Low doses of cholera toxin and its mediator cAMP induce CTLA-2 secretion by dendritic cells to enhance regulatory T cell conversion

Abstract

Immature or semi-mature dendritic cells (DCs) represent tolerogenic maturation stages that can convert naive T cells into Foxp3+ induced regulatory T cells (iTreg). Here we found that murine bone marrow-derived DCs (BM-DCs) treated with cholera toxin (CT) matured by up-regulating MHC-II and costimulatory molecules using either high or low doses of CT (CThi, CTlo) or with cAMP, a known mediator CT signals. However, all three conditions also induced mRNA of both isoforms of the tolerogenic molecule cytotoxic T lymphocyte antigen 2 (CTLA-2α and CTLA-2β). Only DCs matured under CThi conditions secreted IL-1β, IL-6 and IL-23 leading to the instruction of Th17 cell polarization. In contrast, CTlo- or cAMP-DCs resembled semi-mature DCs and enhanced TGF-β-dependent Foxp3+ iTreg conversion. iTreg conversion could be reduced using siRNA blocking of CTLA-2 and reversely, addition of recombinant CTLA-2α increased iTreg conversion in vitro. Injection of CTlo- or cAMP-DCs exerted MOG peptide-specific protective effects in experimental autoimmune encephalomyelitis (EAE) by inducing Foxp3+ Tregs and reducing Th17 responses. Together, we identified CTLA-2 production by DCs as a novel tolerogenic mediator of TGF-β-mediated iTreg induction in vitro and in vivo. The CT-induced and cAMP-mediated up-regulation of CTLA-2 also may point to a novel immune evasion mechanism of Vibrio cholerae.

Tolerogenic Transcriptional Signatures of Steady-State and Pathogen-Induced Dendritic Cells

“…Of note, LPS stimulated DCs produce immunogenic Th1-polarizing IL-12p70, formed by the p35/p40 heterodimer ( Il12a and Il12b genes), but the protein amounts of IL-12p40 secreted by DCs are typically 50–100 times higher than the amount measured for IL-12p70. Similarly, the IL-23 heterodimer secretion, composed of p19/p40 ( Il23a Il12b genes ) is much lower than p40 by cholera toxin stimulation of DCs ( 22 , 23 ). This opens space for speculation on a counterbalancing and thereby tolerogenic role for excessive IL-12p40 production.…”

“…Previously, we performed transcriptional profiling of murine GM-CSF generated BM-DCs and human MoDCs. Selected in vitro maturation protocols for induction of Th1 responses by LPS, Th2 by TNF and Th17 by cholera toxin (CT-DCs) were applied to both human and mouse DCs for the same time period of 6 h ( 22 , 23 ) (GEO data bases GSE106080). Among the clearly immunogenic transcriptomic signatures, we also identified additional molecules at the protein level that exert tolerogenic immune functions.…”

“…Among the clearly immunogenic transcriptomic signatures, we also identified additional molecules at the protein level that exert tolerogenic immune functions. These include IL-10 production by LPS-DCs ( 86 ), Tr1 induction by Trypanosoma-matured or TNF-DCs after three injections ( 22 , 87 ) and Foxp3 + Treg induction via TGF-β plus CTLA-2, a newly identified tolerogenic molecule from CT-DCs ( 23 ).…”

Seq-ing out the Killers of Mice and Men

“…Compared to blood, murine splenic NK cells highly expressed both Fos and Jun as well as many well-known NK effector molecules (Ccl3, Ccl4, Ifng, Xcl1, Prf1). Compared to splenic NKs, murine blood NK cells highly expressed Tgfb1 and Ctla2a, a molecule highly represented in placenta and shown to enhance regulatory T (Treg) cell differentiation when released by dendritic cells (Silva-Vilches et al, 2017). These findings suggest that NK cells may be under a selective pressure to maintain a more quiescent phenotype in circulation until they are recruited to the tissue by an alerting signal to verify the nature of the danger.…”

GM-CSF Monocyte-Derived Cells and Langerhans Cells As Part of the Dendritic Cell Family

“…More recent studies further dissected cDC2 into ESAM + cDC2 inducing Th17 and ESAM + cDC2 specialized to promote Th2 polarization ( 5 ). By contrast, in vitro -generated MoDCs (BM-DCs) appear more versatile in their functional adaptation to generate specific Th1, Th2, and Th17 responses in vitro or after injection into mice depending on their stimulation with LPS or TNF ( 75 ), cholera toxin ( 88 ), or in cross-presenting antigens to CD8 + T cells ( 89 ) or presenting glycolipids on CD1d molecules for polarizing NKT cells into either IFN-γ or IL-4 producing subtypes ( 90 , 91 ). However, indirect cross-priming and NKT cell priming by injected BM-DCs with endogenous spleen DC subsets has been observed ( 92 ), which may point to DC–DC cooperation as observed in lymph nodes after virus infection ( 93 ).…”