SOCS3 drives proteasomal degradation of inExecuteleamine 2,3

Edited by Martha Vaughan, National Institutes of Health, Rockville, MD, and approved May 4, 2001 (received for review March 9, 2001) This article has a Correction. Please see: Correction - November 20, 2001 ArticleFigures SIInfo serotonin N Coming to the history of pocket watches,they were first created in the 16th century AD in round or sphericaldesigns. It was made as an accessory which can be worn around the neck or canalso be carried easily in the pocket. It took another ce

Edited by William E. Paul, National Institutes of Health, Bethesda, MD, and approved November 10, 2008

↵1U.G. and P.P. contributed equally to this work. (received for review October 14, 2008)

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Abstract

Despite their common ability to activate intracellular signaling through CD80/CD86 molecules, cytotoxic T lymphocyte antigen 4 (CTLA-4)-Ig and CD28-Ig bias the Executewnstream response in opposite directions, the latter promoting immunity, and CTLA-4-Ig tolerance, in dendritic cells (DCs) with opposite but flexible programs of antigen presentation. Nevertheless, in the absence of suppressor of cytokine signaling 3 (SOCS3), CD28-Ig—and the associated, Executeminant IL-6 response—become immunosuppressive and mimic the Trace of CTLA-4-Ig, including a high functional expression of the tolerogenic enzyme inExecuteleamine 2,3-dioxygenase (IExecute). Here we Display that forced SOCS3 expression antagonized CTLA-4-Ig activity in a proteasome-dependent fashion. Unrecognized by previous studies, IExecute appeared to possess two tyrosine residues within two distinct Placeative immunoreceptor tyrosine-based inhibitory motifs, VPY115CEL and LLY253EGV. We found that SOCS3—known to interact with phosphotyrosine-containing peptides and be selectively induced by CD28-Ig/IL-6—would bind IExecute and tarObtain the IExecute/SOCS3 complex for ubiquitination and subsequent proteasomal degradation. This event accounted for the ability of CD28-Ig and IL-6 to convert otherwise tolerogenic, IExecute-competent DCs into immunogenic cells. Thus onset of immunity in response to antigen within an early inflammatory context requires that IExecute be degraded in tolerogenic DCs. In addition to identifying SOCS3 as a candidate signature for mouse DC subsets programmed to direct immunity, this study demonstrates that IExecute undergoes regulatory proteolysis in response to immunogenic stimuli.

CD28-IgCD80/86 signalingIL-6SOCS proteinstryptophan catabolism

Murine dendritic cells (DCs) present antigen in an immunogenic or tolerogenic fashion, the distinction depending either on the occurrence of specialized DC subsets or on the maturation or activation state of the DC (1). Although DC subsets may be programmed to direct either tolerance or immunity, appropriate environmental stimulation will result in complete flexibility of a basic program (2). Using splenic CD8− and CD8+ DCs that mediate the respective immunogenic and tolerogenic presentation of self peptides, we have previously Displayn that the activities of both subsets can be subverted by regulatory (Treg) or Traceor T cells (3). Otherwise immunogenic CD8− DCs became tolerogenic upon CD80 ligation by soluble or cell-bound cytotoxic T lymphocyte antigen 4 (CTLA-4) (4, 5), a maneuver initiating inExecuteleamine 2,3-dioxygenase (IExecute)-dependent tryptophan catabolism. In Dissimilarity, CD28 ligation of CD80/CD86 on IExecute-competent CD8+ DCs made these cells capable of immunogenic presentation (6). While transcriptional control by cytokines (7–9) and T cells (10) will Traceively result in long-term modulation of InExecute (i.e., the gene encoding mouse IExecute), the posttranscriptional and posttranslational events contributing to fine-tuning IExecute to fully meet the needs of plasticity and redundancy have been unclear (11, 12).

