to IFN- exposure. n = 7–13 independent DC donors.

View larger version (39K): [in this window] [in a new window]   FIGURE 3. Relative cytokine chain mRNA levels in DC-TNF- and DC-TNF-/IFN- stimulated with CD40L/IFN-/SEA in the absence or the presence of IFN- pretreatment. DC were cultured and stimulated as described in Fig. 2. Relative cytokine mRNA expression was determined by quantitative real-time PCR analysis of total RNA, isolated at the indicated time points. A, Relative p35, p40, p28, p19, EBI3, and IL-18 expression at the onset of stimulation. At time zero, mRNA levels, normalized to GAPDH, were determined for each individual cytokine chain and were expressed relative to GAPDH mRNA level (=1) for each DC condition. B–G, Kinetics of p35, p40, p28, p19, EBI3, and IL-18 mRNA expression during DC stimulation. At the indicated time points, mRNA levels, normalized to GAPDH, were determined for each cytokine in each DC condition. mRNA levels for a given cytokine are expressed relative to DC-TNF- at 0 h mRNA level (= 1) for that cytokine. *, Significant differences (p 0.05) between conditions due to IFN- exposure. n = 4 independent DC donors.

In summary, maturation of DC in the presence of IFN- results in a DC (DC-TNF-/IFN-) with a significantly reduced capacity for activation-induced IL-12 p40, IL-12 p70, EBI3, and IL-27 secretion, and this reduction is, with the exception of p35 transcripts, reflected by changes at the message level. In contrast, in either the absence or the presence of IFN- pretreatment, stimulated DC-TNF-/IFN- secrete significantly more IL-18 than DC matured without IFN- (DC-TNF-). This increase in IL-18 secretion appears to require more than an increase in mRNA expression for IL-18, as IL-18 mRNA levels are equal between DC-TNF- pretreated with IFN- and DC-TNF-/IFN- without IFN- pretreatment (Fig. 3B), while secreted IL-18 levels are at least 4-fold higher in nonpretreated DC-TNF-/IFN- (Fig. 2). It is possible that this additional post-transcriptional requirement for optimal IL-18 secretion may involve an IFN--induced increase in caspase-mediated processing of pro-IL-18, a step necessary for IL-18 secretion (32). Stimulated DC-TNF-/IFN- also express much higher levels of IL-23 p19 mRNA than DC-TNF- (Fig. 3B). Thus, if p40 is not limiting, this suggests that DC-TNF-/IFN- are likely to secrete higher levels of IL-23 protein. Lastly, pretreatment with IFN- in both DC types leads to a significant reduction in IL-12 p40 and p70 secretion (Fig. 2), which is largely consistent with changes in p40, but not p35, message levels (Fig. 3B). Interestingly, although IFN- pretreatment does not alter EBI3 secretion, it does significantly augment IL-27 secretion in both DC-TNF- and DC-TNF-/IFN- (Fig. 2).

Taken together, these data suggest that maturation in the presence of IFN- increases the ability of DC to promote naive Th cell differentiation into IFN--secreting Th1 cells as a result of an enhanced capacity for DC IL-18 and possibly IL-23 secretion. Furthermore, the results indicate that exposure to IFN- during primary stimulation suppresses Th1 cell generation by inhibiting DC secretion of IL-12 p70.

DC maturation with IFN- promotes the IL-18-dependent generation of IFN-- and TNF--secreting Th1 cells

