CD55 Is Essential for CD103+ Dendritic Cell Tolerogenic Responses that Protect against Autoimmunity

Abstract

Recent studies traced inflammatory bowel disease in some patients to deficiency of CD55 [decay-accelerating factor (DAF)], but the mechanism underlying the linkage remained unclear. Herein, we studied the importance of DAF in enabling processes that program tolerance in the gut and the eye, two immune-privileged sites where immunosuppressive responses are continuously elicited. Unlike oral feeding or ocular injection of ovalbumin in wild-type (WT) mice, which induced dominant immune tolerance, identical treatment of DAF^-/- mice or DAF^-/- to WT bone marrow chimeras did not. While 10% to 30% of mesenteric and submandibular lymph node CD4⁺ cells became robust T-regulatory cells (Tregs) in WT forkhead box P3 (Foxp3)-green fluorescent protein mice, few in either site became Tregs with little suppressor activity in DAF^-/- Foxp3-green fluorescent protein mice. Phenotyping of CD103⁺ dendritic cells (DCs) from the ovalbumin-fed DAF^-/- mice showed impaired expression of inducer of costimulation (ICOS) ligand, programmed death receptor 1-ligand 1 (PD1-L1), CxxxC chemokine receptor 1 (Cx3CR1), CCR7, and CCR9. Analyses of elicited DAF^-/- Foxp3⁺ Tregs showed reduced expression of interferon regulatory factor 8 (IRF-8)/aldehyde dehydrogenase 1 family member A2 (Aldh1a2) and glycoprotein A repetitions predominant/latency-associated protein associated with Treg transforming growth factor-β production and presentation, as well as integrin β6/integrin β8 associated with Treg and CD103⁺ DC transforming growth factor-β release. Thus, DAF is required for the properties of CD103⁺ DCs and their naïve CD4⁺ cell partners that together program tolerance.

Figure 1

Decay-accelerating factor (DAF) is required for the ocular tolerogenic response to an anterior chamber (a.c.) antigen. A:Daf1^+/+ and Daf1^–/– mice were preinjected in the a.c. with 50 μg ovalbumin (OVA) or phosphate-buffered saline (PBS). Seven days later, the mice were challenged subcutaneously with 12.5 μg OVA in IFA in the right leg and with PBS in IFA in the left leg; and 48 hours later, delayed-type hypersensitivity (DTH) size was measured. B: Representative DTH lesions from wild-type (WT) and Daf1^–/– mice. C: Splenocytes (6 × 10⁵) from Daf1^+/+ and Daf1^–/– mice preinjected a.c. with OVA or with PBS (7 days earlier, as in the above protocol) were assayed on interferon (IFN)-γ ELISPOT plates in the presence of a varying amount of whole OVA protein or OVA_323-339 peptide. Arrows point to the assays with peptide rather than the whole protein. D: Lymph node cells [effectors (E)] from mice preinjected a.c. with OVA or PBS were mixed with OVA-transfected EL-4 targets at the designated effector to target (E/T) ratios. Percentage cytolysis was determined using Alamar blue. E:Daf1^+/+ and Daf1^–/– mice were preinjected in the a.c. with OVA or PBS and challenged with OVA in CFA subcutaneously at day 7 (as in A–C). Sera harvested at day 17 were assayed for anti-OVA antibody isotypes by isotype enzyme-linked immunosorbent assays. F: IFN-γ ELISPOT assays were performed on splenocytes from groups of Daf1^+/+ and CD97^–/– mice 7 days after a.c. preinjection with OVA or PBS. G and H: IFN-γ ELISPOT (G) and cytotoxicity (H) assays were performed on splenocytes from WT bone marrow (BM) → WT recipients (left panels) or Daf1^–/– BM → WT recipients (right panels) on day 7 after s.c. injection with OVA (day 17 after a.c. injection). I: (F12) Daf1^–/– and (F12) Daf1^–/–C3ar1^–/–C5ar1^–/– mice were studied using the protocol described in F. Data are shown as ratios of IFN-γ–producing cells by OVA a.c. protected mice normalized to PBS a.c. protected controls. n = 6 (A and F); n = 3 per group (G–I). ^∗P < 0.05, ^∗∗P < 0.01; ^†P < 0.05 versus Daf1^–/–. Original magnification, ×40 (B).