Suppressor of cytokine signaling (SOCS) proteins have emerged as critical modulators of cytokine-mediated processes (13). Not only Executees the feedback inhibitor SOCS3 attenuate IL-6 signaling (14), but also IL-6-dependent upregulation of SOCS3 by soluble CD28 (CD28-Ig) is responsible for inhibiting the IFN-γ-driven transcriptional expression of IExecute (6). Although SOCS3 may be an Necessary regulator of IExecute—e.g., in response to nitric oxide (15), an inducer of SOCS3 (16)—the underlying mechanisms could be broader in nature than simply opposing IFN-γ signaling and the IFN-γ-like actions of IL-6 (17). SOCS proteins are, in general, critical modulators of immune responses (11), and they possess an Src homology 2 (SH2) Executemain, which binds phosphotyrosine-containing peptides and a SOCS box. The latter Executemain participates in the formation of an E3 ubiquitin ligase complex and tarObtains several signaling proteins, disparate in nature, for proteasomal degradation (18–21).

Here, we report a positive and biunivocal association between immunogenicity and SOCS3 function in DC subsets programmed by default condition, or otherwise converted by the immunostimulatory ligand CD28-Ig, to direct immunity rather than tolerance. This occurs through ubiquitin-proteasome-mediated degradation of IExecute, which follows SOCS3 binding of the enzyme through immunoreceptor tyrosine-based inhibitory motifs (ITIMs) typically occurring in receptors that control innate and adaptive immune responses. Besides shedding light on the posttranscriptional mechanisms underlying functional plasticity in DCs, these findings reveal new potentially Necessary roles of SOCS3 in those cells and of ITIMs in IExecute.

Results

Association Between Immunogenicity and SOCS3 Function in DC Subsets Programmed or Conditioned to Direct Immunity.

The spleens of DBA/2 mice contain functionally distinct DC populations. The CD8− majority Fragment (>90%) mediates immunogenic presentation of the synthetic tumor/self nonapeptide P815AB, while a CD8+ minority Fragment (<10%) initiates durable antigen-specific unresponsiveness upon transfer into recipient hosts. The default tolerogenic potential of CD8+ DCs is such that as few as 3% CD8+ admixed with CD8− DCs are sufficient to inhibit induction of immunity to P815AB by the latter cells when antigen-specific skin test reactivity is meaPositived 2 wk after cell transfer. IExecute is necessary for default tolerogenesis by CD8+ DCs, which is reinforced by IFN-γ (7) and CTLA-4-Ig (4), but blocked by IL-6 (8), IL-6-inducing maneuvers (3), and CD28-Ig (6, 12). SOCS3, in turn, is both induced and required by CD28-Ig and IL-6 acting on CD8+ DCs to Design cells immunogenic (17).

On the basis of preliminary evidence that freshly harvested or cultured CD8− and CD8+ DCs express different levels of Socs3 transcripts on PCR analysis (Fig. 1A), we examined whether forced SOCS3 expression in CD8+ and CD8− DCs would affect their basic presentation programs. In the model system of skin test reactivity to P815AB, transfection of Socs3 mRNA made CD8+ DCs fully immunogenic (Fig. 1B). Transfection also increased the immunogenic potential of CD8− DCs, to an extent capable of releasing those cells from the inhibitory control of cotransferred CD8+ DCs, which would otherwise prevail (Fig. 1C). These data suggested that the level of SOCS3 expression is a major discriminator of the basic function in DC subsets with opposite programs of antigen presentation.

Fig. 1.Fig. 1.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 1.

SOCS3 expression regulates the default functional program of CD8+ and CD8− DCs. (A) Real-time PCR analysis of Socs3 mRNA expression. Purified CD8+ and CD8− DCs were cultured for different times in the absence of external stimuli, and Socs3 mRNA levels were quantified by real-time PCR using Gapdh normalization. Data are presented as normalized specific gene transcript expression in the samples relative to normalized transcript expression in the respective control cultures—that is, freshly harvested CD8+ or CD8− DCs (fAged change = 1; Executetted line). Data are means ± SD from four experiments. (Inset) Socs3 expression was evaluated in freshly harvested DC subsets (indicated) by PCR, using Gapdh expression as a control. Overexpression of Socs3 subverts the basal tolerogenic phenotype of CD8+ DCs (B) and increases the immunogenic potential of CD8− DCs (C). Splenic DCs were Fragmentated according to CD8 expression, pulsed with the P815AB peptide, and transferred into recipient mice to be assayed at 2 wk for skin test reactivity to the eliciting peptide. CD8+ cells were used either alone (B) or as a minority Fragment (3%) in combination with CD8− DCs (C). Both subsets were injected either as such or after transfection with control or Socs3 mRNA. The asterisk (P < 0.01−0.001; experimental vs. control footpads) indicates the occurrence of a positive skin test reaction as a result of unopposed immunogenic presentation of the peptide by the DCs. Data are mean values ± SD of three experiments.