The differential levels of cytokine secretion by stimulated DC-TNF- and DC-TNF-/IFN- in the absence of IFN- pretreatment (Fig. 2) suggested that the ability of these two DC types to promote the generation of IFN--secreting Th1 cells (Fig. 1B) may depend on different cytokine milieu. To establish the contributions of IL-12 p70, IL-27, and IL-18 in promoting DC-mediated naive Th cell differentiation into Th1 cells, we performed mAb blocking studies in the DC-dependent, naive Th cell differentiation model. A combination of anti-p35 and anti-p40 mAbs was used to target IL-12 p70 and possibly IL-23. An anti-EBI3 mAb was used to inhibit IL-27 activity. The results of blocking studies, depicted in Fig. 4, indicate that for both DC-TNF--generated and DC-TNF-/IFN--generated Th1 cells, IFN- secretion can be significantly inhibited by the addition, during primary stimulation, of either anti-p35/p40 or anti-EBI3 mAbs (Fig. 4, A and D). Furthermore, combining these Abs yielded an additive effect in IFN- blocking. Anti-IL-18 mAb did not block any of the Th1 cell IFN- secretion promoted by DC-TNF- (Fig. 4A). In contrast, DC-TNF-/IFN--promoted Th1 cell IFN- secretion was significantly inhibited by anti-IL-18 mAb, and this effect was additive with anti-p35/p40 and anti-EBI3 blocking (Fig. 4D). mAb blocking of Th1 cell TNF- secretion followed a similar pattern as IFN- blocking, except that neither anti-p35/p40, anti-EBI3, nor anti-IL-18 mAbs alone could block TNF- secretion in DC-TNF--generated Th1 cells (Fig. 4, B and E). Interestingly, none of the anti-IL-12 family or anti-IL-18 cytokine mAbs, alone or in combination, was able to block LT secretion in either DC-TNF-- or DC-TNF-/IFN--generated Th1 cells (Fig. 4, C and F).

We also investigated the effects of mAb blocking on Th cell secretion of IL-10 and IL-13. No significant effects were seen on Th cell IL-10 secretion by the mAbs (data not shown). However, secretion of the Th2-type cytokine, IL-13, was significantly augmented by coculture with either anti-p35/p40 or anti-EBI3 alone, but not anti-IL-18, in DC-TNF- (Fig. 5).

Together these results directly identify functional differences between DC-TNF- and DC-TNF-/IFN- in their use of cytokines to promote Th1 polarization, differences that are reflected phenotypically in their distinct cytokine secretion profiles (Fig. 2). Specifically, while both DC-TNF- and DC-TNF-/IFN- promote Th1 cell generation through their secretion of IL-12 p70 and/or IL-23 as well as IL-27, the generation of Th1 cells by DC-TNF-/IFN- appears to be additionally dependent on secreted IL-18. Thus, these data are consistent with a role for IFN- during DC maturation in promoting the generation of Th1 cells through the enhancement of mature DC IL-18 secretion.

IL-12 p70 prevents the inhibition of naive Th cell differentiation into IFN-- and TNF--secreting Th1 cells that results from exposure to IFN- during primary stimulation

We found that, in contrast to its effects on DC maturation, exposure to IFN- during primary naive Th cell stimulation inhibits DC-dependent generation of Th1 cells (Fig. 1). The results depicted in Figs. 2 and 3 suggested that the basis for this inhibition might be the ability of IFN- to suppress DC secretion of IL-12 p70 and, if IL-12 p40 is limiting, IL-23. The effects of IFN- on IL-27 and IL-18 were not expected to contribute to the inhibition of Th1 cell polarization, as secretion of these cytokines was either increased (IL-27) or not altered (IL-18) by IFN- pretreatment. To further characterize the Th1 inhibitory effect of IFN-, experiments were performed in which exogenous, recombinant IL-12 p70, IL-23, and IL-18 were tested for their ability to prevent IFN--mediated inhibition of Th1 cell generation. Naive Th cells were stimulated in the DC-dependent differentiation model in the absence of IFN-, or with IFN- alone or in the presence of recombinant IL-12, IL-18, or IL-23. Th cell expansion under these different culture conditions was determined after 7 days of culture. The ability of generated effector Th cells to secrete IFN-, TNF-, and LT upon restimulation was subsequently determined by ELISA.