Figure 2

Ocular tolerance requires immunosuppression of the decay-accelerating factor (DAF)–dependent regulatory T cell (Treg). A: Ocular explants from anterior chamber (a.c.)–preinjected mice were placed in RPMI 1640 medium for 48 hours. Supernatants were assayed for transforming growth factor (TGF)-β by enzyme-linked immunosorbent assay (ELISA). B: Wild-type (WT) or Daf1^–/– mice were preinjected a.c. with ovalbumin (OVA) or phosphate-buffered saline (PBS), followed by s.c. challenge with OVA in IFA in the right leg 7 days later. Splenocytes were cultured at 37°C for 48 hours with 100 μg/mL of OVA, and culture supernatants were assayed for TGF-β1, IL-10, and interferon (IFN)-γ by ELISAs. C: Wild-type (WT) or Daf1^–/– mice were preinjected a.c. with OVA or PBS, followed 7 days later by s.c. challenge with OVA in IFA in the right leg. After another 7 days, CD4⁺ cells from draining submandibular lymph node (smLN) and from the spleen were isolated. Sorted Foxp3⁺ cells from each site were incubated for 5 days in increasing ratios with mixtures of dendritic cells (DCs), OVA_323-339, and OT-II cells (prelabeled with CellTracker Red), after which suppression of proliferation was assessed by determining percentage dividers. D: A total of 4 × 10⁶ sorted green fluorescent protein (GFP)^– CD4⁺ cells from (Thy1.2) WT or Daf1^–/– Foxp3-GFP mice were injected intravenously into WT Thy1.1 mice. Two days thereafter, the mice were preinjected a.c. with OVA or PBS and challenged 7 days later with OVA in IFA subcutaneously in the right leg. Seven days after the s.c. challenge, Foxp3⁺ Thy1.2⁺ cells in the spleen were quantified by flow cytometry. E: Foxp3⁺ cells from D were sorted, and the sorted cells were incubated for 5 days at a 4:1 ratio with mixtures of DCs, OVA_323-339, and OT-II cells (prelabeled with CellTracker Red). Suppression of proliferation was assessed by determining percentage dividers. n = 3 (C and E); n = 6 GFP^– CD4⁺ cells from (Thy1.2) WT or Daf1^–/– Foxp3-GFP mice and Foxp3⁺ Thy1.2⁺ cells in the spleen (D). ^∗P < 0.05, ^∗∗P < 0.01.

