IDO Activates Regulatory T Cells and Blocks Their Conversion into Th17-Like T Cells The Journal of Immunology 주 지 민

Introduction TLR 은 infectious microorganisms 에 대한 host defense 중요한 역할. Unmethylated CpG motif 를 포함하는 oligonucleotides 는 TLR9 에 binding 하여 pDC 가 성숙하고 IFN-α 와 IL-6 와 같은 proinflammatory cytokines 을 내놓으며, T cell 을 자극. TLR ligands 는 Tregs 에 의한 suppression 을 억제. IL-6 + TGFβ + IL-23 : naïve CD4 + T cells => effector Th17 T cells

Introduction Paradoxically, some pathogen 의 TLR ligands 는 T cell 반응을 억제. 가능한 mechanism 은 IDO activity 이며, immune counterregulation 에 관여. IDO 를 발현하는 pDC 는 T cell 반응을 억제하며, naïve T cell 을 Treg 으로 분 화시키는 T cell 조절 능력을 가짐. 유도된 IDO 는 TLR ligands 의 proinflammatory effects 를 억제 가능. High dose 의 CpG 를 mice 에 주입하면, splenic CD + 19 pDC 가 IDO 를 발현하 고 T cell suppressor 기능.

The functions of IDO

CpG-induced IDO activates splenic Tregs ? High-dose CpG rapidly activate resting splenic Tregs A -CBA mice were treated with CpGs (50 ㎍ i.v.) -CpGs (●) or control ODNs(○) -FACS-sorted splenic Tregs T cells (BM3) + APCs from CBK-transgenic mice B ->Tregs suppressor activity is not activated during readout assay itself -B6 mice treated with CpGs (100 ㎍ i.v.) -MACS-enriched Tregs responder T cells(A1) + female CBA APCs + cognate (H-Y) peptide C -CpG-treated B6 mice (100 ㎍ i.v.) -FACS-sorted Tregs +A1 responders and CBA APCs -absence (●) or presence (○) of a mixture of anti-PD1, anti-PD-L1, and anti-PD-L2 Abs : suppression

IDO was required for CpG-induced Treg activation? IDO is essential for Treg activation following high-dose CpG treatment -50 ㎍ of CpGs (i.v.) or control ODNs -FACS-sorted splenic Tregs (5000/well) responder BM3 T cells + APCs from CBK mice A CBA or IDO1-KO mice were used as sources of Tregs B Tregs from mice pretreated for 48 h with pellets containing vehicle alone or pellets impregnated with IDO inhibitor (D-1MT) before CpG treatment : suppression

Requirements for TLR9-mediated Treg activation? IFN type I and GCN2 signaling is essential for CpG-mediated Treg activation Sorted splenic Tregs from CpG treated (24 h) mice + A1 T cell with or without PD- 1/PD-L blocking mAbs : No suppression

IDO-activated Tregs inhibited effector T cell responses ? Thy1.2 IDO-activated Tregs suppress alloreactive T cell responses elicited in vivo A.Sorted Tregs from donor mice + MACS-enriched CFSE-labeled OT-1 (Thy1.1) CD8 + T cells => Act- mOVA recipients. B.OT-1 T cell only -> B6 C.OT-1 T cell only -> Act-mOVA D.Untreated B6 Tregs + OT-1 T cell -> Act-mOVA E.CpG-treated B6 Tregs + OT-1 T cell -> Act-mOVA F.CpG-treated IDO1-KO B6 Tregs + OT-1 T cell -> Act-mOVA G.Untreated B6 Tregs H.CpG-treated IDO1-KO B6 Tregs suppression : Polyclonal expansion

IDO attenuated IL-6 expression in mice treated with high CpG doses? Induced IDO activity blocks IL-6 production after high-dose CpG treatment suppressed Not suppressed A -Spleen sections from CpG-treated B6 and IDO1-KO mice were stained with anti-IDO and anti-IL-6 Abs 24 h after injecting CpGs B -Splenic pDCs (gated CD11c + B220 + cells) from CpG-treated B6 (left) and IDO1-KO (right) mice were analyzed to detect intracellular IL-6 expression

CpG-induced IL-6 expression in IDO-deficient mice reprogrammed Tregs to express IL-17? CpG treatment reprograms splenic Tregs to express IL-17 in the absence of IDO A & B : Analyses of total splenocytes C : Analyses of gated CD4 cells => CpG treatment in the absence of IDO stimulated selective IL-17 expression in Tregs (only CD4 + CD25 + ) D :Analyses of total splenocytes from Foxp3 GFP mice treated with CpGs. E : FACS-sorted GFP cells from Foxp3GFP donors -> B6 and IDO1-KO recipients

IL-6 is required to reprogram Tregs into T cells expressing proinflammatory cytokines in the absence of IDO? Treg reprogramming to express IL-17 is dependent on CpG dose and IL-6 => IL-6 is essential for IL17 induction in Tregs when IDO is inactive # : fluorescence intensities % proportions of expressing cytokines

Discussion IDO is the molecular switch.  Active : dominant Treg-mediated suppression  Inactive : CpG-induced inflammatory stimuli drive IL-6-dependent Treg conversion into Th17-like T cells PD-1 pathway is critical for Treg differentiation in vivo. CpG-induced Treg activation of the spleen was also dependent on intact IFNAR signaling, but not IFN-γR signaling.

Discussion IDO-mediated inhibition of IL-6 expression  IDO may directly suppress up-regulation of IL-6 gene expression (Metz) IL-6 + IL-1 + IL-23 reprogram Foxp3 lineage-committed Tregs to express IL- 17 Chronic or excessive IDO => local immune suppression & privilege by blocking innate T cell immunity to pathogens and tumor Ags. Deficiency in IDO => autoimmunity

What I learned from this study

CpG 를 low dose 로 주입하면 TLR9 ligation 이 일어나 IL-6 가 발현. CpG 를 high dose 주입하면 splenic CD + 19 pDC 에서 IDO 가 발현. IL-6 는 IL-1, IL-23 와 함께 Foxp3 lineage Tregs 을 IL-17 을 발현하는 Th17-like cells 로 전환시킨다. IDO 는 IL-6 발현을 억제하고, Tregs 활성화 시켜 T cell response 를 억 제시키고, Tregs 의 Th17 like cells 로 전환을 억제시킨다. IDO 의 응용 ? ->GVHD, GVL, Cancer therapy, Vaccination, Autoimmune disease, etc