Confirming the results depicted in Fig. 1, A and B, coculture with IFN- during primary stimulation inhibited Th cell expansion (Fig. 6A) and IFN-, TNF-, and LT secretion (Fig. 6B) in both DC-TNF-/IFN-- and DC-TNF--generated Th1 cells. None of the exogenous added cytokines could overcome IFN--mediated inhibition of Th cell expansion (Fig. 6A). Inhibition of IFN-, TNF-, and LT secretion was also unaffected by the addition of either recombinant IL-23 or IL-18 during the primary stimulation. In contrast, recombinant IL-12 was able to prevent IFN--mediated inhibition of both IFN- and TNF- in the generated Th1 cells, while having no effect on LT secretion. Furthermore, addition of IL-12 actually promoted Th1 cell IFN- secretion to levels significantly higher than those achieved in the absence of IFN- exposure.

View larger version (39K): [in this window] [in a new window]   FIGURE 6. IL-12 p70 rescues the IFN--inhibited differentiation of naive Th cells into IFN-- and TNF--secreting Th1 cells. Naive Th cells were stimulated with SEA and either DC-TNF- or DC-TNF-/IFN-. Exogenous IFN- was added during primary stimulation at a concentration of 5 ng/ml (1000 U/ml) as indicated, alone or in the presence of recombinant IL-12 p70 (30 pg/ml), IL-23 (1 ng/ml), or IL-18 (1 ng/ml). A, Relative Th cell expansion was determined on day 7, before restimulation; B, Th cytokine secretion was determined by ELISA of culture supernatants harvested 48 h after the initiation of anti-CD3/anti-CD28 restimulation. See Fig. 1 for stimulation details. *, Significant differences (p 0.05) between conditions due to IFN- exposure. n = 4–12 independent donor combinations.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
Type I IFNs can have sharply contrasting regulatory effects on the generation of IFN--secreting Th1 cells. Initially, it was thought that these differences might be explained by species differences in type I IFN receptor signaling due to a minisatellite insertion in murine STAT2, but not human STAT2 (reviewed in Ref. 33). It is now clear, however, that these opposite effects of type I IFNs on Th1 polarization can be found in both human and murine species (reviewed in Ref. 9). In this report we show that the timing of DC exposure to IFN- during the initiation of an immune response is likely to be a critical factor in determining whether IFN- will promote or inhibit Th1 polarization. In addition, we identify mechanisms by which IFN- can positively or negatively influence the generation of Th1 cells, mechanisms based on the ability of IFN- to differentially regulate the expression of DC-derived IL-12 family members and IL-18. Central to our understanding of the regulatory role of IFN- in adaptive immunity has been the development of an in vitro model of monocyte-derived DC-dependent, human naive Th cell differentiation. In this model one can distinguish the effects of IFN- exposure during immature DC maturation from those due to exposure to IFN- during the primary stimulation of naive Th cells by mature DC and SEA.

We found that IFN- exposure during TNF--induced DC maturation results in mature DC (DC-TNF-/IFN-) that strongly promote differentiation into Th1 cells that secrete almost twice the level of IFN- as Th1 cells generated by DC matured with TNF- alone (DC-TNF-; Fig. 1B). mAb blocking studies indicate that IL-18 mediates the increased IFN- secretion by DC-TNF-/IFN--generated Th1 cells (Fig. 4). This observation is supported by results demonstrating that upon CD40L/IFN- stimulation, isolated DC-TNF-/IFN- express significantly more IL-18 mRNA (Fig. 3) and secreted protein (Fig. 2) than DC-TNF-. Of note, stimulated DC-TNF-/IFN- secrete modestly, but significantly, lower levels of IL-12 p40, IL-12 p70, EBI3, and IL-27 compared with DC-TNF- (Fig. 2). However, these decreased levels do not appear to affect the contribution of the IL-12 family members to Th1 cell IFN- or TNF- secretion, as revealed by the finding that blocking with combined anti-p35, -p40, and -EBI3 mAbs results in a similar absolute decrease in the amounts of IFN- and TNF- secreted by DC-TNF-/IFN-- and DC-TNF--generated Th1 cells (Figs. 1B and 4).

