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. 2011 Sep 15;187(6):2966-73.
doi: 10.4049/jimmunol.1101643. Epub 2011 Aug 17.

Ubiquitination of CD86 is a key mechanism in regulating antigen presentation by dendritic cells

Günther Baravalle  1 , Hyesuk Park, Megan McSweeney, Mari Ohmura-Hoshino, Yohei Matsuki, Satoshi Ishido, Jeoung-Sook Shin
Affiliations

Ubiquitination of CD86 is a key mechanism in regulating antigen presentation by dendritic cells

Günther Baravalle et al. J Immunol. .

Abstract

Dendritic cells (DCs) require costimulatory molecules such as CD86 to efficiently activate T cells for the induction of adaptive immunity. DCs maintain minimal levels of CD86 expression at rest, but upregulate levels upon LPS stimulation. LPS-stimulated DCs produce the immune suppressive cytokine IL-10 that acts in an autocrine manner to regulate CD86 levels. Interestingly, the underlying molecular mechanism behind the tight control of CD86 is not completely understood. In this study, we report that CD86 is ubiquitinated in DCs via MARCH1 E3 ubiquitin ligase and that this ubiquitination plays a key role in CD86 regulation. Ubiquitination at lysine 267 played the most critical role for this regulation. CD86 is ubiquitinated in MARCH1-deficient DCs to a much lesser degree than in wild-type DCs, which also correlated with a significant increase in CD86 expression. Importantly, CD86 is continuously ubiquitinated in DCs following activation by LPS, and this was due to the autocrine IL-10 inhibition of MARCH1 downregulation. Accordingly, DCs lacking MARCH1 and DCs expressing ubiquitination-resistant mutant CD86 both failed to regulate CD86 in response to autocrine IL-10. DCs expressing ubiquitination-resistant mutant CD86 failed to control their T cell-activating abilities at rest as well as in response to autocrine IL-10. These studies suggest that ubiquitination serves as an important mechanism by which DCs control CD86 expression and regulate their Ag-presenting functions.

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Conflict of interest statement

Disclosures

The authors have no financial conflicts of interest.

