Cutting edge: 4-1BB controls regulatory activity in dendritic cells through promoting optimal expression of retinal dehydrogenase - PubMed (original) (raw)

Cutting edge: 4-1BB controls regulatory activity in dendritic cells through promoting optimal expression of retinal dehydrogenase

Seung-Woo Lee et al. J Immunol. 2012.

Abstract

Dendritic cells (DC) in the gut promote immune tolerance by expressing retinal dehydrogenase (RALDH), an enzyme that promotes retinoic acid, which aids differentiation of Foxp3+ inducible regulatory T cells (iTreg) in the intestinal mucosa. How RALDH expression is regulated is unclear. We found that 4-1BB (CD137), a member of the TNFR family, together with CD103, marked mesenteric lymph node DC with the highest level of RALDH activity, and ligation of 4-1BB maintained RALDH expression in these gut DC. Moreover, 4-1BB signals synergized with those through TLR2 or GM-CSFR to promote RALDH activity in undifferentiated DC. Correspondingly, 4-1BB-deficient mice were impaired in their ability to generate iTreg in the GALT when exposed to oral Ag, and 4-1BB-deficient mesenteric lymph node DC displayed weak RALDH activity and were poor at promoting iTreg development. Thus, our data demonstrate a novel activity of 4-1BB in controlling RALDH expression and the regulatory activity of DC.

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Figures

Figure 1

Figure 1. 4-1BB is expressed on mesenteric lymph node dendritic cells and correlates with RALDH activity

A, 4-1BB expression on gated CD11c+MHC class IIhi dendritic cells in mesenteric lymph nodes (MDC) and spleen (SDC) isolated from wt mice (8 weeks-old). Shaded histograms represent isotype control. B, MDC stained for CD103 vs. 4-1BB expression (top left). MDC gated into CD103+4-1BB- and CD103+4-1BB+ subsets (top right). MDC gated based on levels of CD103 and 4-1BB (1, orange, CD103lo4-1BB−; 2, green, CD103hi4-1BB−; 3, blue, CD103lo4-1BBlo; 4, red, CD103hi4-1BBhi) (bottom, left and right). RALDH activity was determined by ALDEFLUOR staining (top right, and bottom left and right). C, MDC were stained for 4-1BB, and divided into subsets based on 4-1BB levels (left). RALDH activity was assayed by ALDEFLUOR staining (right) with histograms representing gated subsets: Cyan, 4-1BB−; Orange, 4-1BBlo; Red, 4-1BBhi. Results are representative of three experiments.

Figure 2

Figure 2. 4-1BB is a signaling receptor for RALDH induction in DC

A, MLN DC, isolated by MACS, were cultured for 1 or 2 days in the absence of stimulation with control IgG, or stimulated with an agonist antibody to 4-1BB (25 μg/ml). Activity of RALDH was assayed by ALDEFLUOR staining. Percentages of ALDEFLUOR+ cells are indicated. B–F, MACS- isolated SDC were stimulated with GM-CSF and Zymosan for 48 h. Where indicated, agonist anti-4-1BB (25 μg/ml) or rat IgG was added at the start of culture. B, 4-1BB expression in SDC stimulated with GM-CSF (100 pg/ml) and Zymosan (25 μg/ml). C, Activity of RALDH in SDC was determined by ALDEFLUOR staining when treated with GM-CSF (10 pg/ml), Zymosan (2.5 μg/ml), and Pam-3-cys (1 μg/ml) for 48h. D–E, SDC, pre-treated with Zymosan in the presence of IgG or agonist anti-4-1BB for 48h, were cultured with naïve OT-II CD4 T cells and OVA peptide (0.01 μM) for 4 days. Where indicated, TGF-β (5 ng/ml) and LE540, the pan antagonist of RA receptor, (2 μM) were added. Treg/Th1 development was determined by intracellular staining after restimulation of cells with PMA and ionomycin. Representative flow plot (D) and % Foxp3+ T cells in individual cultures (E) are shown. F, Inhibitors for signaling pathways were added at the start of culture when SDC were stimulated with Zymosan (25 μg/ml). ERK, U0126 (5 μM); PI3K, Ly (5 μM); NFκB (1 μM); Wnt/β-Catenin (5 μM). Results are representative of three experiments.

Figure 3

Figure 3. Defective iTreg conversion to oral antigen in the GALT of 4-1BB-deficient mice

Naïve wt OT-II CD4 T cells (CD45.1+ and CD25-Foxp3−) were adoptively transferred into wt or 4-1BB−/− mice (CD45.2+) that were subsequently given OVA (1%) in the drinking water for five days. Lymphocytes from the indicated organs were analyzed on day 6 for the conversion of naïve OT-II cells into Treg by gating on CD45.1 and staining for intracellular Foxp3. A, concatenate histograms of four individual samples shown in spleen (top) and MLN (bottom). Percentages of Foxp3+ cells in CD4+ populations positive for CD45.1 are shown. B, Percentage of Foxp3+ cells within donor OT-II cells (CD45.1+) in each organ from individual mice. Results are representative of three experiments.

Figure 4

Figure 4. Defective regulatory function of MLN DC from 4-1BB−/− mice

A–B, Activity of RALDH in MDC or SDC from wt or 4-1BB−/− mice was determined by ALDEFLUOR staining. A, Representative RALDH activity displayed as histogram of ALDEFLUOR staining in gated CD11c+ class IIhi DC. B, RALDH activity in MDC from individual mice. Left, percent. Right, MFI. C–F, Naïve wt OT-II CD4 T cells (CD25-Foxp3−) were cultured for 4 days with OVA peptide (1 μM) presented on MDC isolated from wt or 4-1BB−/− mice. Cells were cultured without exogenous cytokines (C) or with TGF-β (5 ng/ml, D–F) for four days to determine default Th1 cell generation by intracellular staining of IFN-γ after restimulation of cells with PMA and ionomycin, and Treg differentiation by intracellular Foxp3 staining in CD4+ T cells, respectively. Percent positive indicated. E–F, Where indicated, cells were cultured with retinoic acid (RA, 100 nM) or neutralizing antibodies against IFN-γ and IL-12 (10 μg/ml) added at the start of culture. G, MDC from wt and 4-1BB−/− mice were stimulated as indicated for 24h. IL-12p70 production was measured by ELISA in culture supernatants. Results are representative of two (F–G) and three experiments (A–E), respectively.

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