In addition to the differential regulation of DC cytokine secretion, other mechanisms may contribute to the greater ability of DC-TNF-/IFN- to promote Th1 cell IFN- secretion, for example, enhanced cell surface expression of functionally relevant molecules such as HLA class II and costimulatory molecules. Indeed, while flow cytometric analysis demonstrated no differences between DC-TNF- and DC-TNF-/IFN- in surface expression of HLA class II, CD40, CD80 (B7-1), or CD123 (IL-3R), there was a significant increase in CD86 (B7-2) expression on DC-TNF-/IFN- (3-fold increase in mean fluorescence intensity; p < 0.05; data not shown).

In contrast to its effect on DC maturation, IFN- exposure during the primary stimulation of naive Th cells results in a strong inhibition of Th1 cell polarization. This effect of IFN- is observed upon naive Th cell stimulation with either DC-TNF- or DC-TNF-/IFN-, and it is characterized by inhibition of Th cell expansion and IFN-, TNF-, LT, and IL-13 secretion, but not IL-10 secretion. The inhibitory effect of IFN- on Th1 cell IFN- and TNF- secretion appears to involve a suppression of DC IL-12 p70, as only rhIL-12, not rhIL-18 or rhIL-23, can overcome the negative effects of IFN- on Th1 secretion of these cytokines (Fig. 6B). Of note, during primary naive Th cell stimulation in the presence of IFN-, the addition of as little as 30 pg/ml of rhIL-12 results in the generation of Th1 cells that secrete twice as much IFN- (but no more TNF-) as Th1 cells generated under control conditions without IFN- (Fig. 6B). It is possible that this high level of, specifically, Th1 IFN- secretion may be due in part to the reported capacity of type I IFNs to enhance high affinity IL-12R1/2 expression on Th cells (34, 35). Such an enhancement would be expected to increase Th cell sensitivity to IL-12.

Not all the inhibitory effects mediated by IFN- can be reversed by exogenous rhIL-12. For example, rhIL-12 could not prevent IFN--mediated suppression of Th cell expansion (Fig. 6A). This result is consistent with recent findings of Dondi et al. (36), demonstrating a role for type I IFN in inhibiting the initial entry of naive Th cells into the cell cycle, possibly as a result of type I IFN maintaining the expression of p27^Kip1, a cyclin-dependent kinase inhibitor that negatively regulates the cell cycle. Recombinant human IL-12 could also not overcome IFN--mediated inhibition of Th1 cell LT secretion. This lack of rescue is consistent with the finding that LT secretion in the absence of IFN- does not appear to be dependent on IL-12 family members or IL-18 (Fig. 4), and it corroborates earlier reports by us in different models (22, 37).

Although the presence of IFN- during primary stimulation inhibits the acquisition of a prototypic Th1-type cytokine secretion profile (i.e., high level IFN-, TNF-, and LT secretion), it does not result in the up-regulation of prototypic Th2-type cytokines, such as IL-4 and IL-5 (Fig. 1B). In fact, low level Th cell secretion of IL-13, the only Th2-type cytokine we could detect, was further suppressed by IFN- (Fig. 1B). Interestingly, IL-10 was the only detectable Th cytokine that was not inhibited by IFN-, and thus, it was relatively more predominant in those Th cells generated in the presence of type I IFN. IL-10 is no longer considered a uniquely Th2-type cytokine, as it can be secreted by other functional T cell subsets, including Th1 (38) and regulatory T cell (39, 40) subsets. Roncarolo et al. (41) reported that IFN- and IL-10 synergize in the generation of Tr1 regulatory T cells. Our results similarly suggest that the presence of IFN- during primary naive Th cell stimulation not only leads to the inhibition of both Th1 and Th2 cells, but may also promote the generation of regulatory T cells. We and others previously reported that IFN- will actually induce human Th cell IL-10 secretion (22, 42, 43) in models where Th cell cytokine secretion profiles were determined in the continued presence of IFN-. In our current model IFN- is only present during primary naive Th cell stimulation, not during secondary stimulation when Th cell cytokine secretion was accessed. Together these findings suggest that while IFN- exposure during primary stimulation will not promote Th cell IL-10 secretion, exposure of differentiated, effector Th cells to IFN- will elevate IL-10 levels.