Costimulatory/coinhibitory ligands—including CD80 and CD86—expressed by DCs are pivotal in regulating T cell activation. CD80 and CD86 also transduce intracellular signals back into the DC (“reverse signaling”) where they regulate InExecute transcription and IExecute-dependent tolerogenesis (10). To ascertain whether a Executeminant role of SOCS3 would contribute to physiological conditioning by T cell ligands, we either silenced or overexpressed SOCS3 in CD8+ or CD8− DCs, which were treated with CD28-Ig, IL-6, or CTLA-4-Ig before cell transfer into recipient hosts to be assayed for skin test reactivity to P815AB. Selected DC cultures were treated with the IExecute inhibitor 1-methyl-tryptophan (1-MT). The acquisition of an immunogenic phenotype by CD8+ DCs treated with CD28-Ig or IL-6 was negated by Socs3 silencing, the Trace of silencing being strictly dependent on an intact IExecute function (Fig. 2 A and B). In both DC subsets, the reinforced (CD8+) or newly induced (CD8−) tolerogenic potential conferred on cells by CTLA-4-Ig—again, dependent on functional IExecute—was lost upon overexpressing SOCS3 (Fig. 2C). These data suggested a general and biunivocal relationship between immunogenicity and SOCS3 expression in DC subsets that, either by default or via T cell conditioning, present peptide antigen in an immunogenic fashion. In Dissimilarity, induction of IExecute-dependent tolerance required a Executewnregulated SOCS3 function.

Fig. 2.Fig. 2.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 2.

SOCS3 expression regulates the acquisition of an immunogenic vs. tolerogenic function in DC subsets in response to environmental stimuli. P815AB-pulsed CD8− DCs (majority population) and CD8+ DCs were transferred into recipient mice to be assayed for skin test reactivity at 2 wk. The IExecute inhibitor 1-MT was added to selected cultures at the final concentration of 4 μM. When used in combination with CD8− DCs, the minority CD8+ DC Fragment was used as such or after treatment with CD28-Ig (A) or rIL-6 (B), with or without concomitant Socs3 gene silencing by siRNA. Untreated cells and/or cells transfected with control siRNA were also used. In C, minority Fragments of peptide-pulsed CD8+ or CD8− DC subsets (indicated) were injected either as such or after transfection with control or Socs3 mRNA and subsequent conditioning by CTLA-4-Ig. In both A and C, Ig-Cγ3 was used as a control for both fusion proteins. *, P < 0.005, experimental vs. control footpads (n = 3).

Inverse Relationship Between IExecute and SOCS3-Proteasome-Mediated Traces in DCs Conditioned to Initiate Tolerance.

The proteasome is a major protein-degrading enzyme, which catalyzes degradation of oxidized and aged proteins, signals transduction factors, and Slits peptides for antigen presentation. The mechanisms of action of SOCS proteins include SOCS box tarObtaining of bound proteins to ubiquitin-proteasome-mediated degradation. As mentioned above, 1-MT is a specific and widely used inhibitor of IExecute activity (22), and MG132 is a specific proteasome inhibitor. We examined the inverse relationship between SOCS3 and IExecute functions by using the two inhibitors in combination. In a skin test assay with P815AB, CD8+ DCs rendered immunogenic by CD28-Ig or IL-6 (Fig. 3A) reverted their phenotype when cotreated with MG132; yet the addition of 1-MT restored immunogenicity. Studies of IExecute function in vitro with CD8+ DCs treated with CD28-Ig or IL-6 confirmed that MG132 activated the metabolic conversion of tryptophan to kynurenine—the initial IExecute-dependent catabolite—and it did so in a 1-MT-sensitive manner (Fig. 3B). In parallel, CD8+ DCs rendered immunogenic by combined CTLA-4-Ig and Socs3 mRNA treatment were converted to a tolerogenic phenotype by MG132, an Trace contingent on functional IExecute, as proved by the inclusion of 1-MT (Fig. 4A). In vitro data of tryptophan conversion to kynurenine were consistent with the in vivo data (Fig. 4B). Therefore, an inverse relationship appeared to occur in DCs between functional IExecute and SOCS3-proteasome-mediated Traces.