Figure 3

Oral tolerance requires immunosuppression of decay-accelerating factor (DAF)–dependent regulatory T cell (Treg). A–E: Data are organized showing wild-type (WT) versus Daf1^–/– mice on the left, and for side-by-side comparison, C3ar1^–/–C5ar1^–/– mice on the right. WT, Daf1^–/–, and C3ar1^–/–C5ar1^–/– OT II mice were fed 20 mg/mL ovalbumin (OVA) or phosphate-buffered saline (PBS) in their drinking water for 5 days, after which mesenteric lymph nodes (mLNs) were isolated and CD4⁺ and CD11c⁺ cells were separated via sorting. A: Percentage of CD4⁺ cells that were CD25⁺ Foxp3⁺ in the initial isolate were quantified by flow cytometry. B and C: After overnight culture of the remainder of the cells with OVA (100 μg/mL), amounts of transforming growth factor (TGF)-β1 (B) and interferon (IFN)-γ (C) released into culture supernatants were assayed by enzyme-linked immunosorbent assays (ELISAs). D: In other animals, delayed-type hypersensitivity responses were measured 48 hours after OVA injection into the footpad. E: Sorted CD25⁺ cells that were Foxp3⁺ in other aliquots from A were incubated for 5 days at increasing T-effector (Teff)/Treg ratios with mixtures of dendritic cells (DCs), OVA_323-339, and WT OT-II cells prelabeled with CellTracker Red. Suppression of CellTracker Red–labeled OT-II cell proliferation was assessed by quantifying percentage dividers. F–I: Sorted Foxp3^– OT-II cells from WT, Daf1^–/–, and C3ar1^–/–C5ar1^–/– OT-II Foxp3–green fluorescent protein mice were adoptively transferred into Thy1.1 recipients. The Thy1.1 recipients were fed 20 mg/mL OVA or an equal volume of PBS in their drinking water for 5 days and immunized subcutaneously with OVA in IFA in the hind leg. Seven days later, mLNs were harvested and the cells were isolated. F: Percentage of CD4⁺ cells that were Foxp3⁺ CD25⁺ in the initial mLN isolate were quantified by flow cytometry. G and H: After 48 hours of culture of the remainder of the cells with OVA, the amounts of TGF-β1 and IFN-γ released into the supernatants were quantified by ELISAs. I: Sorted Foxp3⁺ cells in the initial isolate (F) were incubated for 5 days with mixtures of DCs, OVA_323-339, and sorted WT OT-II cells (prelabeled with CellTracker Red) at varying ratios. Suppression of proliferation was assessed by quantifying percentage dividers. n = 6 mice for each genotype, OVA group, and PBS group (F–I); n = 12 for each recipient group (F–I). ^∗P < 0.05, ^∗∗P < 0.01, and ^∗∗∗P < 0.001.

Figure 4

Decay-accelerating factor (DAF) is required for the tolerogenic properties of CD103⁺ dendritic cells (DCs) and regulatory T cells. A–C: CD11c⁺ DCs harvested from the eyes (A), draining submandibular lymph nodes (smLNs; B), and mesenteric lymph nodes (mLNs; C) of wild-type (WT), Daf1^–/–, and Daf1^–/–C3ar1^–/–C5ar1^–/– mice were assayed for CD40, B7.1, B7.2, major histocompatibility complex (MHC) II, PD-L1, and ICOS-L by flow cytometry (Daf1^–/– versus WT and Daf1^–/–C3ar1^–/–C5ar1^–/– versus WT). D–F: CD11b⁺F4/80⁺ macrophages harvested from the eyes (D), draining smLNs (E), and mLNs (F) of WT, Daf1^–/–, and Daf1^–/–C3ar1^–/–C5ar1^–/– mice were assayed for the same markers. Cells were also assayed for C5ar1 and C5L2. The fact that differences in ICOS-L levels between Daf1^−/− and WT cells reach statistical significance reflects mouse numbers as other studies in the laboratory (M.G.S., J.L., F.A., and M.E.M., unpublished data) reproducibly have documented statistical significance. G and H: Human plasmacytoid DCs (pDCs; G) and CD4 T cells (H) were treated with siRNA targeting DAF or C3ar1 and C5ar1 for 72 hours, after which expression of CD40, B7.1, B7.2, CCR7, CCR9, PD-L1, and ICOS-L on pDCs (G) and expression of CD28, CD40 ligand, CCR7, CCR9, PD-1, and ICOS on CD4⁺ T cells (H) was assessed by flow cytometry. n = 10 per group (A–F); n = 3 (G and H). ^∗∗P < 0.01. MFI, mean fluorescent intensity.