IFN- pretreatment of DC-TNF- and DC-TNF-/IFN- significantly inhibited IL-12 p70 secretion by these DC upon CD40L-dependent stimulation (Fig. 2). The mechanism of this of IL-12 p70 inhibition is not yet clear, but a comparison of mRNA and protein expression data for IL-12 family cytokines (Figs. 2 and 3) suggests that multiple factors, at both the transcriptional and the post-transcriptional level, may contribute to the IFN--mediated decrease in IL-12 p70 secretion. IFN- pretreatment did not affect p35 mRNA expression levels (Fig. 3B), although it induced a significant decrease in p40 mRNA levels (Fig. 3B), which correlated with a suppression of IL-12 p40 secretion (Fig. 2). It should be noted that IL-23 p19 message is expressed at relatively high levels compared with p40 in unstimulated DC (Fig. 3A), and that these levels increase dramatically in IFN--pretreated, CD40L/IFN--stimulated DC (Fig. 3B). Thus, as p40 heterodimerizes to form not only IL-12 p35/p40, but also IL-23 p19/p40, it is possible that IFN- pretreatment may inhibit IL-12 p70 by limiting the amount of p40 chain available to heterodimerize with p35 chain through effects on both inhibiting steady state p40 message and promoting formation of IL-23 p19/p40 heterodimer. In addition, multiple post-translational p35 modifications are required for p35 to heterodimerization with p40, and IL-12 p70 to be secreted (44, 45), and each of these modifications is an additional potential target by which IFN- might mediate the inhibition of IL-12 p70 secretion.

The anti-EBI3 blocking mAb used in the current studies (Fig. 4) recognizes not only IL-27, but also free EBI3 and p35/EBI3 heterodimer (46). To date, neither free EBI3 nor p35/EBI3 has known functional activity. In addition, p35/EBI3 heterodimer has only been reported intracellular in primary cells (i.e., not secreted) (46), and consistent with this observation, we did not detect p35/EBI3 in DC supernatants (data not shown). Together these findings indicate that the effects of anti-EBI3 mAb in blocking Th cell IFN- and TNF- secretion (Fig. 4) and augmenting IL-13 secretion can be attributed to the blocking of IL-27 by this Ab (Fig. 5A). Thus, we show for the first time that IL-27 contributes to DC-dependent, human naive Th1 cell differentiation. This observation is consistent with the reported functional effects of IL-27 derived from studies in APC-independent in vitro models (12) and in IL-27R-deficient mice (18, 47). The combined results, however, appear to contradict findings from a recent report on EBI3-deficient mice, which suggest a role for IL-27 in promoting Th2 cell differentiation through effects on NKT cell secretion of IL-4 (48). It is possible that these paradoxical findings may be explained by distinct functional effects of IL-27 on NKT cells and Th cells and/or by species differences.

Similar to our previous findings with immature DC (28), the results of this study demonstrate that in mature, monocyte-derived DC, IFN- differentially regulates the transcriptional activity of the various IL-12 family chains. Specifically, IFN- exposure during TNF--mediated DC maturation (i.e., DC-TNF-/IFN-) resulted in a decrease in IL-12 p40 and IL-27 p28 mRNA levels in stimulated DC, whereas it enhanced the expression of IL-23 p19 mRNA (Fig. 3B). IL-23 p19 mRNA levels were also enhanced in DC-TNF- by IFN- pretreatment (Fig. 3B). The lower level of IL-12 p40, IL-12 p70, and IL-27 secretion by DC-TNF-/IFN- compared with DC-TNF- appear to reflect the IFN--induced changes in steady state mRNA expression (Fig. 2). At present there are no commercial reagents to determine IL-23 protein levels. Nevertheless, under these IFN--dependent, IL-23 p19-enhancing, IL-12 p40-suppressing culture conditions, there are still significant amounts of IL-12 p40 secreted (Fig. 2). Thus, it is likely that DC matured in the presence of IFN- will secrete higher levels IL-23 p19/p40 cytokine upon stimulation than DC matured in the absence of IFN-.