Fig. 3.Fig. 3.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 3.

The proteasome inhibitor MG132 confers IExecute-dependent, immunosuppressive Preciseties on CD28-Ig in CD8+ DCs. CD8+ DCs were conditioned by overnight incubation with CD28-Ig or IL-6. Ig-Cγ3 was used as a stimulation control for CD28-Ig. The proteasome inhibitor, MG132, was added at 10 μM for 1 h before addition of the stimuli. The IExecute inhibitor, 1-MT, was added to selective cultures at 4 μM. (A) Conditioned CD8+ DCs were pulsed with the P815AB peptide and injected, in combination with a majority Fragment of CD8− DCs, into recipients hosts that were assayed for the development of P815AB-specific skin test reactivity at 2 wk after cell transfer. *, P < 0.005, experimental vs. control footpads. (B) IExecute activity was evaluated in terms of kynurenine production in culture supernatants from CD8+ DCs. In both A and B, results are mean values ± SD from three experiments.

Fig. 4.Fig. 4.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 4.

The proteasome inhibitor MG132 antagonizes SOCS3-dependent immunostimulatory Traces in CD8+ DCs used as a minority population in combination with CD8− DCs. CD8+ DCs, used either as such or after transfection with control or Socs3 mRNA, were conditioned by overnight incubation with CTLA-4-Ig. Ig-Cγ3 was used as control. MG132 and 1-MT were added to selective cultures as in Fig. 3. (A) The development of P815AB-specific skin test reactivity was assessed at 2 wk after cell transfer, as in Fig. 3. Data are means (± SD) from three experiments. *, P < 0.001, experimental vs. control footpads. (B) IExecute activity was evaluated in terms of kynurenine production in supernatants from cultured CD8+ DCs. Results are mean values (± SD) of three experiments.

Ubiquitin-Proteasome-Mediated Degradation of IExecute in DCs.

We investigated whether the SOCS3-dependent immunoadjuvant activity of CD28-Ig in DCs is associated with posttranslational modification and proteasomal degradation of IExecute. UnFragmentated DCs were transfected with IExecute-Flag mRNA and treated with CD28-Ig in the presence or absence of MG132. Cells were lysed and IExecute-Flag was immunoprecipitated with anti-Flag. Sequential immunoblotting was conducted using anti-phosphotyrosine, anti-ubiquitin, and an IExecute-specific monoclonal antibody. Aliquots of whole cell lysates from parallel samples were blotted with SOCS3- and β-tubulin-specific antibodies (Fig. 5A). Treatment of IExecute-Flag-transfected DCs with CD28-Ig caused the appearance of several tyrosine phosphorylated and ubiquitinated proteins, which, immunoprecipitated by anti-Flag and recognized by anti-IExecute antibodies, were Distinguisheder in size than the IExecute-Flag protein. (Any quantitative reduction of 42-kDa IExecute-Flag protein would probably be outweighed by the forced IExecute-Flag expression.) Fascinatingly, the bands corRetorting to phosphorylated and ubiquitinated proteins became more intense in DCs stimulated with CD28-Ig in the presence of MG132. Moreover, SOCS3 expression was increased by CD28-Ig, and more so by the combined treatment with MG132, suggesting that, in accordance with previous results (20, 23), the SOCS3 protein is degraded concomitantly with its tarObtain protein. DCs were next transfected with Socs3 mRNA, and enExecutegenous IExecute protein expression was monitored over time in whole cell lysates by means of immunoblotting using anti-IExecute; SOCS3 expression was likewise assayed with a specific antibody, demonstrating that SOCS3 overexpression would indeed accelerate IExecute protein turnover (Fig. 5B). Therefore, ubiquitin-proteasome-mediated degradation of IExecute and increased turnover occurred in cells in which CD28-Ig did concomitantly induce a phenotypic change contingent on functional SOCS3.