Figures

FIGURE 1
FIGURE 1
CD86 is ubiquitinated in DCs. A, CD86 was immunoprecipitated (IP) from mouse BMDCs, treated with or without PNGase F, and immunoblotted (IB). HC indicates the H chain of anti-CD86 Ab. B, CD86−/− BMDCs were retrovirally transduced to express wild-type CD86 or CD86-EGFP fusion proteins. CD86 was immunoprecipitated (IP), treated with PNGase F, and immunoblotted (IB). C, Splenic DCs were isolated using CD11c magnetic beads. CD86 immunoprecipitates were treated and immunoblotted as described. Asterisk indicates a ladder of proteins reacting with anti-Ub Ab.
FIGURE 2
FIGURE 2
CD86 ubiquitination regulates CD86 expression in steady-state DCs. A, BMDCs cultured from CD86−/− mice were retrovirally transduced to express wild-type CD86 (WT) and CD86 mutant in which all of the five cytoplasmic lysines of CD86 were replaced with arginines (K > R). Each retroviral construct included internal ribosome entry site-EGFP cDNA downstream of the CD86 sequence. CD86 immunoprecipitates (IP) were treated with PNGase F and then immunoblotted (IB). Cell lysates were also immunoblotted. B, Flow cytometry analysis of DCs expressing WT CD86 and CD86 (K > R) mutant.
FIGURE 3
FIGURE 3
Ubiquitination via lysine 267 is sufficient for the regulation of CD86 in DCs. A, Amino acid sequence of the cytoplasmic domain of mouse CD86. Lysine residues (K) are numbered and highlighted in bold. B, Flow cytometry analysis of CD86−/− BMDCs expressing wild-type CD86 and a series of CD86 mutants. Wild-type CD86 is designated as KKKKK to indicate five lysines at the cytoplasmic sites numbered. Each of CD86 mutants is designated as a combination of K and R to indicate the specific site(s) where lysine is replaced with arginine (R). The relative surface expression of CD86 in the cells expressing CD86 wild-type and mutants is shown as mean fluorescence intensity (MFI). Data are representative of three independent experiments. C, Western blot analysis of CD86 immunoprecipitates and lysates prepared from DCs expressing wild-type CD86 (WT) or CD86 (K > R), CD86 (RKRRR), and CD86 (KRKKK) mutants.
FIGURE 4
FIGURE 4
MARCH1-mediated ubiquitination regulates CD86 in DCs. A, Flow cytometry analysis of BMDCs generated from wild-type (WT) mice (solid gray) or MARCH1−/− mice (full line). B, BMDCs cultured from WT or MARCH1−/− mice (M1KO) were processed for immunoprecipitation of CD86 followed by Western blot analysis. C, Flow cytometry analysis of surface CD86 on MARCH1−/− BMDCs transduced or untransduced with retrovirus encoding wild-type MARCH1 or mutant MARCH1 (I66A, W97A).
FIGURE 5
FIGURE 5
Lysine 267 is sufficient for the MARCH1-mediated regulation of CD86. A, Splenic B blasts generated from CD86−/− mice were transduced with two groups of retroviruses. One retroviral group encodes MARCH1, -2, or -8 (B–D) along with mCherry. The other group encodes wild-type CD86 or various CD86 mutants along with GFP. Surface levels of CD86 expression of mCherry+GFP+ cells are shown as mean fluorescence intensities (MFI). Data are representative of two independent experiments.
FIGURE 6
FIGURE 6
LPS-induced autocrine IL-10 facilitates CD86 ubiquitination and turnover in DCs. A, Flow cytometry of wild-type BMDCs following indicated treatment. B, Transcripts of CD86 in BMDCs following indicated treatments. Values are relative to actin mRNA, and data are depicted as fold difference of that observed in untreated BMDCs. Data represent the mean ± SEM of three independent experiments. C, Western blot analysis of CD86 immunoprecipitates and cell lysates generated from wild-type mice. D, The relative amounts of ubiquitinated CD86 are expressed as fold difference of that observed in LPS-treated BMDCs. Data represent individual values of three independent experiments and mean values. E, BMDCs were first treated as indicated and then surface biotinylated followed by a chase for 16 h at 37°C. CD86 was immunoprecipitated and analyzed as described earlier. F, The relative amounts of biotinylated CD86 are expressed as a percentage of the total amount of biotinylated CD86 at t = 0. Data are representative of two independent experiments.
FIGURE 7
FIGURE 7
Regulation of CD86 by autocrine IL-10 depends on MARCH1 expression. A, Transcripts of MARCH1 in BMDCs following indicated treatments. Values are relative to actin mRNA, and data are depicted as fold difference of that observed in untreated BMDCs. Data represent the mean ± SEM of three independent experiments. B, Flow cytometry of wild-type and MARCH1−/− BMDCs following indicated treatment. C, Western blot analysis of CD86 immunoprecipitates and cell lysates generated from MARCH1-deficient mice. *p < 0.05.
FIGURE 8
FIGURE 8
Control of CD86 expression by autocrine IL-10 requires CD86 ubiquitination. Flow cytometry of CD86−/− BMDCs transduced with retrovirus encoding wild-type CD86 (A), CD86 (K > R) (B), or CD86 (RKRRR) (C) and treated as indicated. Western blots of CD86−/− BMDCs expressing wild-type CD86 (D), CD86 (K > R) (E), or CD86 (RKRRR) (F) treated as indicated.
FIGURE 9
FIGURE 9
CD86 ubiquitination plays a significant role in controlling DC Ag-presenting functions. In vitro T cell activation assay using immature BMDCs (A) or BMDCs treated as indicated (B). BMDCs cultured from CD86−/− mice were retrovirally transduced to express wild-type CD86 or CD86 (K > R) mutant. DCs were loaded with increasing concentrations of the OVA-specific peptide, SIINFEKL, and cocultured with naive CD8+ T cells isolated from OT-I transgenic mice. Sixteen to 18 h later, IL-2 in the supernatant was determined by ELISA. Data, expressed as the mean ± SEM, are representative of five independent experiments.
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