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. 2002 Dec 16;196(12):1627-38.
doi: 10.1084/jem.20021598.

Efficient targeting of protein antigen to the dendritic cell receptor DEC-205 in the steady state leads to antigen presentation on major histocompatibility complex class I products and peripheral CD8+ T cell tolerance

Laura Bonifaz  1 , David Bonnyay, Karsten Mahnke, Miguel Rivera, Michel C Nussenzweig, Ralph M Steinman
Affiliations

Efficient targeting of protein antigen to the dendritic cell receptor DEC-205 in the steady state leads to antigen presentation on major histocompatibility complex class I products and peripheral CD8+ T cell tolerance

Laura Bonifaz et al. J Exp Med. .

Abstract

To identify endocytic receptors that allow dendritic cells (DCs) to capture and present antigens on major histocompatibility complex (MHC) class I products in vivo, we evaluated DEC-205, which is abundant on DCs in lymphoid tissues. Ovalbumin (OVA) protein, when chemically coupled to monoclonal alphaDEC-205 antibody, was presented by CD11c+ lymph node DCs, but not by CD11c- cells, to OVA-specific, CD4+ and CD8+ T cells. Receptor-mediated presentation was at least 400 times more efficient than unconjugated OVA and, for MHC class I, the DCs had to express transporter of antigenic peptides (TAP) transporters. When alphaDEC-205:OVA was injected subcutaneously, OVA protein was identified over a 4-48 h period in DCs, primarily in the lymph nodes draining the injection site. In vivo, the OVA protein was selectively presented by DCs to TCR transgenic CD8+ cells, again at least 400 times more effectively than soluble OVA and in a TAP-dependent fashion. Targeting of alphaDEC-205:OVA to DCs in the steady state initially induced 4-7 cycles of T cell division, but the T cells were then deleted and the mice became specifically unresponsive to rechallenge with OVA in complete Freund's adjuvant. In contrast, simultaneous delivery of a DC maturation stimulus via CD40, together with alphaDEC-205:OVA, induced strong immunity. The CD8+ T cells responding in the presence of agonistic alphaCD40 antibody produced large amounts of interleukin 2 and interferon gamma, acquired cytolytic function in vivo, emigrated in large numbers to the lung, and responded vigorously to OVA rechallenge. Therefore, DEC-205 provides an efficient receptor-based mechanism for DCs to process proteins for MHC class I presentation in vivo, leading to tolerance in the steady state and immunity after DC maturation.