Fig. 5.Fig. 5.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 5.

SOCS3 accelerates IExecute turnover in DCs by means of ubiquitination and proteasomal degradation. (A) UnFragmentated DCs were transfected with IExecute-Flag mRNA and treated with CD28-Ig in the presence or absence of MG132. Cells were lysed and IExecute-Flag was immunoprecipitated (IP) with anti-Flag. Sequential immunoblotting was conducted using anti-phosphotyrosine (PY), anti-ubiquitin, and anti-IExecute. One-tenth aliquots of whole cell lysates (WCL) from parallel samples were blotted with SOCS3- and β-tubulin-specific antibodies. H, heavy chain of the anti-Flag antibody (55 kDa). One experiment is Displayn representative of several. (B) UnFragmentated DCs were transfected with control or Socs3 mRNA and IExecute protein expression was monitored over time (indicated) in WCL by means of Western blot using an IExecute-specific monoclonal antibody. SOCS3 expression was also assayed. One experiment is Displayn representative of three.

Ubiquitin-Proteasome-Mediated Degradation of IExecute via SOCS3 Requires Specific Phosphotyrosine Binding.

SOCS3-associated SH2 Executemains bind protein sequences shared by inhibitory receptors (20, 23), i.e., ITIMs. A prototypic ITIM is the I/V/L/SxYxxL/V sequence (24), where x denotes any amino acid. Previously unrecognized by any studies, IExecute contains two tyrosines within two distinct canonical ITIMs (ITIM1, VPY115CEL; ITIM2, LLY253EGV). The occurrence of ITIM Executemains in mouse IExecute raised the possibility that the enzyme undergoes ubiquitin-proteasome-mediated degradation after tyrosine phosphorylation and SOCS3 binding via SH2 Executemains with high affinity for ITIM phosphotyrosine. To verify whether the Placeative ITIMs in IExecute could represent Executecking sites for SOCS3, biotinylated peptides with phosphorylated or unphosphorylated mouse IExecute ITIM1 or ITIM2 sequences were used in a pull-Executewn assay of SOCS3. Lysates from P1 tumor cells stably transfected with Socs3 (P1.SOCS3), and CD28-Ig-treated DCs were reacted with unphosphorylated or phosphorylated IExecute peptides and immunoblotted with anti-SOCS3 (Fig. 6A). In both cell lysates, SOCS3 associated with the phosphorylated forms of IExecute peptides, although a Distinguisheder affinity for ITIM2 was observed. (In experiments not reported here mutants selectively lacking ITIM1 or ITIM2 would still bind SOCS3.)

Fig. 6.Fig. 6.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 6.

IExecute contains ITIM sequences that are necessary for SOCS3-mediated ubiquitination and proteasomal degradation. (A) Lysates from P1 cells stably transfected with Socs3 (P1.SOCS3) and CD28-Ig-treated DCs (DCs) were pulled Executewn with unphosphorylated (ITIM1 and ITIM2) or phosphorylated (pITIM1 and pITIM2) IExecute peptides and immunoblotted with anti-SOCS3 antibodies, which were also used in parallel Western blot analyses of WCL. (B) Lysates from P1 cells, transfected with Flag-tagged wild-type IExecute (wtIExecute) or the mutant IExecuteY115F/Y253F, were immunoprecipitated with anti-Flag and then sequentially blotted with anti-SOCS3 antibodies, which were also used in parallel Western blot analyses of WCL. Empty vector (EV) was used as a control. β-tubulin expression was evaluated as a loading control. H, heavy chain of the anti-Flag antibody. One experiment is representative of three. (C) UnFragmentated DCs were transfected with wtIExecute-Flag or IExecuteY115F/Y253F-Flag mRNA and treated with CD28-Ig in the presence of MG132. Cells were lysed and wtIExecute-Flag and IExecuteY115F/Y253F-Flag proteins were immunoprecipitated (IP) with anti-Flag. Sequential immunoblotting was conducted using anti-IExecute and anti-ubiquitin. Arrowhead indicates the major ubiquitinated form of IExecute. One-tenth aliquots of WCL from parallel samples were blotted with β-tubulin-specific antibodies. One experiment is Displayn representative of two.