Based on our findings, we propose a model for the role of type I IFNs in regulating Th cell differentiation and effector function during microbial infections. This model envisions that upon initiation of a localized infection in the periphery, TLR-induced expression of type I IFNs will contribute to the maturation of resident myeloid-derived DC, which, after migrating to draining secondary lymphoid organs, will strongly promote naive Th cell differentiation into Th1 cells. Subsequently, type I IFN-induced cytokines will augment the local inflammatory immune response; for example, local induction of the chemokine IP-10 by type I IFNs will preferentially recruit the newly generated Th1 cells to the site of infection (49), whereas type I IFN-induced IL-23 will promote effector T cell secretion of IFN- (11, 20) and IL-17 (50). The model further envisions that upon progression of the microbial infection beyond localized peripheral boundaries, a systemic elevation in type I IFN levels will inhibit the DC-dependent Th1 cell differentiation occurring in draining lymphoid organs and, as a result, will help prevent an excessive and potentially harmful Th1 cellular response from occurring. Indeed, studies of certain chronic viral infections support a role for type I IFNs in immunosuppression (51, 52). It should be kept in mind, however, that systemically elevated levels of type I IFN will still promote a selection of antimicrobial cellular immune responses, including, for example, enhancement of CTL and NK cell function (52) and memory/effector T cell survival (53). In addition, the direct antiviral effects of type I IFN will continue (4). These latter aspects may explain some of the benefits of IFN- treatment of patients with viral hepatitis (54).

IFN- treatment of RR MS has been shown to slow disease progression. The mechanism by which IFN- induces this clinical effect is still not known. However, various studies of MS patients suggest that IFN- therapy reduces the number of circulating IFN--secreting Th cells and down-regulates IL-12 secretion in stimulated PBMC, while up-regulating IL-10 secretion (24, 26). These observations are consistent with our present findings, in that exposure to IFN- during primary naive Th stimulation resulted in 1) an inhibition in the generation of IFN--secreting Th1 cells through a mechanism appearing to involve suppression of DC IL-12 secretion, and 2) a relative increase in Th cell IL-10 secretion.

In the murine EAE model of RR MS, relapses occur as a result of the phenomenon of epitope spreading (55), in which effector Th cells with new anti-CNS specificities are generated during the progression of the disease. Treatment of mice in this model with murine recombinant IFN- prevents disease progression (56) as a result of a decrease in epitope spreading (57). Work with IL-12 p35- and p40-deficient mice has revealed that although IL-12 p35 is critical for the initiation of EAE, disease progression requires not IL-12 p35 (58), but, rather, IL-p40 (59). Only recently has it become clear, through the generation of IL-23 p19-deficient mice, that this dependence on IL-12 p40 is due to the fact that IL-23 p19/p40 is required for disease progression in EAE (20). This finding has been interpreted as evidence for an effector site role for IL-23 in promoting CNS inflammation. Based on this recent finding on the role of IL-23 in EAE and the results presented herein, we propose that while IFN- treatment may inhibit epitope spreading in the RR form of MS through mechanisms that involve both down-regulation of IL-12 and induction of IL-10-secreting regulatory T cells, such therapy may be detrimental in treating primary progressive forms of MS due to the potential enhancement of IL-23.

	Footnotes

 
¹ This work was supported by Grant 1R01AI44209 from the National Institute of Allergy and Infectious Diseases, National Institutes of Health (to G.A.v.S.).