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Figures

Figure 1.
Figure 1.
Conjugation and delivery of OVA via monoclonal antibodies. (A) OVA conjugated and unconjugated IgGs resolved by SDS-PAGE and stained with Coomassie blue. (B) Immunoblotting with anti-OVA antibody to detect OVA in the Ig:OVA conjugates and in CD11c+ lymph node DCs (106/lane), the latter at 12 and 24 h after subcutaneous injection of 1 μg of αDEC-205:OVA or isotype control:OVA. (C) The subcutaneous injection of 1 μg of αDEC-205:OVA per footpad, but not 10 μg of soluble OVA, targets CD11c+ lymph node DCs. 106 CD11c+ cells from the draining nodes per lane were immuno-blotted 12–72 h after injection. (D) αDEC-205:OVA uptake occurs primarily in CD11c+ DCs in the draining lymph nodes. As in C, but cell fractions were prepared from draining lymph nodes, distal (Dist.) nodes and spleen (Spl.).
Figure 2.
Figure 2.
Targeting of αDEC-205:OVA to CD11c+ lymph node DCs in vitro. (A) αDEC-205:OVA elicits stronger presentation than OVA alone in a dose-dependent manner. CD11c+ cells from C57BL/6 lymph nodes were cultured 18 h in graded doses of αDEC-205:OVA or OVA alone, washed, and cocultured with OT-I or OT-II T cells before measuring uptake of [3H]thymidine at 48–72 h to assess T cell proliferation. (B) Presentation of peptides derived from αDEC-205:OVA is restricted to CD11c+ lymph node cells, and not the CD19+ or CD11c−CD19− (double negative) fractions. As in A, but with αDEC-205:OVA or the isotype:OVA conjugate at 10 μg/ml. (C) Presentation of peptides derived from αDEC-205:OVA is TAP dependent. As in B, but CD11c+ cells were prepared from C57BL/6 or TAP−/− mice. (D) Bone marrow DCs are unable to present αDEC-205:OVA on MHC class I products. Cells from d6 cultures were harvested and cultured with antibody:OVA conjugates for 6 h at 10 μg/ml, washed, and cocultured with T cells as in panel A. Data are representative of three experiments.
Figure 3.
Figure 3.
Targeting of αDEC-205:OVA to lymph node CD11c+ DCs in vivo. (A) Only CD11c+ lymph node DCs efficiently present exogenous αDEC-205:OVA, and to a lesser extent OVA, to OT-I T cells. C57BL/6 mice were injected with 4.0 μg (1.0 μg/footpad) of αDEC-205:OVA conjugate or 400 μg (100 μg/footpad) of soluble OVA subcutaneously 4 and 24 h before sacrifice. The CD11c+ and CD11c−CD5− (B cell) fractions were MACS® sorted from lymph nodes and evaluated for presentation to OT-I T cells as in Fig. 2 A. Peptide controls were performed with the highest titration of APCs (DCs, left; B cells, right) for each group. (B) As in panel A but CD11c+ DC's were studied 1 and 4 d after injection of 4.0 μg (1.0 μg/footpad) of antibody:OVA conjugates subcutaneously. (C) Presentation by DCs of OVA peptides from C57BL/6 but not TAP−/− mice given 4.0 μg (1.0 μg/footpad) of IgG:OVA conjugates subcutaneously 4 d earlier. (D) αDEC-205:OVA elicits better presentation of OVA derived peptides than other DC-targeted conjugates, each injected with 4.0 μg (1.0 μg/footpad) of IgG:OVA conjugates subcutaneously 4 d earlier. (E) αDEC-205:OVA induces stronger in vivo proliferation of OT-I T cells than OVA alone. C57BL/6 mice were injected intravenously with 2 × 106 CFSE-labeled OT-I T cells and then graded doses of IgG:OVA conjugates or OVA subcutaneously 24 h later. 3 d after conjugate injection, lymph nodes were harvested and the expansion of CD8+Vα2Vβ5.1/5.2 cells evaluated by flow cytometry for CFSE dilution. Each panel represents two or more experiments.
Figure 4.
Figure 4.
Maturation of DCs in vivo by agonistic αCD40 but not by αDEC-205:OVA. (A) C57BL/6 mice were injected subcutaneously with PBS or 4.0 μg (1.0 μg/footpad) of αDEC-205:OVA conjugate with or without αCD40 (100 μg FGK45.5 subcutaneously), 1 and 3 d before sacrifice. CD11c+ cells were sorted by MACS® from lymph nodes and evaluated by flow cytometry for expression of CD80, CD86, and MHC class II. Prior to injection of the OVA conjugate and αCD40, the mice were given PBS (−) or OT-I (+) cells. The bold symbols are mean fluorescence indices of the CD11c+ cells in the presence of a maturation stimulus, while the gray-bold at day 3 indicate a significant increase, consistent with maturation. (B) Illustrative FACS® data showing the maturation of the DEC-205hi CD11c+ cells and DEC-205loCD11c+ cells, in mice treated 3 d before with PBS and αCD40 as in panel A.
Figure 5.
Figure 5.
Contrasting responses of OT-I T cells to αDEC-205:OVA in the presence or absence of αCD40-induced DC maturation. (A) αCD40 has little impact on αDEC-205:OVA induced proliferation of OT-I T cells. As in Fig. 3 E, but mice were or were not given αCD40 (100 μg FGK45.5 subcutaneously). (B) Differential IL-2 and IFN-γ production by OT-I T cells in response to isotype:OVA or αDEC-205:OVA with or without αCD40. As in panel A, but lymph node suspensions were restimulated in vitro with the cognate OT-I peptide for 5 h in the presence of brefeldin A (5 μg/ml) before staining for intracellular cytokine. Data are representative of three experiments.
Figure 6.
Figure 6.
Deletion of OT-I T cells in response to αDEC-205:OVA in steady state. (A) C57BL/6 mice were given CD45.1+ OT-I T cells and antigen as described in 3E with or without αCD40. 3 and 12 d later, lymph nodes, spleen, and blood were harvested and evaluated for OT-I T cells (CD45.1+Vβ5.1/5.2 +) by flow cytometry. (B) Data on the number of OT-I cells, expressed as the mean percentage of CD8+ T cells from three experiments of the type shown in panel A. (C) αCD40-rescued OT-I T cells are primed and secrete IFN-γ. C57BL/6 mice were given OT-I T cells and antigen as described in panel A. 12 d after antigen administration, lymph nodes were harvested and OT-I T cells evaluated for IFN-γ secretion as described in Fig. 5 B. (D) OT-I T cells are not present in a peripheral non-lymphoid tissue, after presentation of αDEC-205:OVA by DCs in the steady state, in the absence of αCD40 stimulation. As in panel A, but the lung was harvested 10 d after antigen administration and the OT-I cells were evaluated for expression of CD62L and CD45.1.
Figure 7.
Figure 7.
αDEC-205:OVA induces peripheral tolerance to OVA in the steady state. (A) C57BL/6 mice were given CD45.1+ OT-I T cells and antigen as described in 3E with or without αCD40. 12 d after antigen administration, mice were boosted with 50 μg of OVA protein in complete CFA. After 3 d, lymph nodes were harvested and OT-I T cells evaluated for secretion of IL-2 (top) or IFN-γ (bottom) as in Fig. 5 B. (B) C57BL/6 mice were treated as in panel A, but 3 d after administration of OVA in CFA, mice were injected intravenously with a mixture of CFSE-labeled syngeneic splenocytes pulsed with (CFSEhi) or without (CFSElo) the OT-I cognate peptide (3 × 106 of each). 12 h later the loss of CFSEhi cells in lymph nodes was evaluated as a measure of specific CTL activity. Naive mice do not exhibit any loss of CFSE labeled cells (not shown). The results are representative of three experiments.
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