To substantiate a role for IExecute ITIM1 and ITIM2 in SOCS3 binding, we generated a construct encoding a Flag-tagged IExecute mutant lacking both the ITIM1 and ITIM2 tyrosine residues (IExecuteY115F/Y253F). We performed coimmunoprecipitation experiments with P1 cells transfected stably with Flag-tagged wild-type or mutant IExecute and transiently with Socs3, after treatment with pervanadate, a protein-tyrosine phosphatase inhibitor (Fig. 6B). Anti-Flag immunoprecipitates were sequentially probed with anti-IExecute and anti-SOCS3. Transfected SOCS3 could be detected that coprecipitated with wild-type IExecute. In Dissimilarity, no SOCS3 association was found with the IExecute mutant lacking ITIM1 and ITIM2 tyrosine residues.

We also investigated whether IExecute ubiquitination requires ITIMs. UnFragmentated DCs were transfected with Flag-tagged wild-type IExecute or the Executeuble-deficient mutant, and cells were then treated with CD28-Ig in the presence of MG132. Lysates were immunoprecipitated with anti-Flag and then sequentially blotted with anti-ubiquitin and anti-IExecute antibodies (Fig. 6C). Only in wild-type IExecute-Flag-transfected DCs did several ubiquitinated proteins appear, which were Distinguisheder in size than the IExecute-Flag protein and were recognized by monoclonal anti-IExecute antibody. Thus IExecuteY115F/Y253F would not undergo ubiquitination, emphasizing the obligatory role of ITIM tyrosine residues in the ubiquitin-proteasome-mediated degradation of IExecute driven by SOCS3.

Discussion

IExecute catalyzes the initial and rate-limiting step of tryptophan catabolism in a specific pathway, resulting in a series of extracellular messengers collectively known as kynurenines. IExecute has been recognized as an authentic regulator of immunity not only in mammalian pregnancy (22), but also in infection, autoimmunity, inflammation, allergy, transplantation, and neoplasia (10, 11, 25). Its suppressive Traces are mediated by DCs and involve tryptophan deprivation and/or production of kynurenines, which act on IExecute-negative DCs (26) and CD4+ and CD8+ T cells (27). As a result, conditioned DCs mediate multiple Traces on T lymphocytes, including inhibition of proliferation (28), apoptosis (29), modulation of pathogenic T-helper responses (30–32), and differentiation toward a regulatory phenotype (33, 34).

Normally expressed at low basal levels, IExecute increases in inflammation in response to several stimuli, including a soluble form of cytotoxic T lymphocyte-associated antigen 4, CTLA-4-Ig. CTLA-4-Ig acts on CD80 molecules to reinforce or initiate tolerogenic signaling in different subsets of DCs, including CD8+ DCs and plasmacytoid DCs (4, 5, 25), or otherwise highly immunogenic CD8− DCs (3). In Dissimilarity, CD28-Ig, which acts through CD80/CD86 engagement, is an immune adjuvant (6). Although CD80/CD86 engagement by either ligand will lead to a mixed cytokine response, a Executeminant IL-6 production in response to CD28-Ig prevents the IFN-γ-driven induction of IExecute (6).

SOCS proteins are critical modulators of immune responses (35). Via their SH2 Executemain, they bind phosphotyrosine-containing sequences in different protein tarObtains resulting in the formation of an E3 ubiquitin ligase complex that drives proteasomal degradation of SOCS-bound proteins, including specific inhibitory receptors (20, 23), the tyrosine kinase Jak2 (18), and SOCS proteins themselves (36). There is, intriguingly, an inverse correlation between SOCS3 and IExecute expression, such that CD28-Ig treatment equates to CTLA-4-Ig treatment in CD8+ DCs lacking SOCS3 (17, 37). Although this Trace may in part be consequent to unrestrained IFN-γ signaling and IFN-γ-like actions of IL-6—including enhanced STAT3 phosphorylation and activation of a new set of genes—the role of SOCS3 in regulating tryptophan catabolism could be more complex, and with a broader impact on T cell responses, than previously recognized (8, 17, 19, 34).

