SOCS3 drives proteasomal degradation of indoleamine 2,3-dioxygenase (IDO) and antagonizes IDO-dependent tolerogenesis

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 downstream 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, dominant IL-6 response-become immunosuppressive and mimic the effect of CTLA-4-Ig, including a high functional expression of the tolerogenic enzyme indoleamine 2,3-dioxygenase (IDO). Here we show that forced SOCS3 expression antagonized CTLA-4-Ig activity in a proteasome-dependent fashion. Unrecognized by previous studies, IDO appeared to possess two tyrosine residues within two distinct putative immunoreceptor tyrosine-based inhibitory motifs, VPY 115CEL and LLY253EGV. We found that SOCS3-known to interact with phosphotyrosine-containing peptides and be selectively induced by CD28-Ig/IL-6 -would bind IDO and target the IDO/SOCS3 complex for ubiquitination and subsequent proteasomal degradation. This event accounted for the ability of CD28-Ig and IL-6 to convert otherwise tolerogenic, IDO-competent DCs into immunogenic cells. Thus onset of immunity in response to antigen within an early inflammatory context requires that IDO 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 IDO undergoes regulatory proteolysis in response to immunogenic stimuli.CD28-Ig ͉ CD80/86 signaling ͉ IL-6 ͉ SOCS proteins ͉ tryptophan catabolism M urine 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 Ϫ and CD8 ϩ DCs that mediate the respective immunogenic and tolerogenic presentation of self peptides, we have previously shown that the activities of both subsets can be subverted by regulatory (Treg) or effector T cells (3). Otherwise immunogenic CD8 Ϫ DCs became tolerogenic upon CD80 ligation by soluble or cell-bound cytotoxic T lymphocyte antigen 4 (CTLA-4) (4, 5), a maneuver initiating indoleamine 2,3-dioxygenase (IDO)-dependent tryptophan catabolism. In contrast, CD28 ligation of CD80/CD86 on IDO-competent CD8 ϩ DCs made these cells capable of immunogenic presentation (6). While transcriptional control by cytokines (7-9) and T cells (10) will effectively result in long-term modulation of Indo (i.e., the gene encoding mouse IDO), the posttranscriptional and posttranslational events contributing to fine-tuning IDO to fully meet the needs of plasticity and redundancy have been unclear (11, 12).Suppressor of cytokine signaling (S...

Uptake of apoptotic DC converts immature DC into tolerogenic DC that induce differentiation of Foxp3+ Treg

“…Therefore, it is likely that RhoA induces PI3K which phosphorylates mTOR resulting in release of eIF4E, which further results in increased translation of TGF-b1 mRNA. Some studies have indicated that another mechanism whereby DC can acquire tolerogenic potential is through induction of IDO [28,29]. Our results show no upregulation of IDO upon uptake of apoptotic DC by viable DC, indicating that induction of IDO is likely not the underlying mechanism for tolerance induction (data not shown).…”

Distinct roles of immunoreceptor tyrosine‐based motifs in immunosuppressive indoleamine 2,3‐dioxygenase 1

“…Phosphorylated ITIMs allow IDO1 to associate with different molecular partners responsible for different downstream effects 7, 8. Because the known molecular partners of IDO1 ( i.e .…”

“…The same analysis was conducted on Y115F, Y253F and Y115_253F transfectants in which a phenylalanine (F) replaces the phosphorylable tyrosine of IDO1's ITIMs, abrogating a single or both docking sites of the enzyme (Fig. 3C) 7, 8. Using WCLs from the same transfectant cells in combination with IDO1‐specific antibody, we performed immunoprecipitation experiments to obtain IDO1 precipitates to be assayed by immunoblotting for their relative amounts of molecular partners ( i.e .…”

“…All purification procedures of splenic Ido1 −/− pDCs and CD8 − DCs (hereafter referred to as cDCs, for conventional DCs) were as described 7, 8, 22, 23, 24, 25. Plasmid constructs coding for WT or IDO1 mutants (Y115E, Y253E and Y115_253E, in which either one or both phosphorylable tyrosines were replaced by a glutamate residue) were used as templates for in vitro transcription of mRNA, using the mMESSAGE mMACHINE T7 Ultra Kit (Ambion, Waltham, MA USA).…”

“…Plasmid constructs coding for WT or IDO1 mutants (Y115E, Y253E and Y115_253E, in which either one or both phosphorylable tyrosines were replaced by a glutamate residue) were used as templates for in vitro transcription of mRNA, using the mMESSAGE mMACHINE T7 Ultra Kit (Ambion, Waltham, MA USA). In vitro transcribed‐mRNA (2 μg) was transfected into Ido1 −/− pDCs by means of DOTAP (Roche, Basel, Switzerland), as described 7, 13. Control treatments consisted of Ido1 −/− pDCs transfected with a control mRNA supplied by the manufacturer (Ambion).…”