Figure 5

The expression of T-cell and dendritic cell (DC) decay-accelerating factor (DAF) regulates cytokine and surface protein expression. A: A total of 1 × 10⁶ CD11c⁺ cells from the submandibular lymph node (smLN) of wild-type (WT), Daf1^–/–, and Daf1^–/–C3ar1^–/–C5ar1^–/– mice were incubated in RPMI 1640 media for 24 hours, after which IL-10 and transforming growth factor (TGF)-β1 were assayed by internal staining. B: A total of 2.5 × 10⁵ WT dendritic cells (DCs) were incubated for 24 hours with 1 × 10⁶ CD4⁺ T cells from WT, Daf1^–/–, and Daf1^–/–C3ar1^–/–C5ar1^–/– mice and anti-CD3/28 (1 μg/mL each), after which IL-10 and TGF-β1 were assayed by enzyme-linked immunosorbent assays. C: A total of 2.5 × 10⁵ WT DCs were incubated for 24 hours with 1 × 10⁶ WT, Daf1^–/–, and Daf1^–/–C3ar1^–/–C5ar1^–/– CD4⁺ T cells and anti-CD3/28, after which B7-1, B7-2, CD40, PD-L1, and ICOS-L expression levels on the DCs were assayed by flow cytometry (Supplemental Figure S2B). D: A total of 1 × 10⁶ CD62L^hiCD25^– CD4⁺ cells from WT, ICOS^–/–, or PD-1^–/– mice were incubated for 3 days with 1 × 10⁵ CD11c⁺ WT DCs + anti-CD3/28 (1 μg/mL each) or IL-2 and C3aR-A/C5aR-A (10 μg/mL each), after which percentage of Foxp3⁺ cells was assayed by intracellular staining. E: A total of 1 × 10⁵ CD11c⁺ WT or PD-L1^–/– DCs were incubated for 3 days with 1 × 10⁶ CD62L^hiCD25^– CD4⁺ cells from WT mice + anti-CD3/28 (1 μg/mL each) or IL-2 and C3aR-A/C5aR-A (10 μg/mL each), after which percentage Foxp3⁺ cells was assayed by intracellular staining. F: A total of 1 × 10⁶ sorted Daf1^−/−, WT, and C3ar1^–/–C5ar1^−/− Foxp3-GFP CD4⁺ cells were incubated for 2 hours without or with anti-CD3/CD28 Dynabeads in the presence of IL-2 and Shp-1, and Shp-2 mRNA level was quantitated by real-time quantitative PCR. The results are shown in comparison to WT cells set as 100%. As in the above studies, each group contained three mice and experiments were repeated three times. n = 6 (A and C); n = 5 (B). ^∗∗P < 0.01. KO, knockout; MFI, mean fluorescent intensity.

Figure 6

CD103⁺ dendritic cell (DC) expression of decay-accelerating factor (DAF) is required for tolerance. A: CD11c⁺ CD103⁺ DCs were harvested from mesenteric lymph nodes (mLNs) of wild-type (WT) and Daf1^–/– Foxp3–green fluorescent protein mice and assayed for expression of CD40, B7.1, B7.2, ICOS-L, and PD-L1 by flow cytometry. B: CD11c⁺CD103⁺ DCs harvested from mesenteric LNs of WT, Daf1^–/–, and C3ar1^–/–C5ar1^–/– mice were assayed for expression of CxCR3, CCR7, and CCR9 by flow cytometry. C: CD11c⁺CD103⁺ DCs were harvested from submandibular lymph nodes (LNs) of WT, Daf1^–/–, and C3ar1^–/–C5ar1^–/– mice and assayed for expression of CD40, B7.1, B7.2, ICOS-L, and PD-L1 by flow cytometry. D: CD11c⁺CD103⁺ DCs harvested from submandibular LNs of WT, Daf1^–/–, and C3ar1^–/–C5ar1^–/– mice were assayed for expression of CxCR3, CCR7, and CCR9 by flow cytometry. E: A total of 2.5 × 10⁵ WT DCs were incubated for 24 hours with 1 × 10⁶ CD4⁺ T cells from WT and Daf1^–/–C3ar1^–/–C5ar1^–/– mice and anti-CD3/28 (1 μg/mL each), after which cells were assayed for TGFB2, ALDH1A2, and IRF8 mRNA expression by real-time quantitative PCR (qPCR). F: Foxp3⁺ CD4⁺ T cells from WT, Daf1^–/–, and C3ar1^–/–C5ar1^–/– mice fed ovalbumin or phosphate-buffered saline were assayed for glycoprotein A repetitions predominant (GARP) expression by flow cytometry. G: Sorted Foxp3^– CD4⁺ T cells from WT, Daf1^–/–, and C3ar1^–/–C5ar1^–/– mice were activated for 3 days with anti-CD3/28 Dynabeads + C3aR-A/C5aR-A (10 μg/mL each), after which sorted Foxp3⁺ cells were assayed for latency-associated protein (LAP) expression by flow cytometry. H: Sorted Foxp3^– CD4⁺ T cells from WT, Daf1^–/–, and C3ar1^–/–C5ar1^–/– mice were activated for 3 days with anti-CD3/28 Dynabeads + C3aR-A/C5aR-A (10 μg/mL each), after which sorted Foxp3⁺ cells were assayed for integrin β6 (Itg-β6) and Itg-β8 mRNA expression by qPCR. I: CD103⁺CD11c⁺ cells from the mLN of WT, Daf1^–/–, and C3ar1^–/–C5ar1^–/– mice were assayed for Itg-β6 expression by flow cytometry. n = 10 (A–D). ^∗P < 0.05, ^∗∗P < 0.01. MFI, mean fluorescent intensity.