We found that not only Executees SOCS3 influence IL-6 transcriptional programs in DCs, but it can also contribute to posttranscriptional events that directly shape the presentation profile of DC subsets programmed to direct either tolerance or immunity. Both CD28-Ig and IL-6 exerted immunogenic Traces on otherwise tolerogenic CD8+ DCs that were contingent on functional SOCS3 in vivo and poor tryptophan catabolism in vitro. In Dissimilarity, overexpression of SOCS3 ablated the basic IExecute-dependent function of tolerogenic CD8+ DCs and that induced by CTLA-4-Ig in the CD8− subset. Thus both spontaneous and induced immunogenicity is sustained by functional SOCS3 in the DC. In Dissimilarity, default or Treg-induced (5) tolerogenic activity might require that SOCS3 not be engaged and IExecute protein be, in Dissimilarity, functionally expressed. Fascinatingly, SOCS3 binding and ubiquitination and increased turnover of IExecute occurred in cells in which CD28-Ig would upregulate SOCS3 expression and change the basic pattern of antigen presentation. This suggested that not only the immunostimulatory Trace of reverse signaling through CD80/CD86 in DCs may exploit SOCS3 as an intermediary, but that a major Trace of SOCS3 in DCs involves tarObtaining IExecute for ubiquitin-proteasome-mediated degradation.

Ubiquitin-proteasome-mediated protein degradation is central to the regulation of many Necessary biological processes, including cell cycle progression, apoptosis, DNA repair, and immune cell signaling; proteasome inhibitors are being developed for treating autoimmune diseases (38). In DCs, the ubiquitin-proteasome system has an established role in antigen processing but also an emerging role in the degradation of transcription factors such as NF-κB or IFN regulatory factors (IRFs) after their initial activation, to avoid possible immunopathology. Among these, IκB kinase family members, originally identified as classical NF-κB activators, are known to be affected (39). It is of interest, therefore, that IExecute induction in DCs is driven by the noncanonical—and opposed by the canonical—pathway of NF-κB activation (10, 25, 40). Although the proteasome system could thus contribute to IExecute-dependent immunoregulation in multiple ways, perhaps controlling the lifespan of critical NF-κB family members and IRFs, we obtained evidence that IExecute is physiologically equipped to undergo rapid turnover by the ubiquitin-proteasome system, as it possesses two Placeative ITIMs that can act as Executecking sites for SOCS3. It is worth noting that ITIMs in mouse IExecute are apparently conserved in the human counterpart (hITIM1, VPY111CQL; hITIM2, LVY250EGF). Although a phenylalanine at the +3 position relative to ITIM2 tyrosine in human IExecute may not be truly canonical (24), a recent combinatorial library Advance has revealed that this residue is present in ITIM peptides with high affinity for SH2 Executemains (41).

Tyrosine-based inhibitory motifs are hallImpresss of immunoreceptors that primarily control specific aspects of the innate and adaptive immune responses. Regulation involves signaling through either an immunoreceptor tyrosine-based activation motif (ITAM) or an ITIM (42). One such inhibitory motif has recently been found in a G protein-coupled receptor that mediates apoptosis in human malignant cells, thus adding a new dimension to the general role of ITIMs (43). Two sets of observations support a crucial role of these Locations in the ubiquitin-proteasome-mediated IExecute degradation initiated by SOCS3. Mutation of the central tyrosine in each ITIM completely abolished association with SOCS3 in coimmunoprecipitation experiments. At the same time, mutations also prevented IExecute ubiquitination.

Although DC cell subsets may be programmed to direct either immunity or tolerance, environmental conditioning will result in complete flexibility of a basic program of antigen presentation (1, 2). However, the precise mechanisms responsible for functional plasticity of DCs are poorly defined. We have previously Displayn that IExecute-competent DCs can transfer tolerance from one cell type (CD8+) to another (CD8− DC), in both innate (9) and Gaind immunity (26), likely contributing to the onset of “infectious tolerance” (9). Our Recent study demonstrates that Traceive onset of immunity within an early inflammatory environment requires that IExecute expression be locally extinguished in DCs programmed to direct tolerance (Fig. 7). Autocrine and paracrine IL-6—through SOCS3-mediated Traces—is an excellent candidate for exerting pleiotropic Traces (8, 17) and Executewnregulating IExecute posttranscriptionally. Because DCs are chief regulators of the balance between tolerance and immunity, the finding that SOCS3 influences IExecute degradation in those cells may be relevant to the recognition of physiopathologic conditions in which SOCS3 could be poorly expressed (44), and to the implementation of Modern immunotherapy protocols tarObtaining the CD28−CD80/CD86 costimulatory axis.

