Plasmacytoid dendritic cells from mouse tumor-draining lymph nodes directly activate mature Tregs via indoleamine 2,3-dioxygenase
Madhav D. Sharma, … , Andrew L. Mellor, David H. Munn
Madhav D. Sharma, … , Andrew L. Mellor, David H. Munn
Published September 4, 2007
Citation Information: J Clin Invest. 2007;117(9):2570-2582. https://doi.org/10.1172/JCI31911.
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Research Article Oncology

Plasmacytoid dendritic cells from mouse tumor-draining lymph nodes directly activate mature Tregs via indoleamine 2,3-dioxygenase

Abstract

A small population of plasmacytoid DCs (pDCs) in mouse tumor-draining LNs can express the immunoregulatory enzyme indoleamine 2,3-dioxygenase (IDO). We show that these IDO+ pDCs directly activate resting CD4+CD25+Foxp3+ Tregs for potent suppressor activity. In vivo, Tregs isolated from tumor-draining LNs were constitutively activated and suppressed antigen-specific T cells immediately ex vivo. In vitro, IDO+ pDCs from tumor-draining LNs rapidly activated resting Tregs from non–tumor-bearing hosts without the need for mitogen or exogenous anti-CD3 crosslinking. Treg activation by IDO+ pDCs was MHC restricted, required an intact amino acid–responsive GCN2 pathway in the Tregs, and was prevented by CTLA4 blockade. Tregs activated by IDO markedly upregulated programmed cell death 1 ligand 1 (PD-L1) and PD-L2 expression on target DCs, and the ability of Tregs to suppress target T cell proliferation was abrogated by antibodies against the programmed cell death 1/PD-L (PD-1/PD-L) pathway. In contrast, Tregs activated by anti-CD3 crosslinking did not cause upregulation of PD-Ls, and suppression by these cells was unaffected by blocking the PD-1/PD-L pathway. Tregs isolated from tumor-draining LNs in vivo showed potent PD-1/PD-L–mediated suppression, which was selectively lost when tumors were grown in IDO-deficient hosts. We hypothesize that IDO+ pDCs create a profoundly suppressive microenvironment within tumor-draining LNs via constitutive activation of Tregs.

Authors

Madhav D. Sharma, Babak Baban, Phillip Chandler, De-Yan Hou, Nagendra Singh, Hideo Yagita, Miyuki Azuma, Bruce R. Blazar, Andrew L. Mellor, David H. Munn

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Figure 1

Treg activation by DCs from TDLNs.

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Treg activation by DCs from TDLNs. (A) Contralateral LNs and TDLNs from ...
(A) Contralateral LNs and TDLNs from mice with B16F10 and B78H1–GM-CSF tumors (day 7–11). B16-OVA tumors were identical to B16F10. Red color identifies IDO by immunohistochemistry. One representative of 3–6 experiments per cell line. Original magnification, ×200. (B) TDLNs and contralateral LNs were stained for CD4 and intracellular Foxp3. Numbers indicate quadrant percentages. Representative of 6 experiments using B16-OVA and B78H1–GM-CSF. (C) Tregs (CD4+CD25+) from TDLNs and contralateral LNs were sorted and added to readout assays, which were comprised of 1 × 105 A1 T cells plus CBA DCs plus H-Y peptide. Proliferation (incorporation of [3H]thymidine deoxyribose, [3HTdR]) is shown for a representative experiment. In all similar figures, the ratio of Tregs to A1 cells is shown below the axis (bars show SD of replicate wells). The lower graph shows data from 8 independent experiments using the tumor types shown (cpm were normalized to the proliferation in control assays receiving no Tregs, to permit comparison across experiments). (D) CD11c+ DCs were harvested from TDLNs, pulsed with OVA peptide, and injected subcutaneously into recipient mice preloaded with OT-I. One group of mice received implantable sustained-release 1MT pellets at 5 mg/day (IDO blocked), while the other received vehicle control pellets (IDO active). After 4 days, the LNs draining the site of DC injection were harvested and the Tregs sorted and tested in vitro for spontaneous suppressor activity in readout assays (A1 T cells + CBA DCs). Representative of 3 experiments; bars show SD of replicate wells.