² Address correspondence and reprint requests to Dr. Jean Maguire van Seventer or Dr. Gijs A. van Seventer, Department of Environmental Health, Boston University School of Public Health, 715 Albany Street, Talbot 2E, Boston, MA 02118. E-mail addresses: jvsevent@bu.edu and gvsevent{at}bu.edu

³ Abbreviations used in this paper: TLR, Toll-like receptor; CD40L, CD40 ligand; DC, dendritic cell; EAE, experimental allergic encephalomyelitis; LT, lymphotoxin; MS, multiple sclerosis; rh, recombinant human; RR, relapsing remitting; SEA, Staphylococcus enterotoxin A.

Received for publication July 8, 2003. Accepted for publication September 15, 2003.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. Barton, G. M., R. Medzhitov. 2002. Control of adaptive immune responses by Toll-like receptors. Curr. Opin. Immunol. 14:380.[Medline]
2. Zou, W., J. Borvak, S. Wei, T. Isaeva, D. T. Curiel, T. J. Curiel. 2001. Reciprocal regulation of plasmacytoid dendritic cells and monocytes during viral infection. Eur. J. Immunol. 31:3833.[Medline]
3. Le Bon, A., D. F. Tough. 2002. Links between innate and adaptive immunity via type I interferon. Curr. Opin. Immunol. 14:432.[Medline]
4. Bogdan, C.. 2000. The function of type I interferons in antimicrobial immunity. Curr. Opin. Immunol. 12:419.[Medline]
5. Ito, T., R. Amakawa, M. Inaba, S. Ikehara, K. Inaba, S. Fukuhara. 2001. Differential regulation of human blood dendritic cell subsets by IFNs. J. Immunol. 166:2961.[Abstract/Free Full Text]
6. Santini, S. M., C. Lapenta, M. Logozzi, S. Parlato, M. Spada, T. Di Pucchio, F. Belardelli. 2000. Type I interferon as a powerful adjuvant for monocyte-derived dendritic cell development and activity in vitro and in Hu-PBL-SCID mice. J. Exp. Med. 191:1777.[Abstract/Free Full Text]
7. Buelens, C., E. J. Bartholome, Z. Amraoui, M. Boutriaux, I. Salmon, K. Thielemans, F. Willems, M. Goldman. 2002. Interleukin-3 and interferon cooperate to induce differentiation of monocytes into dendritic cells with potent helper T-cell stimulatory properties. Blood 99:993.[Abstract/Free Full Text]
8. Luft, T., K. C. Pang, E. Thomas, P. Hertzog, D. N. J. Hart, J. Trapani, J. Cebon. 1998. Type I IFNs enhance the terminal differentiation of dendritic cells. J. Immunol. 161:1947.[Abstract/Free Full Text]
9. Biron, C. A.. 2001. Interferons and as immune regulators: a new look. Immunity 14:661.[Medline]
10. Murphy, K. M., W. Ouyang, J. D. Farrar, J. Yang, S. Ranganath, H. Asnagli, M. Afkarian, T. L. Murphy. 2000. Signaling and transcription in T helper development. Annu. Rev. Immunol. 18:451.[Medline]
11. Oppmann, B., R. Lesley, B. Blom, J. C. Timans, Y. Xu, B. Hunte, F. Vega, N. Yu, J. Wang, K. Singh, et al 2000. Novel p19 protein engages IL-12p40 to form a cytokine, IL-23, with biological activities similar as well as distinct from IL-12. Immunity 13:715.[Medline]
12. Pflanz, S., J. C. Timans, J. Cheung, R. Rosales, H. Kanzler, J. Gilbert, L. Hibbert, T. Churakova, M. Travis, E. Vaisberg, et al 2002. IL-27, a heterodimeric cytokine composed of EBI3 and p28 protein, induces proliferation of naive CD4⁺ T cells. Immunity 16:779.[Medline]
13. Okamura, H., H. Tsutsi, T. Komatsu, M. Yutsudo, A. Hakura, T. Tanimoto, K. Torigoe, T. Okura, Y. Nukada, K. Hattori. 1995. Cloning of a new cytokine that induces IFN- production by T cells. Nature 378:88.[Medline]