Supplemental Figure S1

Representative flow cytometry plots. A: Gating strategy for isolating cells for assaying regulatory T cells. B: Top panel: Gating strategy for analyzing dendritic cells (DCs) from the mesenteric lymph nodes (mLNs). Arrows designate the cells gated to eliminate doublets that were analyzed. Bottom panels: Representative flow plots for CD11c⁺ DCs harvested from the mLN of wild-type (WT) and Daf1^–/–C3ar1^–/–C5ar1^–/– mice assayed for expression of CD40, B7.1, B7.2, major histocompatibility complex (MHC) II, PD-L1, and ICOS-L by flow cytometry. C: CD11c⁺ DCs were harvested from the inguinal LN of WT, Daf1^–/–, and Daf1^–/–C3ar1^–/–C5ar1^–/– mice and assayed for expression of CD40, B7.1, B7.2, MHC II, PD-L1, and ICOS-L by flow cytometry. D: CD11b⁺F4/80⁺ macrophages harvested from mesenteric LNs of WT, Daf1^–/–, and C3ar1^–/–C5ar1^–/– mice were assayed for expression of CxCR3, CCR7, and CCR9 by flow cytometry. n = 10 (B–D). ^∗P < 0.05. FSC, forward scatter; MFI, mean fluorescent intensity; SSC, side scatter.

Supplemental Figure S2

Comparative transforming growth factor (TGF)-β, IL-10, and interferon (IFN)-γ production by Daf1^−/−, wild-type (WT), and C3ar1^–/–C5ar1^−/− CD4⁺ cells and effects of C3a/C5a signaling on the phenotypes of dendritic cells (DCs) from each genotype. A and B: A total of 1 × 10⁶ splenocytes from WT, Daf1^–/–, and Daf1^–/–C3ar1^–/–C5ar1^–/– mice were incubated in RPMI 1640 media for 24 hours, after which IFN-γ, IL-10, and TGF-β1 expression levels were assayed by internal staining. A: Left panel: Representative histograms. Right panel: Mean fluorescent intensity (MFI). C: Phenotypes of DCs from each genotype homeostatically (left and middle panels) and the inability of anti-C3a/anti-C5a monoclonal antibody blockade to reverse the maturity of Daf1^−/− DCs (right panel). D: Effects of C3a/C5a or C3ar1/C5ar1 antagonists on costimulatory and coinhibitory molecule expression by WT CD11c⁺ F40^– DCs. n = 6 (A, right panel, B, left and right panels, and D). ^∗P < 0.05, ^∗∗P < 0.01. MHC, major histocompatibility complex.

Supplemental Figure S3

Representative flow cytometry plots. Representative flow plots from CD103⁺CD11c⁺ (A) and wild-type (WT), Daf1^–/–, and C3ar1^–/–C5ar1^–/– (B) dendritic cells assayed for B7-1, B7-2, CD40, PD-L1, and ICOS-L expression levels by flow cytometry. smLN, submandibular lymph node.