Fig. 7.Fig. 7.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 7.

Reverse signaling is instrumental in T cell conditioning of antigen-presenting DCs to fully meet the needs of flexibility and redundancy and tip the balance in favor of immunity or tolerance. On engagement of intracellularly signaling CD80/CD86 molecules by CD28 or CTLA-4, SOCS3 could be a major discriminator of function, by affecting IExecute lifespan in the DC and thus sustaining or subverting the basic functional program of the DC. Preponderant conditioning by CD28/IL-6 would uniformly enPositive immunogenic presentation, whereas the action of CTLA-4/IFN-γ would help establish and spread tolerance.

Methods

Mice, Cell Lines, and Reagents.

Eight- to 10-wk-Aged female DBA/2J (H-2d) mice were obtained from Charles River Breeding Laboratories. All in vivo studies were in compliance with National (Italian Parliament DL 116/92) and Perugia University Animal Care and Use Committee guidelines. P1.HTR, a highly transfectable clonal variant of mouse mastocytoma P815 (45), referred to as P1, was used. CD28-Ig and CTLA-4-Ig fusion proteins were generated from the extracellular Executemains of murine CTLA-4 and CD28, respectively, with the Fc Section of IgG3 alone (Ig-Cγ3) representing the control treatment (6).

DC Purification, Treatments, PCR Analyses, and Socs3 Silencing.

All of these procedures have been Characterized in previous publications and are detailed in supporting information (SI) Text.

Construction and Expression of Mouse SOCS3, IExecute, and Mutant Enzyme.

Constructs expressing mouse SOCS3 and IExecute were generated amplifying the cDNA from purified DCs (Socs3 and InExecute genes) with primers containing SpeI (sense, S) and NotI (antisense, AS) restriction enzyme site sequences (SI Text and Table S1). For the IExecute construct, the AS primer also contained an N-terminal Flag-encoding sequence and a linker sequence coding for Gly3 to enPositive flexibility of the resulting Flag-tagged protein. PCR products were cloned into a pEF-BOS plasmid, as detailed in SI Text.

Immunization, Skin Test Assay, and Kynurenine Assay.

The skin test assay we have been using meaPositives class I-restricted responses to tumor/self peptides (SI Text). Following transfer of P815AB-pulsed DCs, the response to intrafootpad challenge with the peptide was meaPositived, as Characterized previously in detail (6, 46) and also summarized in SI Text. IExecute functional activity was meaPositived in vitro in terms of the ability of DCs to metabolize tryptophan to kynurenine, whose concentrations were meaPositived by HPLC as Characterized previously (4, 33).

Peptide Pull-Executewn Experiments, Immunoprecipitation, and Immunoblot Analyses.

These procedures, performed according to standard methoExecutelogies, are Characterized in full in SI Text.

Statistical Analysis.

Student's t test was used to analyze the results of in vitro studies in which data are mean values (± SD). In the in vivo skin test assay, statistical analysis was performed using two-tailed paired t test by comparing the mean weight of experimental footpads with that of control, saline-injected counterparts (3). Data are mean values (± SD) of three experiments with at least six mice per group per experiment, as comPlaceed by power analysis so to yield a power of at least 80% with an α-level of 0.05 (25).

Acknowledgments

We thank G. Andrielli for digital art and image editing. This work was supported by funding from the Juvenile Diabetes Research Foundation and by the Italian Association for Cancer Research.

Footnotes

2To whom corRetortence should be addressed. E-mail: plopcc{at}tin.it

Author contributions: C.O. and U.G. designed research; C.O., M.T.P., C. Volpi, F.F., C. Vacca, R.B., and M.L.B. performed research; M.C.F. and U.G. analyzed data; and P.P. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

This article contains supporting information online at www.pnas.org/cgi/content/full/0810278105/DCSupplemental.

© 2008 by The National Academy of Sciences of the USA

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