14. Trinchieri, G.. 2003. Interleukin-12 and the regulation of innate resistance and adaptive immunity. Nat. Rev. Immunol. 3:133.[Medline]
15. Watford, W. T., J. J. O’Shea. 2003. Autoimmunity: a case of mistaken identity. Nature 421:706.[Medline]
16. Parham, C., M. Chirica, J. Timans, E. Vaisberg, M. Travis, J. Cheung, S. Pflanz, R. Zhang, K. P. Singh, F. Vega, et al 2002. A receptor for the heterodimeric cytokine IL-23 is composed of IL-12R1 and a novel cytokine receptor subunit, IL-23R. J. Immunol. 168:5699.[Abstract/Free Full Text]
17. Sprecher, C. A., F. J. Grant, J. W. Baumgartner, S. R. Presnell, S. K. Schrader, T. Yamagiwa, T. E. Whitmore, P. J. O’Hara, D. F. Foster. 1998. Cloning and characterization of a novel class I cytokine receptor. Biochem. Biophys. Res. Commun. 246:82.[Medline]
18. Chen, Q., N. Ghilardi, H. Wang, T. Baker, M. H. Xie, A. Gurney, I. S. Grewal, F. J. de Sauvage. 2000. Development of Th1-type immune responses requires the type I cytokine receptor TCCR. Nature 407:916.[Medline]
19. Robinson, D. S., A. O’Garra. 2002. Further checkpoints in Th1 development. Immunity 16:755.[Medline]
20. Cua, D. J., J. Sherlock, Y. Chen, C. A. Murphy, B. Joyce, B. Seymour, L. Lucian, W. To, S. Kwan, T. Churakova, et al 2003. Interleukin-23 rather than interleukin-12 is the critical cytokine for autoimmune inflammation of the brain. Nature 421:744.[Medline]
21. Segal, B. M., B. K. Dwyer, E. M. Shevach. 1998. An interleukin (IL)-10/IL-12 immunoregulatory circuit controls susceptibility to autoimmune disease. J. Exp. Med. 187:537.[Abstract/Free Full Text]
22. McRae, B. L., R. T. Semnani, M. P. Hayes, G. A. van Seventer. 1998. Type I IFNs inhibit human dendritic cell IL-12 production and Th1 cell development. J. Immunol. 160:4298.[Abstract/Free Full Text]
23. Bartholome, E. J., F. Willems, A. Crusiaux, K. Thielemans, L. Schandene, M. Goldman. 1999. Interferon- inhibits Th1 responses at the dendritic cell level: relevance to multiple sclerosis. Acta Neurol. Belg. 99:44.[Medline]
24. Byrnes, A. A., J. C. McArthur, C. L. Karp. 2002. Interferon- therapy for multiple sclerosis induces reciprocal changes in interleukin-12 and interleukin-10 production. Ann. Neurol. 51:165.[Medline]
25. McRae, B. L., B. A. Beilfuss, G. A. van Seventer. 2000. IFN- differentially regulates CD40-induced cytokine secretion by human dendritic cells. J. Immunol. 164:23.[Abstract/Free Full Text]
26. Furlan, R., A. Bergami, R. Lang, E. Brambilla, D. Franciotta, V. Martinelli, G. Comi, P. Panina, G. Martino. 2000. Interferon- treatment in multiple sclerosis patients decreases the number of circulating T cells producing interferon- and interleukin-4. J. Neuroimmunol. 111:86.[Medline]
27. Devergne, O., A. Coulomb-L’Hermine, F. Capel, M. Moussa, F. Capron. 2001. Expression of Epstein-Barr virus-induced gene 3, an interleukin-12 p40-related molecule, throughout human pregnancy: involvement of syncytiotrophoblasts and extravillous trophoblasts. Am. J. Pathol. 159:1763.[Abstract/Free Full Text]
28. van Seventer, J. M., T. Nagai, G. A. van Seventer. 2002. Interferon- differentially regulates expression of the IL-12 family members p35, p40, p19 and EBI3 in activated human dendritic cells. J. Neuroimmunol. 133:60.